bluuebunny/crossref_metadata_2025 huggingface dataset:
The Suprapharmacologic Dosing of Antithrombin Concentrate for Staphylococcus Aureus-Induced Disseminated Intravascular Coagulation in Guinea Pigs: Substantial Reduction in Mortality and Morbidity
Authors: Kessler Craig M. , Tang ZhaoCheng , Jacobs Helena M. , Szymanski Linda M.
Published: October 2019

An animal model of gram-positive septicemia was developed to evaluate the effects of antithrombin (AT) concentrates on morbidity, mortality, and laboratory consequences of disseminated intravascular coagulation (DIC). DIC was induced in guinea pigs by infusing Staphylococcus aureus (SA) isolated from blood cultures of patients with DIC (DIC-SA) or without DIC (non–DIC-SA). The non–DIC-SA animals and animals infused with sterile saline served as controls. Varying doses of AT were administered either 30 minutes or 24 hours after infusion of SA. DIC was confirmed within 4 hours by changes in prothrombin time, activated partial thromboplastin time, fibrinogen, fibrinogen-fibrin degradation products, and AT activity. Clinical bleeding was also evident. Mortality of untreated DIC-SA animals was 36% within 24 hours and up to 75% by 72 hours. Intervention with any dose of AT between 125 and 1,000 IU/kg 30 minutes after DIC-SA infusion was associated with 100% survival (P ≤ .05 in the 250 IU/kg group) and sustained increases in AT activity and fibrinogen concentrations (P ≤ .05). When AT was administered in combination with low molecular weight heparin (LMWH) or if LMWH was adminstered alone, mortality from DIC-SA was slightly, but not significantly reduced compared with untreated DIC-SA. Gross hemorrhage was observed premortem and at autopsy in all of the DIC-SA animals but in substantially fewer animals that received AT (P ≤ .001 in the 250, 500, and 1,000 IU/kg groups). In contrast, groups treated with LMWH, alone or with AT, experienced hemorrhage and appeared to develop pathologic DIC. Fibrin formation in end-organs was detected in all guinea pigs in the untreated DIC-SA group and in the groups treated with 125 IU/kg AT and LMWH alone. AT doses between 250 and 1,000 IU/kg administered 30 minutes after DIC-SA infusion prevented fibrin formation in end-organs (P ≤ .001 in the 250 and 1,000 IU/kg groups). AT administered 24 hours after DIC-SA could not reverse pre-existing histopathologic evidence of DIC but favorably affected survival, which reached statistical significance in the 1,000 IU/kg AT group (P ≤ .025). In summary, suprapharmacologic doses of AT concentrate significantly decreased morbidity and mortality and ameliorated adverse changes in laboratory measures induced by DIC-SA in this guinea pig model and were not associated with untoward hemorrhagic complications. These findings provide justification for studying the use of AT therapy in patients with DIC-SA.
Mechanisms of Stroke in Sickle Cell Disease: Sickle Erythrocytes Decrease Cerebral Blood Flow in Rats After Nitric Oxide Synthase Inhibition
Authors: French James A. , Kenny Dermot , Scott J. Paul , Hoffmann Raymond G. , Wood James D. , Hudetz Antal G. , Hillery Cheryl A.
Published: October 2019

The etiology of stroke in sickle cell disease is unclear, but may involve abnormal red blood cell (RBC) adhesion to the vascular endothelium and altered vasomotor tone regulation. Therefore, we examined both the adhesion of sickle (SS)-RBCs to cerebral microvessels and the effect of SS-RBCs on cerebral blood flow when the nitric oxide (NO) pathway was inhibited. The effect of SS-RBCs was studied in the rat cerebral microcirculation using either a cranial window for direct visualization of infused RBCs or laser Doppler flowmetry (LDF ) to measure RBC flow. When fluorescently labeled human RBCs were infused into rats, SS-RBCs had increased adhesion to rat cerebral microvessels compared with control AA-RBCs (P = .01). Next, washed SS-RBCs or AA-RBCs were infused into rats prepared with LDF probes after pretreatment (40 mg/kg intravenously) with the NO synthase inhibitor, N-ω-nitro-L-arginine methyl ester (L-NAME), or the control isomer, D-NAME. In 9 rats treated with systemic L-NAME and SS-RBCs, 5 of 9 experienced a significant decrease in LDF and died within 30 minutes after the RBC infusion (P = .0012). In contrast, all control groups completed the experiment with stable LDF and hemodynamics. Four rats received a localized superfusion of L-NAME (1 mmol/L) through the cranial window followed by infusion of SS-RBCs. Total cessation of flow in all observed cerebral microvessels occurred in 3 of 4 rats within 15 minutes after infusion of SS-RBCs. We conclude that the NO pathway is critical in maintaining cerebral blood flow in the presence of SS-RBCs in this rat model. In addition, the enhanced adhesion of SS-RBCs to rat brain microvessels may contribute to cerebral vaso-occlusion either directly, by disrupting blood flow, or indirectly, by disturbing the vascular endothelium.
Expression of the T-Cell Activation Antigen, OX-40, Identifies Alloreactive T Cells in Acute Graft-Versus-Host Disease
Authors: Tittle Thomas V. , Weinberg Andrew D. , Steinkeler Cara N. , Maziarz Richard T.
Published: October 2019

