bluuebunny/crossref_metadata_2025 huggingface dataset:
The Combined Study of Agglutination, Hemolysis and Erythrophagocytosis
Authors: BONNIN JAMES A. , SCHWARTZ LAWRENCE
Published: October 2019

1. A method for the combined demonstration of erythrophagocytosis, agglutination, and hemolysis was developed and applied to the study of a series of antierythrocytic antibodies under varying conditions of temperature and pH, using normal, trypsinized, and PNH corpuscles. Erythrophagocytosis was observed supravitally and in fixed preparations.

2. Only those antibodies which were potentially or actually hemolytic produced phagocytosis under the experimental conditions employed. The conditions necessary for the production of the two phenomena were similar except in the case of anti-A. In titration experiments, erythrophagocytosis occurred in higher serum dilutions than did hemolysis.

3. All the antibodies producing opsonization required the presence of thermolabile components of fresh serum for optimal activity. Except with anti-A, appreciable erythrophagocytosis was not produced by heat-inactivated sera. Hemolysis produced by systems other than antibody and complement was unaccompanied by phagocytosis.

4. It is suggested that hemolysis and erythrophagocytosis may both result from the same type of alteration of the red cell surface.
Serum Concentrations of Vitamin B12 in Patients Suffering from Leukemia
Authors: BEARD MARION F. , PITNEY W. R. , SANNEMAN E. H.
Published: October 2019

1. Vitamin B12 serum concentrations have been investigated in various types Of leukemia.

2. The concentration in chronic myeloid leukemia is approximately 15 times normal. In chronic lymphatic leukemia it is normal. In acute leukemia it is variable.

3. In chronic myeloid leukemia, the serum vitamim B12 exists in the bound form and the binding capacity for added B12 is increased.

4. Electrophoretic studies indicate that serum vitamims B12 in chronic myeloid leukemia is bound in the alpha globulin fractions.

5. Some correlation was found between serum vitamin B12 concentrations and total white cell counts in chronic myeloid leukemia. The possible significance of this is discussed.
A Serologic and Genetic Analysis of an r'r' (dCe/dCe) Patient Producing Anti-D and Anti-c
Authors: LEVINE PHILIP , COOPER MARY B. , KOCH ELIZABETH A.
Published: October 2019

A most unusual combination of antibodies, anti-D and anti-c produced by a transfusion and pregnancy, respectively, was identified by cross-absorption experiments. The immunized mother (propositus), whose husband was of genotype dce/dce, possessed the very rare genotype dCe/dCe, each of her parents, who were related to each other as fourth cousins, contributing the rare chromosome dCe.

The critically affected male infant was successfully treated by early replacement transfusion with the mother’s plasma-free red cells and her sibling’s rare compatible blood. The infant made a complete recovery although the preceding pregnancy ended in a macerated fetus.
Mild PTC (Plasma Thromboplastin Component) Deficiency Occurring in Two Brothers
Authors: BERGSAGEL D. E. , SETNA S. S. , CARTWRIGHT G. E. , WINTROBE M. M.
Published: October 2019

1. Thirteen cases of plasma thromboplastin component (PTC) deficiency or Christmas disease are reviewed and summarized. Of these cases, only in one were the whole blood coagulation time and prothrombin consumption normal.

2. Two cases of a mild bleeding disorder, occurring in brothers with slightly prolonged whole blood coagulation times and normal prothrombin consumption are described.

3. In these two cases, the thromboplastin generation test revealed the deficiency of a serum factor essential for normal thromboplastin generation. A mild deficiency of PTC was demonstrated by the correction of the serum deficiency by the addition to the patients’ serum of a partially purified preparation of PTC.

4. The differentiation of PTC deficiency from hemophilia is discussed.

5. Mild bleeding disorders due to a moderate reduction of antihemophilic globulin (AHG) or PTC can be differentiated by the use of the thromboplastin generation test.
A Recombinant Human scFv Anti-Rh(D) Antibody With Multiple Valences Using a C-Terminal Fragment of C4-Binding Protein
Authors: Libyh M. Tonye , Goossens D. , Oudin S. , Gupta N. , Dervillez X. , Juszczak G. , Cornillet P. , Bougy F. , Reveil B. , Philbert F. , Tabary T. , Klatzmann D. , Rouger P. , Cohen J.H.M.
Published: October 2019