The OX-40 molecule is expressed on the surface of recently activated T lymphocytes. The presence of OX-40 on CD4+ T cells was analyzed in a rat haplo-identical (parental → F1) bone marrow transplant model of acute graft-versus-host disease (aGVHD). Increased numbers of activated CD4+ T cells that expressed the OX-40 antigen were detected in peripheral blood soon after transplantation before the earliest sign of disease. The peak of OX-40 expression occurred 12 days posttransplantation with a range of 18% to 36% of circulating T cells and remained 10-fold above background, never returning to baseline. A slight increase in OX-40 expression (range, 1% to 6%) was also detected on peripheral blood lymphocytes from control syngeneic F1 → F1 recipients. OX-40+ T cells were isolated from spleen, skin, lymph node, and liver tissue of rats undergoing aGVHD, but not in syngeneic transplants. OX-40+ T cells isolated from these tissues were of donor origin and were shown to be allo-reactive. These data raise the possibility of using the OX-40 antibody to detect and deplete selectively the T cells that cause aGVHD.
Altered Myeloid Development and Acute Leukemia in Transgenic Mice Expressing PML-RARα Under Control of Cathepsin G Regulatory Sequences
Authors: Grisolano Jay L. , Wesselschmidt Robin L. , Pelicci Pier Giuseppe , Ley Timothy J.
Published: October 2019

Acute promyelocytic leukemia (APML) is characterized by abnormal myeloid development, resulting an accumulation of leukemic promyelocytes that are often highly sensitive to retinoic acid. A balanced t(15; 17) (q22; q21) reciprocal chromosomal translocation is found in approximately 90% of APML patients; this translocation fuses the PML gene on chromosome 15 to the retinoic acid receptor α (RARα) gene on chromosome 17, creating two novel fusion genes, PML-RARα and RARα-PML. The PML-RARα fusion gene product, which is expressed in virtually all patients with t(15; 17), is thought to play a direct role in the pathogenesis of APML. To determine whether PML-RARα is sufficient to cause APML in an animal model, we used the promyelocyte-specific targeting sequences of the human cathepsin G (hCG) gene to direct the expression of a PML-RARα cDNA to the early myeloid cells of transgenic mice. Mice expressing the hCG–PML-RARα transgene were found to have altered myeloid development that was characterized by increased percentages of immature and mature myeloid cells in the peripheral blood, bone marrow, and spleen. In addition, approximately 30% of transgene-expressing mice eventually developed acute myeloid leukemia after a long latent period. The splenic promyelocytes of mice with both the nonleukemic and leukemic phenotypes responded to all-trans retinoic acid (ATRA) treatment, which caused apoptosis of myeloid precursors. Although low-level expression of the hCG–PML-RARα transgene is not sufficient to directly cause acute myeloid leukemia in mice, its expression alters myeloid development, resulting in an accumulation of myeloid precursors that may be susceptible to cooperative transforming events.
Interactions Between c-kit and Stem Cell Factor Are Not Required for B-Cell Development In Vivo
Authors: Takeda Shunichi , Shimizu Takeyuki , Rodewald Hans-Reimer
Published: October 2019

The receptor-type tyrosine kinase, c-kit is expressed in hematopoietic stem cells (HSC), myeloid, and lymphoid precursors. In c-kit ligand-deficient mice, absolute numbers of HSC are mildly reduced suggesting that c-kit is not essential for HSC development. However, c-kit− HSC cannot form spleen colonies or reconstitute hematopoietic functions in lethally irradiated recipient mice. Based on in in vitro experiments, a critical role of c-kit in B-cell development was suggested. Here we have investigated the B-cell development of c-kitnull mutant (W/W ) mice in vivo. Furthermore, day 13 fetal liver cells from wild type or W/W mice were transferred into immunodeficient RAG-2−/− mice. Surprisingly, transferred c-kit− cells gave rise to all stages of immature B cells in the bone marrow and subsequently to mature conventional B2, as well as B1, type B cells in the recipients to the same extent as transferred wild type cells. Hence, in contrast to important roles of c-kit in the expansion of HSC and the generation of erythroid and myeloid lineages and T-cell precursors, c-kit− HSC can colonize the recipient bone marrow and differentiate into B cells in the absence of c-kit.
Activation of JAK2 in Human Vascular Endothelial Cells by Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Soldi Raffaella , Primo Luca , Brizzi Maria Felice , Sanavio Fiorella , Aglietta Massimo , Polentarutti Nadia , Pegoraro Luigi , Mantovani Alberto , Bussolino Federico
Published: October 2019

Besides the regulation of hematopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the expression of a functional program in endothelial cells (ECs) related to angiogenesis and to their survival in the bone marrow microenvironment. ECs express specific GM-CSF high-affinity binding sites, which mediate the proliferative and migratory response. We now report that ECs express the α and β subunits of GM-CSF receptor (GM-CSFR), and that GM-CSF is able to activate the Janus kinase (JAK)2, a member of the cytosolic tyrosine kinase family, which is known to mediate signals of several non–tyrosine kinase receptors. JAK2 tyrosine phoshorylation, as well as activation of its catalytic activity, is induced by subnanomolar concentrations of GM-CSF and occurs within 3 minutes of stimulation and persists at least for 10 minutes. The effect is specific as inferred by the lack of effect of heat-inactivated GM-CSF or neutralized by specific antibodies and by the finding that interleukin-5, which utilizes a specific α chain and the same β chain of GM-CSFR, does not phosphorylate JAK2. Furthermore, we show that the amount of JAK2 physically associated with GM-CSFR β chain is increased after GM-CSF stimulation and that GM-CSF triggers both β chain and JAK2 tyrosine phosphorylation. Taken together, these results suggest that biologic activities of GM-CSF in vascular endothelium may, in part, be elicited by GM-CSFR–mediated JAK2 activation.
Hepatocyte Growth Factor/Scatter Factor Promotes Adhesion of Lymphoma Cells to Extracellular Matrix Molecules Via α4β1 and α5β1 Integrins
Authors: Weimar Iris S. , de Jong Daphne , Muller Egbert J. , Nakamura Toshikazu , M.H.H. van Gorp Joost , de Gast Gijsbert C. , Gerritsen Winald R.
Published: October 2019