Monomeric recombinant molecules prove generally unsatisfactory for in vivo use. Most biological systems are indeed multivalent either structurally, associating different chains, or functionally, when cross-linked by their ligands. Mimicking natural molecules for immune intervention implies the need for multimerizing systems to create multivalent molecules capable of interfering with physiological processing. A multivalent anti-Rh(D) recombinant protein has been designed by reconstructing the antibody binding site of a human monoclonal anti-Rh(D) antibody as a single chain Fv mini antibody, then multimerizing it by inserting at its C-terminal end the C-terminal part of the C4 binding protein (C4bp) alpha chain, which is responsible for the octamer multimerization of that molecule. This soluble multivalent recombinant molecule was functional, bound red blood cells (RBCs), agglutinated them, and did not activate complement. This demonstration model opens the way for future in vivo use of multivalent molecules associating antibody valences and other functional molecules for cell targeting, imaging, or removal of cells such as Rh(D)-positive RBCs for preventing Rh alloimmunization.
Evidence for Copurification of HERV-K–Related Transcripts and a Reverse Transcriptase Activity in Human Platelets From Patients With Essential Thrombocythemia
Authors: Boyd Mark T. , Foley Brian , Brodsky Isadore
Published: October 2019

We have previously reported that particles resembling retroviral particles and possessing an RNA-directed DNA polymerase activity can be prepared from platelets. Furthermore, we and others have shown that these particles are present at higher levels in patients with essential thrombocythemia and polycythemia vera. We show here that these particles package RNA molecules that encode HERV-K–related pol genes. A subset of the RNA molecules that are packaged are likely to encode the RNA directed DNA polymerase activity and, because these RNAs possess long/full-length open reading frames for the reverse transcriptase and RNaseH (also for part of the integrase domains in genomic clones) of HERV-K, we propose that these transcripts are indeed strong candidates for encoding the enzyme activity found in these particles. Moreover, by using a modification of the polymerase chain reaction-based reverse transcriptase assay in which activated DNA is added during cDNA synthesis to suppress DNA polymerase-mediated RNA-directed DNA synthesis, we have found that the particle-associated enzyme behaves like a retroviral reverse transcriptase, further supporting the conclusion that retrovirus-like, perhaps HERV-K sequences, encode this enzyme activity.
Folate Deficiency Delays the Onset But Increases the Incidence of Leukemia in Friend Virus-Infected Mice
Authors: Koury Mark J. , Park Don J. , Martincic Danko , Horne Donald W. , Kravtsov Vladimir , Whitlock James A. , del Pilar Aguinaga Maria , Kopsombut Prapaporn
Published: October 2019

Clinical studies have indicated that folate deficiency may enhance the development of various malignancies. In animal studies that examined the effect of folate deficiency on malignancies, conflicting results have been reported. In some studies, folate deficiency increased the development and growth of malignant tumors; in others, it decreased the development and growth of malignancies. We examined the effect of transient folate deficiency on the development of leukemia in mice infected with the anemia-inducing strain of Friend leukemia virus. Friend virus disease can be considered as a model for human acute leukemias that are preceded by a preleukemic period. These include leukemias that develop in patients who received previous chemotherapy and/or radiation therapy, as well as patients with chronic granulocytic leukemia or myelodysplasia. Folate deficiency around the time of Friend virus-infection delayed the onset but increased the incidence of leukemia. The rates of rearrangement of the Spi-1 (PU.1 ) oncogene by provirus integration and alteration of the p53 tumor-suppressor gene were the same in leukemia cell lines derived from folate-deficient mice as they were in cell lines from control mice. These results indicate that folate deficiency did not exert its enhancement of leukemogenesis through changes in either Spi-1 or p53, even though these two genes have been found to be the most frequently altered ones in Friend virus-induced leukemias. Our results suggest that folate deficiency may enhance the development of acute leukemia in patients who are at high risk for this disease.
A Common Prothrombin Variant (20210 G to A) Increases the Risk of Myocardial Infarction in Young Women
Authors: Rosendaal F.R. , Siscovick D.S. , Schwartz S.M. , Psaty B.M. , Raghunathan T.E. , Vos H.L.
Published: October 2019

Using specimens from a population-based case control study among women ages 18 to 44 years in western Washington, we assessed the relationship between carriership of a genetic clotting factor II variant (20210 G→A) and myocardial infarction (MI). The factor II variant was previously shown to be present in 1% to 2% of the population, to increase the levels of factor II, and to be associated with venous thrombotic disease. Personal interviews and blood samples were obtained from 79 women with a first myocardial infarction and 381 control women identified through random-digit telephone dialing. Polymerase chain reaction (PCR) method was used to determine the factor II genotypes. The factor II 20210 G to A transition was present more often in women with MI (5.1%) than among control women (1.6%). The age-adjusted odds ratio for MI was 4.0 (95% confidence interval 1.1 to 15.1). The relative risk was high when another major cardiovascular risk factor was also present, such as smoking (odds ratio 43.3, 95% confidence interval 6.7 to 281), and the risk seemed limited to those with other risk factors. These results, in which the effect of major coronary risk factors is enhanced fourfold to sixfold by the prothrombin variant, are similar to those previously reported for another genetic clotting abnormality, factor V Leiden. We conclude that factor II 20210 G to A increases the risk of myocardial infarction in young women, especially in the women with other major risk factors for coronary heart disease.
Influence of Interleukin-3 and Other Growth Factors on α4β1 Integrin-Mediated Adhesion and Migration of Human Hematopoietic Progenitor Cells
Authors: Schofield Karen P. , Rushton Graham , Humphries Martin J. , Dexter T. Michael , Gallagher John T.
Published: October 2019

The mechanisms by which hematopoietic progenitor cells are normally anchored in stromal niches and yet can be mobilized by specific growth factors are poorly understood. It is likely, however, that integrins and their extracellular matrix (ECM) ligands play a key role in this process, and recent evidence suggests that integrin function is modulated by signals originating from activated growth factor receptors. We have now examined this further by studying the role of growth factors on α4β1 integrin-mediated adhesion of human CD34+ hematopoietic progenitor cells to specific recombinant fibronectin fragments coated onto tissue culture dishes. Cells were prepared from cord blood and peripheral blood harvests. During a 30-minute adhesion assay a mean of 74% of CD34 cells attached to the so-called H120 fragment of fibronectin, which contains the strongest α4β1 integrin-binding sequence. The level of cell adhesion was significantly reduced by low concentrations of interleukin-3 (IL-3) (2.5 to 10 ng/mL), whereas stem cell factor (SCF ) and granulocyte colony-stimulating factor (G-CSF ) at these concentrations did not affect adherence of the cells. Migratory behavior of CD34 cells was examined using fibronectin fragments adsorbed onto a Transwell filter. The H120 fragment supported much higher levels of cell migration than the H0 fragment of fibronectin, which contains a weak α4β1 integrin binding sequence. Over a 16-hour assay, migration of peripheral blood progenitor cells was increased slightly by SCF and by G-CSF. However, a marked stimulation was observed with IL-3, which significantly increased migration. Similar effects were noted with cord blood cells, although a small proportion of cells were able to migrate in the absence of growth factors. These results indicate that there is a highly selective and functional link between the α4β1 integrin and IL-3/IL-3–receptor that could affect the position of stem and progenitor cells in the marrow stroma and influence their growth and development.
FcγRIII-Mediated Regulation of Hematopoiesis in Murine Bone Marrow Cells by Interleukin-3 and CD95 (Fas/Apo-1)
Authors: Yoshikawa Hideshi , Sakihama Toshiko , Nakajima Yasuo , Tasaka Kachio
Published: October 2019

The interleukin-3 (IL-3)–dependent murine bone marrow–derived cell line FDC-P2/185-4 (185-4) undergoes apoptosis when IL-3 is withdrawn from culture medium. Previous results from our studies indicated that a high concentration of aggregated mouse IgG prevented apoptosis of 185-4 cells through FcγRIII by an autocrine mechanism, producing IL-3. But after 24 hours, 185-4 cells expressed CD95 (Fas/Apo-1) on their surfaces on stimulation via FcγRIII. In addition, this CD95 was functional and apoptosis was induced by anti-CD95 monoclonal antibody (MoAb). We investigated how these conflicting effects were induced by FcγRIII stimulation within the context of cell survival and death. The results showed that IL-3 was induced by calcium ionophore and that the IL-3 induced by FcγRIII stimulation was blocked by EGTA or FK506, but not by staurosporine (protein kinase C [PKC] inhibitor), indicating the important role of calcium-calcineurin in this system. On the other hand, the CD95 expression induced by FcγRIII stimulation was blocked by staurosporine, but not by EGTA or FK506, and phorbol myristate acetate (PMA) induced CD95 expression in the same manner as FcγRIII, indicating the involvement of PKC in the CD95 expression induced by FcγRIII stimulation. Thus, FcγRIII-mediated stimulation even while promoting immediate survival of the bone marrow cells, also triggers mechanisms that will facilitate their eventual deletion at the end of the response. These results suggest that a balance between cell survival and death is maintained to avoid unlimited cell growth caused by FcγRIII-ligand interaction in hematopoiesis during inflammation.
In Vitro Negative Selection of Viral Superantigen-Reactive Thymocytes by Thymic Dendritic Cells
Authors: Ferrero Isabel , Anjuère Fabienne , Robson MacDonald H. , Ardavı́n Carlos
Published: October 2019

Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)–encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vβ elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR αβ P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.
Long-Term Malignant Hematopoiesis in Human Acute Leukemia Bone Marrow Biopsies Implanted in Severe Combined Immunodeficiency Mice
Authors: Legrand Faezeh , Khazaal Ibrahim , Peuchmaur Michel , Fenneteau Odile , Cavé Hélène , Rohrlich Pierre , Vilmer Etienne , Péault Bruno
Published: October 2019

Bone marrow (BM) trephine biopsies from 15 pediatric patients with acute lymphoid (ALL) or myeloid (AML) leukemia were engrafted subcutaneously into severe combined immunodeficiency (SCID) mice conditioned by 200 cGy total-body irradiation. Implants were harvested 5 to 19 weeks later for histologic, cytologic, and/or flow cytometric analysis of the residing marrow. Eighteen of 19 grafts contained viable human leukemic cells to various extents as assessed by one or more of these methods. Thirteen of 14 implants analyzed by flow cytometry included high numbers of tumor cells, accounting for 85% to 100% of the total nucleated cells in seven of them. Histologically, engrafted marrow samples exhibited areas of blastic infiltration, and tumor-specific gene rearrangements were retrieved in long-term engrafted biopsies. Importantly, engrafted mice remained perfectly healthy even 5 months posttransplantation, and no human tumor cell dissemination was detected in the hematolymphoid and nonhematopoietic tissues at the time of autopsy. These results demonstrate that human malignant hematopoiesis can be sustained long-term in its original, intact marrow stromal environment transplanted in appropriately conditioned immunodeficient mice.
The Hemoglobin-Deficit Mouse: Analysis of Phenotype and Hematopoiesis in the Transplant Model
Authors: Bloom Michael L. , Simon-Stoos Karen L.
Published: October 2019

The mouse mutant hemoglobin deficit (gene symbol hbd ) is characterized by a severe microcytic anemia that is inherited in an autosomal-recessive manner. To assess the mutation's effect on hematopoiesis, unfractionated bone marrow (BM) from either a mutant C57BL6/J-hbd/hbd, Gpi1b/Gpi1b (phenotype symbol HBD), or normal C57BL6/J -+hbd/+hbd, Gpi1b/Gpi1b mouse was injected intravenously into irradiated congenic C57BL6/J-+hbd/+hbd, Gpi1a/Gpi1a, Igha/Igha, Thy1a/Thy1a mice. The congenic recipients of mutant or normal marrow obtained complete red blood cell (RBC) and leukocyte reconstitution, with the exception of one recipient of HBD marrow. After 24 weeks posttransplantation, the normal recipients of HBD marrow obtained a microcytic anemia similar to the donor. These results suggest that the HBD phenotype is caused by a BM defect. We observed that the erythroid lineage derived from donor HBD marrow repopulated more slowly than the normal marrow at 4 weeks posttransplantation. To determine if this difference was a result of an erythropoietic defect, competitive repopulation was performed using either mutant or normal marrow competed against normal congenic marrow. For the erythroid lineage, no significant contribution from HBD marrow was observed. To assess if the RBC block was based on a deficiency of myeloid progenitors, both in vitro and in vivo assays were performed: absolute numbers of bone progenitors were increased, suggesting that the defect results in a late block to erythroid differentiation.
In Vitro Generation of Allospecific Human CD8+ T Cells of Tc1 and Tc2 Phenotype
Authors: Halverson David C. , Schwartz Gretchen N. , Carter Charles , Gress Ronald E. , Fowler Daniel H.
Published: October 2019