Hepatocyte growth factor (HGF )/scatter factor (SF ) is the ligand for a tyrosine kinase cell surface receptor encoded by the MET protooncogene (c-MET). HGF/SF can induce proliferation and motility in epithelial cells and promotes invasion of carcinoma cells and NIH3T3 fibroblasts transfected with both HGF/SF and c-MET genes. Our results show that HGF/SF and c-MET also play a role in adhesion and invasion of human lymphoma cells. c-MET mRNA is expressed in hemopoietic cells, such as hemopoietic progenitor cells (CD34+ cells) in bone marrow (BM) and mobilized peripheral blood, immature B cells in cord blood and BM, and germinal center B-centroblasts. In normal peripheral blood B cells, which are c-MET−, c-MET expression was induced by PMA, ConA, HGF/SF, and Epstein-Barr virus (EBV) infection. Using immunohistochemistry, we detected c-MET on the cell surface of large activated centroblasts in lymph nodes from patients with B-non–Hodgkin's lymphoma and Hodgkin's disease. In the latter group, c-MET expression correlated well with the presence of EBV. Because HGF/SF and c-MET promote metastasis of carcinoma cells, we studied the effects of c-MET stimulation by HGF/SF of B-lymphoma cells on properties relevant for metastasis, ie, adhesion, migration, and invasion. HGF/SF stimulated adhesion of the c-MET+ B-cell lines to the extracellular matrix molecules fibronectin (FN) and collagen (CN) in a dose dependent manner. However, adhesion to laminin was not affected by HGF/SF. Adhesion to FN was mediated by β1-integrins α4β1 (VLA4) and α5β1 (VLA5) since blocking antibodies against β1- (CD29), α4- (CD49d), or α5- (CD49e) integrin subunits, completely reversed the effect of HGF/SF. Furthermore, HGF/SF induced adhesion was abrogated by addition of genistein, which blocks protein tyrosine kinases, including c-MET. Addition of HGF/SF resulted in a sixfold increase in migration of c-MET B-lymphoma cells through Matrigel, compared to medium alone. In rat fibroblast cultures, HGF/SF doubled the number of c-MET+ B-lymphoma cells that invaded the fibroblast monolayer. In these adhesion, migration and invasion assays HGF/SF had no effect on c-MET− cell lines. In conclusion, c-MET is expressed or can be induced on immature, activated, and certain malignant B cells. HGF/SF increased adhesion of c-MET+ B-lymphoma cells to FN and CN, mediated via β1-integrins α4β1 and α5β1 , and furthermore promoted migration and invasion.
Thrombopoietin Enhances Proliferation and Differentiation of Murine Yolk Sac Erythroid Progenitors
Authors: Era Takumi , Takahashi Tomomi , Sakai Katsuya , Kawamura Kazuo , Nakano Toru
Published: October 2019

Thrombopoietin (TPO), the ligand for the receptor proto-oncogene c-Mpl, has been cloned and shown to be the critical regulator of proliferation and differentiation of megakaryocytic lineage. Initially, TPO was not considered to have the activity on hematopoietic lineages other than megakaryocytes. Recently, however, TPO was reported to enhance the in vitro erythroid colony formation from human bone marrow (BM) CD34+ progenitors or from mouse BM cells in combination with other cytokines. We examined the effects of TPO on the colony formation of hematopoietic progenitors in mouse yolk sac. TPO remarkably enhanced proliferation and differentiation of erythroid-lineage cells in the presence of erythropoietin (Epo). This effect was observed even in the absence of Epo. Compared with adult BM, yolk sac turned out to have relatively abundant erythroid and erythro-megakaryocytic progenitors, which responded to TPO and Epo stimulation. TPO similarly stimulated erythroid colony formation from in vitro differentiation-induced mouse embryonic stem (ES) cells whose hematopoietic differentiation status was similar to that of yolk sac. These findings help to understand the biology of hematopoietic progenitors of the early phase of hematopoiesis. Yolk sac cells or in vitro differentiation-induced ES cells would be good sources to analyze the TPO function on erythropoiesis.
Studies of a Second Family With the Quebec Platelet Disorder: Evidence That the Degradation of the α-Granule Membrane and Its Soluble Contents Are Not Secondary to a Defect in Targeting Proteins to α-Granules
Authors: Hayward Catherine P.M. , Cramer Elisabeth M. , Kane William H. , Zheng Shilun , Bouchard Madeleine , Massé Jean-Marc , Rivard Georges E.
Published: October 2019