We have previously shown that allospecific murine CD8+ T cells of the Tc1 and Tc2 phenotype could be generated in vitro, and that such functionally defined T-cell subsets mediated a graft-versus-leukemia (GVL) effect with reduced graft-versus-host disease (GVHD). To evaluate whether analogous Tc1 and Tc2 subsets might be generated in humans, CD8+ T cells were allostimulated in the presence of either interleukin-12 (IL-12) and transforming growth factor-beta (TGF-β) (Tc1 culture) or IL-4 (Tc2 culture). Tc1-type CD8 cells secreted the type I cytokines IL-2 and interferon gamma (IFN-γ), whereas Tc2-type cells primarily secreted the type II cytokines IL-4, IL-5, and IL-10. Both cytokine-secreting populations effectively lysed tumor targets when stimulated with anti–T-cell receptor (TCR) antibody; allospecificity of Tc1- and Tc2-mediated cytolytic function was demonstrated using bone marrow–derived stimulator cells as targets. In addition, both Tc1 and Tc2 subsets were capable of mediating cytolysis through the fas pathway. We therefore conclude that allospecific human CD8+ T cells of Tc1 and Tc2 phenotype can be generated in vitro, and that these T-cell populations may be important for the mediation and regulation of allogeneic transplantation responses.
Effect of Diagnosis (Refractory Anemia With Excess Blasts, Refractory Anemia With Excess Blasts in Transformation, or Acute Myeloid Leukemia [AML]) on Outcome of AML-Type Chemotherapy
Authors: Estey Elihu , Thall Peter , Beran Miloslav , Kantarjian Hagop , Pierce Sherry , Keating Michael
Published: October 2019

In current medical practice, patients with refractory anemia with excess blasts in transformation (RAEB-t), and especially patients with RAEB, receive chemotherapy regimens (AML Rx) administered to patients with acute myeloid leukemia (AML) less often than do patients with AML. These entities are distinguished primarily by marrow blast percentage (5% to 19% RAEB, 20% to 29% RAEB-t, and ≥30% AML). The poor prognosis of many RAEB or RAEB-t patients, if untreated, led us to give them AML Rx using the same plan as for AML. The purpose of this analysis was to see if diagnosis (RAEB, RAEB-t, or AML) affected outcome. We treated 372 patients with AML (acute promyelocytic leukemia [APL] excluded), 106 with RAEB-t, and 52 with RAEB. AML Rx produced a 62% complete remission (CR) rate in RAEB, essentially identical to the rates in RAEB-t and AML, but event-free survival (EFS) from CR and from start of treatment (start of Rx), as well as overall survival, were poorer in RAEB than in AML or RAEB-t, with AML and RAEB-t being identical. However, patients with RAEB or RAEB-t were more likely to have poor prognostic characteristics, in particular complex abnormalities involving chromosomes 5 and/or 7. Multivariate analyses indicated that, when considered together with cytogenetics and other patient characteristics, a diagnosis of RAEB rather than AML or RAEB-t had no effect on EFS from start of Rx, EFS from CR, survival, or achievement of CR. These analyses suggested a trend for patients with RAEB-t to have better EFS from start of Rx than patients with AML or RAEB (P = .08; relative risk, 0.80; 95% confidence interval, 0.62 to 1.03), but there were no differences with respect to the other outcomes. Our data suggest that the propriety of administering AML Rx to patients with RAEB or RAEB-t who have poor prognosis without treatment is identical to the propriety of treating AML in this fashion. Deterrents to standard AML Rx in these patients could justifiably include cytogenetics, age, etc, but not a diagnosis of RAEB or RAEB-t per se.
The Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Exists as a Preformed Receptor Complex That Can Be Activated by GM-CSF, Interleukin-3, or Interleukin-5
Authors: Woodcock Joanna M. , McClure Barbara J. , Stomski Frank C. , Elliott Michael J. , Bagley Christopher J. , Lopez Angel F.
Published: October 2019

The granulocyte-macrophage colony-stimulating factor (GM-CSF ) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor α chain (GMRα) and to the common β chain of the GM-CSF, IL-3, and IL-5 receptors (βc ) immunoprecipitated both GMRα and βc from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMRα and βc , as shown by the ability of soluble βc to associate with membrane-anchored GMRα or soluble GMRα. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMRα-associated βc . These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.
Mice Lacking Both Granulocyte Colony-Stimulating Factor (CSF) and Granulocyte-Macrophage CSF Have Impaired Reproductive Capacity, Perturbed Neonatal Granulopoiesis, Lung Disease, Amyloidosis, and Reduced Long-Term Survival
Authors: Seymour John F. , Lieschke Graham J. , Grail Dianne , Quilici Cathy , Hodgson George , Dunn Ashley R.
Published: October 2019

Mice lacking granulocyte colony-stimulating factor (G-CSF) are neutropenic with reduced hematopoietic progenitors in the bone marrow and spleen, whereas those lacking granulocyte-macrophage colony-stimulating factor (GM-CSF) have impaired pulmonary homeostasis and increased splenic hematopoietic progenitors, but unimpaired steady-state hematopoiesis. These contrasting phenotypes establish unique roles for these factors in vivo, but do not exclude the existence of additional redundant functions. To investigate this issue, we generated animals lacking both G-CSF and GM-CSF. In the process of characterizing the phenotype of these animals, we further analyzed G-CSF– and GM-CSF–deficient mice, expanding the recognized spectrum of defects in both. G-CSF–deficient animals have a marked predisposition to spontaneous infections, a reduced long-term survival, and a high incidence of reactive type AA amyloidosis. GM-CSF–deficient mice have a modest impairment of reproductive capacity, a propensity to develop lung and soft-tissue infections, and a similarly reduced survival as in G-CSF–deficient animals. The phenotype of mice lacking both G-CSF and GM-CSF was additive to the features of the constituent genotypes, with three novel additional features: a greater degree of neutropenia among newborn mice than in those lacking G-CSF alone, an increased neonatal mortality rate, and a dominant influence of the lack of G-CSF on splenic hematopoiesis resulting in significantly reduced numbers of splenic progenitors. In contrast to newborn animals, adult mice lacking both G-CSF and GM-CSF exhibited similar neutrophil levels as G-CSF–deficient animals. These findings demonstrate that the additional lack of GM-CSF in G-CSF–deficient animals further impairs steady-state granulopoiesis in vivo selectively during the early postnatal period, expand the recognized roles of both G-CSF and GM-CSF in vivo, and emphasize the utility of studying multiply deficient mouse strains in the investigation of functional redundancy.
Cytoplasmic Localization of a Functionally Active Fanconi Anemia Group A–Green Fluorescent Protein Chimera in Human 293 Cells
Authors: Kruyt Frank A.E. , Waisfisz Quinten , Dijkmans Lonneke M. , Hermsen Mario A.J.A. , Youssoufian Hagop , Arwert Fré , Joenje Hans
Published: October 2019

Hypersensitivity to cross-linking agents and predisposition to malignancy are characteristic of the genetically heterogeneous inherited bone marrow failure syndrome, Fanconi anemia (FA). The protein encoded by the recently cloned FA complementation group A gene, FAA, has been expected to localize in the nucleus as based on the presence of sequences homologous to a bipartite nuclear localization signal (NLS) and a leucine repeat motif. In contrast to this expectation, we show here that a functionally active FAA-green fluorescent protein (GFP) hybrid resides in the cytoplasmic compartment of human kidney 293 cells. In accordance with this finding, disruption of the putative NLS by site-directed mutagenesis failed to affect both subcellular localization and the capacity to complement hypersensitivity to the cross-linking agent mitomycin C in FA-A lymphoblasts. Furthermore, the N-terminal part of FAA with the putative NLS at amino acid position 18 to 35 showed no nuclear translocation activity when fused to GFP, while the first 115 N-terminal amino acids appeared to be indispensable for the complementing activity in FA-A cells. Similarly, mutagenesis studies of the putative leucine repeat showed that, even though this region of the protein is important for complementing activity, this activity does not depend on an intact leucine zipper motif. Finally, fusion of the NLS motif derived from the SV40 large T antigen to FAA could not direct the hybrid protein into the nucleus of 293 cells, suggesting that FAA is somehow maintained in the cytoplasm via currently unknown mechanisms. Thus, like the first identified FA protein, FAC, FAA seems to exert its function in the cytoplasmic compartment suggesting FA proteins to be active in a yet to be elucidated cytoplasmic pathway that governs hematopoiesis and protects against genomic instability.
Induction of Apoptosis Without Differentiation by Retinoic Acid in PLB-985 Cells Requires the Activation of Both RAR and RXR
Authors: Monczak Yury , Trudel Michel , Lamph William W. , Miller Wilson H.
Published: October 2019