We recently described a Quebec family with an autosomal dominant bleeding disorder characterized by mildly reduced-low normal platelet counts, an epinephrine aggregation defect, multimerin deficiency, and proteolytic degradation of several, soluble α-granular proteins. Similar clinical features led us to investigate a second family with an unexplained, autosomal dominant bleeding disorder. The affected individuals had reduced to normal platelet counts, absent platelet aggregation with epinephrine, and multimerin deficiency. Their platelet α-granular proteins factor V, thrombospondin, von Willebrand factor, fibrinogen, fibronectin, osteonectin, and P-selectin were proteolyzed and comigrated with the degradation products found in patients from the other family. However, their platelet albumin, IgG, external membrane glycoproteins, CD63 (a lysosomal and dense granular protein), calpain, and plasma von Willebrand factor were normal, indicating restriction in the proteins proteolyzed. Electron microscopy studies indicated preserved α-granular ultrastructure, despite degradation of soluble and membrane α-granular proteins. Immunoelectron microscopy studies of the patients' platelets indicated that fibrinogen, von Willebrand factor, P-selectin, multimerin, and factor V were within α-granules, with normal to reduced labeling for these proteins. Pathologic proteolysis of α-granular contents, rather than a defect in targeting proteins to α-granules, may be the cause of the protein degradation in the Quebec platelet disorder.
Molecular Mechanism of a Mild Phenotype in Coagulation Factor XIII (FXIII) Deficiency: A Splicing Mutation Permitting Partial Correct Splicing of FXIII A-Subunit mRNA
Authors: Mikkola Hanna , Muszbek Laszlo , Laiho Elina , Syrjälä Martti , Hämäläinen Eija , Haramura Gizela , Salmi Toivo , Peltonen Leena , Palotie Aarno
Published: October 2019

Congenital factor XIII (FXIII) deficiency is potentially a severe bleeding disorder, but in some cases, the symptoms may be fairly mild. In this study, we have characterized the molecular mechanism of a mild phenotype of FXIII A-subunit deficiency in a Finnish family with two affected sisters, one of whom has even had two successful pregnancies without regular substitution therapy. In the screening tests for FXIII deficiency, no A-subunit could be detected, but by using more sensitive assays, a minute amount of functional A-subunit was seen. 3H-putrescine incorporation assay showed distinct FXIII activity at the level of 0.35% of controls, and also the fibrin cross-linking pattern in the patients clotted plasma showed partial γ-γ dimerization. In Western blot analysis, a faint band of full-length FXIII A-subunit was detected in the patients' platelets. The patients have previously been identified as heterozygotes for the Arg661 → Stop mutation. Here we report a T → C transition at position +6 of intron C in their other allele. The transition affected splicing of FXIII mRNA resulting in low steady state levels of several variant mRNA transcripts. One transcript contained sequences of intron C, whereas two transcripts resulted from skipping of one or two exons. Additionally, correctly spliced mRNA lacking the Arg661 → Stop mutation of the maternal allele could be detected. These results demonstrate that a mutation in splice donor site of intron C can result in several variant mRNA transcripts and even permit partial correct splicing of FXIII mRNA. Further, even the minute amount of correctly processed mRNA is sufficient for producing protein capable of γ-γ dimerization of fibrin. This is a rare example of an inherited functional human disorder in which a mutation affecting splicing still permits some correct splicing to occur and this has a beneficial effect to the phenotype of the patients.
CD2 Regulates the Positive Selection and Function of Antigen-Specific CD4−CD8+ T Cells
Authors: Teh Soo-Jeet , Killeen Nigel , Tarakhovsky Alexander , Littman Dan R. , Teh Hung-Sia
Published: October 2019

The CD2 glycoprotein has been implicated in both positive and negative regulation of T-cell mitogenesis. To study the involvement of CD2 in T-lymphocyte development and immune responses, we have analyzed two lines of CD2-null mice, each expressing a distinct class I major histocompatibility complex (MHC)-restricted T-cell receptor (TCR). In both situations, the absence of CD2 appeared to promote the positive selection of cells in a manner that is similar to that which occurs in the absence of CD5. Consistent with this, compound homozygotes that lacked both CD2 and CD5 showed evidence of enhanced positive selection even in the absence of a transgenic TCR. Despite the observed enhancement of positive selection, the lack of CD2 was associated with defects in proliferative responses and interferon-γ production when transgenic thymocytes and mature T lymphocytes were stimulated with the appropriate antigens. These findings raise the possibility that impaired sensitivity to selecting ligands in the thymus may provide a selective advantage that improves the efficiency of positive selection for certain TCRs. Furthermore, the results highlight the potential for a differential role for CD2 in thymocyte selection and T-cell immune responses.
Characterization of T-Cell Clones Derived From Peripheral Blood Lymphocytes of a Patient With Transfusion-Associated Graft-Versus-Host Disease: Fas-Mediated Killing by CD4+ and CD8+ Cytotoxic T-Cell Clones and Tumor Necrosis Factor β Production by CD4+ T-Cell Clones
Authors: Nishimura Motoko , Uchida Shigeharu , Mitsunaga Shigeki , Yahagi Yuji , Nakajima Kazunori , Tadokoro Kenji , Juji Takeo
Published: October 2019

Transfusion-associated graft-versus-host disease (TA-GVHD) is one of the most serious adverse effects of blood transfusion. It is generally thought to be caused by the infused lymphocytes. Donor-derived cytotoxic T lymphocytes (CTLs) directed against the recipient's HLAs, which have escaped the recipient's immune system and are proliferating, are considered to attack recipient organs and tissues. Despite the seriousness of the disease, the precise mechanism of its development remains unclear and no definitive treatment has been developed. With the aim of developing an effective treatment, we established and characterized T-cell clones from peripheral blood lymphocytes (PBLs) of a TA-GVHD patient. Three types of clones were established. Type I clones were CD8+ and specifically lyse cells that express HLA B52. Type II clones were CD4+, specifically lysed cells that express HLA DR15, and proliferated in response to stimulation with cells that express DR15. Type III clones were also CD4+, showed no cytotoxic activity toward any HLA-expressing cells, and proliferated in response to stimulation with cells that express DR15. Furthermore, we found that the Fas/Fas-ligand (Fas-L) system is involved in the cytotoxicity of the type I and II clones and that the type III clones produce and secrete a large amount of tumor necrosis factor β (TNFβ) after antigen stimulation. Based on our results, these three types of clones can be classified into two categories: those that have the ability to induce GVHD directly by cytolysis and that show no cytotoxic activity and those that have the ability to cause GVHD indirectly through secretion of cytotoxic lymphokines.
Vascular Endothelial Growth Factor, a Possible Paracrine Growth Factor in Human Acute Myeloid Leukemia
Authors: Fiedler Walter , Graeven Ullrich , Ergün Süleyman , Verago Sandra , Kilic Nerbil , Stockschläder Marcus , Hossfeld Dieter K.
Published: October 2019