Retinoic acid (RA) induces differentiation, followed by apoptosis in acute promyelocytic leukemia (APL) cells, both in vitro and in patients. One problem in understanding these mechanisms is to distinguish molecular events leading to differentiation from those leading to apoptosis. We have identified a leukemic cell line, PLB-985, where RA directly induces apoptosis with no morphologic, genetic, or cell-surface marker evidence of differentiation. These cells differentiate following dimethyl sulfoxide (DMSO), but not RA, treatment. Two-color flow cytometry showed no alteration of the cell cycle after RA treatment, and cell-surface marker analysis of CD11a, CD11b, and CD13 showed no modulation typical of differentiating cells. RNA expression of myeloblastin and transglutaminase, genes regulated by RA-induced differentiation in NB4 cells, was unchanged by RA treatment. Instead, RA induced apoptosis, as shown by typical apoptotic morphological features, genomic DNA laddering, and positive labeling in the TUNEL assay. We found that induction of apoptosis in this model requires a different pattern of retinoid receptor binding and transcriptional activation than is seen in APL cells. As previously described, treatment with retinoid receptor-selective ligands showed that stimulation of RAR alone is sufficient to induce differentiation and apoptosis in NB4 cells, and that stimulation of RXR has no effect on the parameters analyzed. In PLB-985 cells, on the other hand, apoptosis was induced only upon costimulation of both RAR and RXR. Stimulation of either receptor alone had no effect on the cells. Consistent with these findings, bcl-2 RNA and protein levels were downregulated after stimulation of both RAR and RXR, but not with an RAR-specific ligand alone, as in NB4 cells. The expression of several other bcl-2 family members (bcl-X, ich-1, bax, bag, and bak ) and retinoid receptors (RARα, RXRα, and RXRβ) was not affected by treatment with RAR- and/or RXR-activating retinoids; RARβ RNA was undetectable before and after retinoid treatment. Thus, our cell model provides a useful tool in determining the genetic events mediating apoptosis as a response to RA, unobscured by events implicated in differentiation.
Generation of Virtually Pure and Potentially Proliferating Dendritic Cells From Non-CD34 Apheresis Cells From Patients With Multiple Myeloma
Authors: Tarte Karin , Lu Zhao Yang , Fiol Genevieve , Legouffe Eric , Rossi Jean-François , Klein Bernard
Published: October 2019

Defects in immune response are often reported in patients with multiple myeloma (MM). Because dendritic cells (DCs) are key effectors in promoting cellular immunity and are potential vectors for immunotherapy, we have evaluated the ability of MM patients' apheresis cells to generate DCs in short-term cultures. We report here the obtaining of a virtually pure population of DCs (89.7% ± 6%, n = 18) after culturing adherent apheresis cells for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF ) and interleukin-4 (IL-4). These cells exhibited all the phenotypic characteristics (CD1a+, HLA-DR+, CD80+, CD40+, CD14−) and the MLR stimulating capacity of mature DCs. The number of DCs reached 12.1% of the initial apheresis cell number put into culture. As DC precursors involved in this model were CD34− cells, the unabsorbed cells resulting from clinical-grade CD34 purification were a reliable source of DCs, even after freezing. The proliferation of DC precursors could be increased 10-fold by adding IL-3 and tumor necrosis factor-α together with GM-CSF and IL-4. Thus, CD34− apheresis cells from patients with MM offer an interesting source for generating pure, functional, and potentially proliferating DCs.
Successful Reconstitution of Human Hematopoiesis in the SCID-hu Mouse by Genetically Modified, Highly Enriched Progenitors Isolated From Fetal Liver
Authors: Humeau Laurent , Chabannon Christian , Firpo Meri T. , Mannoni Patrice , Bagnis Claude , Roncarolo Maria-Grazia , Namikawa Reiko
Published: October 2019

Highly purified CD34++CD38−Lin− hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli β-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the β-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38−Lin− cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.
Regulated Expression of the Eph-Related Receptor Tyrosine Kinase Hek11 in Early Human B Lymphopoiesis
Authors: Aasheim Hans-Christian , Terstappen Leon W.M.M. , Logtenberg Ton
Published: October 2019

Members of the large Eph family of receptor tyrosine kinases (RTKs) display temporally and spatially restricted expression patterns during embryogenesis, suggesting a role in various developmental processes. We have begun to investigate the expression of members of this receptor family during human hematopoiesis, in particular B lymphopoiesis. Expression of Eph RTKs in cells of the B-lymphoid lineage was assessed by using degenerate oligonucleotide primers based on stretches of conserved nucleic acid sequences in members of the Eph family. First, the content of Eph-family RTKs was assessed in freshly sorted fetal bone marrow pro–B cells. This population was found to harbor transcripts of the Hek8 and Hek11 members of this gene family. Subsequent analysis of expression of these genes in B cells representing various differentiation and ontogenic stages showed that the Hek8 transcript is constitutively present in all fetal and adult B-lineage cells, with high levels of expression in peripheral blood B cells. In contrast, the Hek11 transcript was exclusively found in fetal bone marrow pro–B cells and pre–B cells, but not in more mature fetal B-lineage cells. All adult B-lineage cells, from early pro–B cells to end-stage plasma cells, lacked Hek11 transcripts. The developmentally regulated expression of Hek11 during fetal B lymphopoiesis suggests a role for this gene in pre/pro–B cell expansion and/or differentiation and defines a difference in progenitor B cell populations isolated from fetal versus adult human bone marrow.
Protease Activation Is Required for Glucocorticoid-Induced Apoptosis in Chronic Lymphocytic Leukemic Lymphocytes
Authors: Chandra Joya , Gilbreath Joyce , Freireich Emil J , Kliche Kay-Oliver , Andreeff Michael , Keating Michael , McConkey David J.
Published: October 2019