Vascular endothelial growth factor (VEGF ) is a multifunctional cytokine involved in angiogenesis, inflammation, and wound healing. It is secreted by a variety of tumor cell lines, including hematopoietic lines. Therefore, we investigated expression of VEGF and its receptors on fresh leukemic blasts. VEGF-specific transcripts were detected by polymerase chain reaction (PCR) in 20 of 28 patients with de novo acute myeloid leukemia (AML) and in 3 of 5 patients with secondary AML. Using immunocytochemistry, we found VEGF protein in 2 leukemic cell lines and in 8 AML patients, in concordance with PCR results. Supernatants of fresh leukemic cells from 24 AML patients contained significantly more VEGF than supernatants from bone marrow cells of 9 normal donors or of CD34-enriched cells from 3 normal volunteer donors as determined by an enzyme-linked immunosorbent assay. VEGF possesses two high-affinity receptors, KDR and FLT1. Using a sensitive nested PCR assay, we detected expression of FLT1 in 10 of 20 patients with de novo AML and 3 of 5 patients with secondary AML. KDR was expressed in 4 of 22 patients with de novo AML and 1 of 4 with secondary AML. To study possible paracrine growth stimulation of AML blasts, endothelial cells from human umbilical cords were incubated with increasing concentrations of VEGF. A dose-dependent increase of granulocyte-macrophage colony-stimulating factor secretion from endothelial cells was identified.
Sorting of the Specific Granule Protein, NGAL, During Granulocytic Maturation of HL-60 Cells
Authors: Le Cabec Véronique , Calafat Jero , Borregaard Niels
Published: October 2019

The different types of human neutrophil granules (azurophil, specific, and gelatinase granules) are formed sequentially during maturation of neutrophils from the promyelocyte stage to the band cell stage. The promyelocytic HL-60 cells can maturate to segmented granulocytes but are incapable of activating the transcription of any known intragranular protein, normally located in specific or gelatinase granules. To study the sorting of granule proteins during maturation, we transfected HL-60 cells with the specific granule protein NGAL, inserted under control of a cytomegalovirus promoter. We previously showed that NGAL is sorted to azurophil granules and colocalizes with myeloperoxidase in undifferentiated HL-60 cells. We show here that, when such transfected HL-60 cells differentiate into granulocytes, newly synthesized NGAL is not retained in granules but is constitutively secreted. This indicates that highly specific mechanisms must exist that are responsible for diverting transport vesicles into storage granules, and that HL-60 cells not only lack the ability to activate transcription of specific granule proteins, but also lose the ability to form storage granules during maturation.
A Model for Human B-Chronic Lymphocytic Leukemia in Human/Mouse Radiation Chimera: Evidence for Tumor-Mediated Suppression of Antibody Production in Low-Stage Disease
Authors: Shimoni Avichai , Marcus Hadar , Canaan Allon , Ergas David , David Magda , Berrebi Alain , Reisner Yair
Published: October 2019

B-chronic lymphocytic leukemia (BCLL) is a lymphoproliferative disease that is characterized by clonal expansion of CD5+ B cells. BCLL is associated with secondary immunodeficiency and hypogammaglobulinemia. It has been suggested that T-cell dysregulation may play a role in the hypogammaglobulinemia and in the increased incidence of autoimmunity in BCLL patients. We attempted to transfer human peripheral blood mononuclear cells (PBMC) from BCLL patients in different stages of the disease into immunodeficient mice. PBMC from BCLL patients in stage 0, stages I to II, and stages III to IV were transplanted into the peritoneal cavity of lethally irradiated Balb/c or beige/nude/Xid (BNX) mice radioprotected with bone marrow (BM) from severe combined immunodeficiency (SCID) mice. Different engraftment profiles were found in the chimeric mice 2 weeks after transplantation of PBMC according to the disease stage of the BCLL donors. Infusion of PBMC from donors in stage 0 led to marked engraftment of human T cells, whereas the human tumor cells could hardly be detected. In contrast, chimeric mice receiving PBMC from patients in stage III to IV disease exhibited engraftment with a dominance of tumor cells, compared with a miniscule level of T cells. The ability of the engrafted cells to produce human Ig was also found to be correlated with the disease stage of the donor, although all donors had the same magnitude of hypogammaglobulinemia. Total human Ig production in the chimeric mice was normal in mice receiving PBMC from donors in stage 0, whereas in chimeric mice engrafted with PBMC from donors in stages III to IV almost no human Igs could be detected. This differential reconstitution of antibody production in the mouse model according to the stage of the patient's disease will allow further studies on possible cellular interactions between malignant and immune cells in BCLL.
Factor V Leiden (Resistance to Activated Protein C) Increases the Risk of Myocardial Infarction in Young Women
Authors: Rosendaal F.R. , Siscovick D.S. , Schwartz S.M. , Beverly R.K. , Psaty B.M. , Longstreth W.T. , Raghunathan T.E. , Koepsell T.D. , Reitsma P.H.
Published: October 2019

Factor V Leiden (factor V Arg506Gln), the genetic defect underlying resistance to activated protein C, is the most common risk factor for venous thrombosis. The relationship between this genetic abnormality and arterial disease is still unresolved. To assess whether factor V Leiden increases the risk of myocardial infarction (MI), we conducted a population-based case-control study among women 18 to 44 years of age in western Washington state. We included 84 women with first MI and 388 control women, ie, women residing in the same area in the same age range without MI (n = 388). The control women were contacted by random digit dialing. Data on risk factor status were collected via personal interview, and data on the factor V genotype via polymerase chain reaction techniques. The factor V Leiden mutation was found more often in women with MI (10%) than among controls (4%). The odds ratio for MI was 2.4 [95% confidence interval (CI) 1.0 to 5.9]. The risk was increased fourfold (CIgs 1.2 to 12.1) when adjusted for major cardiovascular risk factors. Among nonsmokers the factor V Leiden mutation had little effect (odds ratio 1.1, CI95 0.1 to 8.5), whereas it had a large effect among smokers (odds ratio 3.6, CI95 0.9 to 14.4), which, because smoking was itself a strong risk factor for MI, led to an odds ratio for smoking carriers of the mutation that was 32-fold increased compared with nonsmoking noncarriers. We conclude that factor V Leiden increases the risk of MI in young women. This effect seems to be confined largely to current smokers.
Differential Effect of E-Selectin Antibodies on Neutrophil Rolling and Recruitment to Inflammatory Sites
Authors: Ramos Carroll L. , Kunkel Eric J. , Lawrence Michael B. , Jung Unsu , Vestweber Dietmar , Bosse Roland , McIntyre Kim W. , Gillooly Kathleen M. , Norton Christine R. , Wolitzky Barry A. , Ley Klaus
Published: October 2019

The selectins are inducible adhesion molecules critically important for the inflammatory response. We investigate here the functional effects of three monoclonal antibodies (MoAbs) raised against murine E-selectin (9A9, 10E6, and 10E9.6) on neutrophil recruitment in vivo, leukocyte rolling and circulating leukocyte concentrations in vivo, and adhesion of myeloid cells to E-selectin transfectants and recombinant E-selectin–IgG fusion protein in vitro. MoAbs 9A9 and 10E6 map to the lectin and epidermal growth factor (EGF)-like domains of murine E-selectin, whereas 10E9.6 binds to the consensus repeat region. 10E9.6 blocked neutrophil recruitment in a model of thioglycollate-induced peritonitis in Balb/c mice by more than 90% but had no effect in C57BL/6 mice. 9A9 and 10E6 blocked neutrophil recruitment in this assay only when combined with a P-selectin antibody, 5H1. Neither 9A9 nor 10E9.6 alone blocked leukocyte rolling in tumor necrosis factor-α–treated venules of Balb/c mice, but 9A9 almost completely inhibited leukocyte rolling when combined with the function-blocking murine P-selectin MoAb, RB40.34. In contrast, 10E9.6 had no effect on leukocyte rolling in RB40.34-treated Balb/c or C57BL/6 mice. 10E9.6 did not affect adhesion of myeloid cells to E-selectin transfectants or attachment, rolling, and detachment of myeloid cells to murine E-selectin–IgG fusion protein. However, adhesion was completely blocked in the same assays by 9A9. Taken together, these results indicate that E-selectin serves a function, other than rolling, that appears to be critically important for neutrophil recruitment to inflammatory sites in Balb/c mice.
Stable Mixed Hematopoietic Chimerism in DLA-Identical Littermate Dogs Given Sublethal Total Body Irradiation Before and Pharmacological Immunosuppression After Marrow Transplantation
Authors: Storb Rainer , Yu Cong , Wagner John L. , Deeg H. Joachim , Nash Richard A. , Kiem Hans-Peter , Leisenring Wendy , Shulman Howard
Published: October 2019

Postgrafting cyclosporine (CSP) given for 35 days resulted in establishment of stable marrow grafts from DLA-identical canine littermates after otherwise suboptimal, but nevertheless, lethal conditioning with 450 cGy of total body irradiation (TBI). We now asked whether sustained allografts could be achieved after sublethal TBI or without TBI. Five groups of recipients were studied. Dogs in group 1 were given 200 cGy TBI and postgrafting CSP, 15 mg/kg twice daily by mouth on days −1 to 35 posttransplant. Dogs in group 2 were given 200 cGy TBI and methotrexate (MTX), 0.4 mg/kg intravenously (IV) on days 1, 3, 6, and 11 along with CSP. Dogs in group 3 were given 200 cGy TBI and CSP along with mycophenolate mofetil (MMF ), 10mg/kg twice daily subcutaneously (SC) on days 0 to 27 after transplant, a novel immunosuppressive combination. Dogs in group 4 were given 100 cGy TBI and MMF/CSP. Dogs in group 5 were not given TBI and they received MMF/CSP posttransplant. Allografts were assessed by (Ca)n dinucleotide repeat polymorphism studies in cells from peripheral blood, lymph nodes, and marrow. Dogs in group 1 had transient mixed donor-host hematopoietic chimerism for no more than 4 weeks. Three of six dogs in group 2 had transient mixed chimerism for 3 to 11 weeks, and three have remained stable mixed chimeras for up to 60 weeks now. Four of five dogs in group 3 have remained stable mixed chimeras for 54 to 57 weeks now, while one lost the allograft after 12 weeks. All dogs in groups 4 and 5 rejected their allografts after 2 to 12 weeks. In summary, the establishment of stable mixed hematopoietic chimerism following nonmyelosuppressive and nontoxic conditioning programs has remained a difficult goal. Here we present evidence in a large random-bred animal species that this goal may be achievable with pharmacological immunosuppression postgrafting, capable of inhibiting both host-versus-graft (HVG) and graft-versus-host (GVH) reactions in the setting of DLA-identical grafts.
Gene Cloning of Rat and Mouse Platelet Glycoprotein V: Identification of Megakaryocyte-Specific Promoters and Demonstration of Functional Thrombin Cleavage
Authors: Ravanat Catherine , Morales Martine , Azorsa David O. , Moog Sylvie , Schuhler Simone , Grunert Pierre , Loew Damarys , Van Dorsselaer Alain , Cazenave Jean-Pierre , Lanza François
Published: October 2019

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V–IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (≈1,700 nucleotides) entirely contained in one exon and a single intron (≈900 nucleotides) in the 5′ untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8–amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2 -terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2 -terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2 -terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.
Variable Immortalizing Potential and Frequent Virus Latency in Blood-Derived T-Cell Clones Infected With Human T-Cell Leukemia Virus Type I
Authors: Richardson J.H. , Höllsberg P. , Windhagen A. , Child L.A. , Hafler D.A. , Lever A.M.L.
Published: October 2019

Human T-cell leukemia virus type I (HTLV-I)-infected T cells expanded in vitro by single-cell cloning provide a unique system for investigating virus-cell interactions in nonimmortalized T cells. By analysis of clones generated randomly from the blood of virus carriers, we confirm that CD4 T cells are the major reservoir of HTLV-I in vivo and show that most infected cells contain a single integrated provirus. Contrary to the situation in HTLV-I immortalized cell lines, the HTLV-I provirus was found to be transcriptionally silent in a high proportion of randomly generated T-cell clones and could not be reactivated by mitogenic stimulation. The spontaneous proliferation previously documented in HTLV-I–infected T-cell clones was not observed in silently infected cells, and therefore correlates directly with the expression of tax and other viral genes. The only cytokine mRNA found to be significantly elevated in the virus-producing clones was interleukin-6; however, receptor-blocking experiments argue against a role for IL-6 in the virus-induced cell proliferation. We observed a striking variation in the ability of individual HTLV-I–producing clones to immortalize fresh peripheral blood lymphocytes. This ability did not correlate with the levels of viral mRNA expression, gag p24 production, spontaneous proliferation, or tax-transactivation, possibly suggesting a role for host cell factors as determinants of viral infectivity or immortalization. Studies to elucidate the basis of this phenotypic heterogeneity should enhance our understanding of viral spread and pathogenesis.
Hodgkin’s Specific Cells in Bone Marrow Aspirations
Authors: BAYED EDWIN D. , PAULSON GORDON S. , HARGRAVES MALCOLM M.
Published: October 2019

The literatutre concerning the observations of Sternberg-Reed cells in smears of bone marrow aspirate has been briefly reviewed and the cytology of node imprint preparations has been noted.

Two cases of Hodgkin’s disease in which Sternberg-Reed cells were observed in marrow smears are recorded with accompanying photomicrographs. No such cells were seen in the course of routine examination of more than six thousand, five hundred other bone marrow smears or in a special review of thirty cases of proved Hodgkin’s disease.
Oxygen Consumption of Erythrocytes from Patients with Various Thyroid Conditions Related to Their Respective Serum Protein—Bound Iodine Concentrations
Authors: ANGELONE LUIS , WATKINS DAVID H. , ANGERER CLIFFORD A.
Published: October 2019

Blood was obtained from patients who were divided into four groups: (A) euthyroid, (B) untreated hyperthyroid, (C) iodine-treated hyperthyroid and (D) hypothyroid. The erythrocytes from each patient group were washed and resuspended in Krebs-phosphate solution for study by the Warburg respirometric technic. When the erythrocytes obtained from patient groups B, C and D are compared with group A (control), a +92 per cent, +23 per cent and -15 per cent change is noted, respectively, in their mean QOO2 values when QOO2 values for erythrocytes for each patient in each patient group (excluding group C) are plotted relative to their respective serum PBI concentration (µg. per cent), a curve of exponential order appears to be the curve of best probable fit for these data. The mean QOO2 value for the iodine-treated hyperthyroid group falls on the ordinate of this curve (fig. 1) between the euthyroid and untreated hyperthyroid groups, though the mean serum PBI value (abscissa), of course, has no meaning for this group.
Hematological Effects of Certain 2,4-Diaminopyrimidines, Antagonists of Folic Acid Metabolism
Authors: HAMILTON LEONARD , PHILIPS FREDERICK S. , STERNBERG STEPHEN S. , CLARKE DONALD A. , HITCHINGS GEORGE H. , WHEELOCK BARBARA , DEBLIEUX ANN
Published: October 2019

1 . The administration of 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (50-197) and 2,4-diamino-5-(3',4'-dichlorophenyl) pyrimidine (50-11) to mice and rats produced delayed effects characteristic of folic acid deficiency; 50-197 amid 2,4-diamino-5-p-chlorophenyl-6-ethylpyrimidine (50-63) produced similar effects in dogs. LD50 values have been calculated.

2. The diaminopyrimidines caused ileitis amid colitis with diarrhea in mice, rats, and dogs, and stomatitis in dogs; animals lost weight and became dehydrated. Microscopic changes found in gastrointestinal tract, lymphatic tissue, and bone marrow have been described.

3. The hematologic manifestations observed were (a) granulocytopenia and reticulocytopenia in rats and dogs; there was a relatively less severe lymphocytopenia, (b) alterations in dog marrow which included an appreciable proportion of erythroid development via the megaloblast series, numerous and varied mitotic abnormalities, nuclear fragments, giant metamyelocytes, hypersegmented polymorphonuclear cells and a change its nuclear chromatin of myeloid series corresponding to megaloblastosis in erythroid development.

4. Doses of the diaminopyrimidines in excess of the LD50 produced convulsions in mice and rats (50-197 and 50-11); acute convulsant effects were also produced in cats given 50-197 and in dogs given 50-63. This acute convulsant actions could be prevented by means of anesthetic barbiturates. It is a property of alkyl substituted 2,4-diaminopyrimidines and 2-methyl-4-aminopyrimidines and is not specifically related to anti-folic activity.

5. The delayed death of some, but not all, mice following the diaminopyrimidines could be prevented by the simultaneous administration of citrovorum factor. This suggests, on the analogy of 4-amino-PGA and PGA, that there might be another factor or factors its the folic-folinic acid reaction chain as yet unidentified in cells.
Polycythemia Vera Terminating with Myeloblastic Leukemia
Authors: WILLIAMS MARJORIE J. , MENDEL JULIUS L.
Published: October 2019

A case of somewhat atypical polycythemia vera with marked leukocytosis and terminal myeloblastic leukemia has been studied hematologically and biochemically. The biochemical studies consisted of repeated estimations of the leukocytic alkaline and acid phosphatase activities during the last nine months of the patient’s life. These studies suggested that, except during the terminal myeloblastic leukemia, there was a leukemoid reaction rather than chronic myelogenous leukemia associated with polycythemia. The significance of these findings is briefly discussed and it is suggested that the widespread application of similar biochemical studies to cases of polycythemia vera with marked leukocytosis might be of value its helping to determine the true incidence of myelogenous leukemia in this disease.
Sideroblasts
Authors: KAPLAN EUGENE , ZUELZER WOLF W. , MOURIQUAND CLAUDE
Published: October 2019

Bone marrow specimens from a large group of infants and children were studied with the prussian blue stain. Iron-staining granules were present in the normoblasts of all the subjects studied, including normal individuals and those with a large variety of hematologic disorders. The term sideroblast was proposed for normoblasts with nonstaining inclusions. The profound decrease in sideroblasts in iron deficiency anemia and their prompt increase following both iron therapy in vivo and the addition of iron to marrow cultures in vitro indicated that the estimation of normoblast iron granules provides a sensitive index of the availability of iron. In thalassemia and in lead poisoning, hemoglobindeficient erythrocytes were found despite the presence of abundant iron granules within the normoblasts.

The presens of stainable nonhemoglobin iron in red cell precursors is a normal phenomenon and provides a useful adjunct for the study of iron metabolism.
Erythroleukemia, with Special Emphasis on the Acute or Incomplete Variety
Authors: MARTIN WILLIAM J. , BAYRD EDWIN D.
Published: October 2019

Five cases of erythroleukemia of the acute or incomplete variety form the basis of this report. Four of the patients were women. The ages of these five patients ranged from 19 to 73 years at time of admission to the Mayo Clinic. Studies were made on the blood and peripheral marrow for varying periods throughout the course of the disease. All five patients had anemia and extreme immaturity of the erythrocytes in the peripheral blood and bone marrow. Leukopenia was eventually present in four cases as was thrombopenia. Splenectomy was performed on one patient.

One patient lived six years after the condition developed, two died within thirteen months of the time of diagnosis, one was not followed, and the fifth was still living at last report.

Relatively few cases of erythroleukemia have been reported in the literature and most of those reported have been cases of the chronic variety. The erythrocytic picture in the chronic variety differs from that in the acute variety. In the acute type acute erythremia is associated with leukemia and in the chronic type polycythemia is associated with it. Either the erythrocytic abnormality or the leukocytic may appear first. Our studies and those reported in the literature suggest excessive activity of the blast cell in two directions.
Hemolysis and Alimentary Lipemia
Authors: SWANK ROY L. , ROTH ESTHER S.
Published: October 2019

1. Following ingestion of cream fat, cod liver oil, and glyceryl trioleate, hemolysis occurred when lipemic whole blood samples were shaken. The degree of hemolysis was roughly proportional to the degree of visible lipemia of the plasma. Blood samples from fasting dogs similarly shaken were not hemolyzed. The amount of hemolysis increased with increasing speed or duration of shaking.

2. This hemolysis was not due to an altered fragility of the erythrocytes per se, but to the chylomicra in the plasma or to substances in the plasma associated closely with the chylomicra.

3. Injections of heparin were followed by a rapid disappearance of lipemia and in many instances by a parallel decrease in hemolysis. At times, however, an early and transient increase in the amount of hemolysis followed injection of heparin in cod liver oil and trioleate-fed dogs. It was also shown that following heparin injection there was a slight increase in the amount of hemolysis produced by the particulate-free, usually inactive, subnatant plasma. Protamine injection caused a return of visible lipemia and an increase in the amount of hemolysis on shaking.

4. Incubation studies with whole blood and with plasma showed that the amount of hemolysis on shaking rapidly increased with incubation of preheparin lipemic blood. After heparin injection, incubation brought about either a decrease in hemolysis on shaking, a slight increase, or an increase followed by a decrease.

5. Possible mechanisms of the hemolysis are discussed.