Recent work has demonstrated that glucocorticoids, nucleoside analogues, and other cancer chemotherapeutics induce apoptosis in chronic lymphocytic leukemia (CLL) cells. In this study, we investigated the involvement of protease activation in these responses using selective peptide inhibitors of the interleukin-1β converting enzyme (ICE)/caspase family and a Ca2+-activated protease we recently implicated in thymocyte apoptosis. Apoptosis was associated with proteolytic cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and increased caspase protease activity, and cell-permeant caspase antagonists [zVAD(OMe)fmk and Boc-D(OBzl)cmk] blocked apoptosis in response to the glucocorticoid methylprednisolone or the nucleoside analogue fludarabine, indicating that caspase activation was required for these responses. However, a peptide-based inhibitor of the Ca2+-dependent lamin protease (zAPFcmk) also completely suppressed DNA fragmentation and the cleavage of lamin B1 . Strikingly, treatment of cells with zAPFcmk alone led to characteristic PARP cleavage, depletion of the precursor forms of two ICE family proteases (CPP32 and ICH-1), and phosphatidylserine exposure, suggesting that blockade of the lamin protease led to activation of the ICE family. Our results implicate the lamin protease as a target for Ca2+ during chemotherapy-induced apoptosis in CLL lymphocytes, and they identify a novel functional interaction between the protease and members of the ICE family.
Morbidity and Mortality Due to Liver Disease in Children Undergoing Allogeneic Bone Marrow Transplantation: A 10-Year Prospective Study
Authors: Locasciulli Anna , Testa Marina , Valsecchi Maria Grazia , Vecchi Laura , Longoni Daniela , Sparano Paola , Rovelli Attilio , Uderzo Cornelio , Masera Giuseppe , Alberti Alfredo
Published: October 2019

We have conducted a long-term prospective study of children undergoing bone marrow transplantation (BMT) to assess morbidity and mortality for liver disease. One hundred eleven consecutive children were enrolled between June 1985 and June 1995 and were followed-up for a median of 5.5 years after BMT. Before transplant 48/111 children (43%) had abnormal alanine aminotransferase (ALT), none were HBsAg+ and 4/111 were anti-HCV+. After BMT 4/111 patients (3.6%) died of liver failure. No relationship was found between pretransplant hepatitis B (HBV) or C (HCV) infection or elevated transaminases and development of severe liver damage. Eighty-two out of one hundred and eleven patients (74%) had abnormalities of ALT after BMT, transient (n = 54) or persistent (n = 28). None developed clinical signs or symptoms of end stage liver disease or of cirrhosis during follow-up. No significant difference in prevalence of liver disease, was found between children with normal or abnormal ALT at BMT (relative risk [RR] = 1.04). HCV infection could be implicated in the etiology of chronic liver disease in 14/28 patients; 2 other patients were found infected by HBV alone (1 case) or combined with HCV (1 case). In the remaining 12 the etiology of chronic liver disease could not be defined. Posttransplant hepatitis B occurred in 4/111 children (3.6%), including a recipient from a donor who had been previously vaccinated against HBV, while no patient who had been vaccinated developed hepatitis B. The rate of posttransplant seroconversion to anti-HCV was 15%.
The Cytoplasmic Domain of Stem Cell Antigen CD34 Is Essential for Cytoadhesion Signaling But Not Sufficient for Proliferation Signaling
Authors: Hu Mickey C.-T. , Chien Shu L.
Published: October 2019

CD34 is widely used as a marker in the identification and purification of human hematopoietic stem and progenitor cells; however, its function within hematopoiesis is largely unknown. We have investigated the contribution of cytoplasmic domain of CD34 in cytoadhesion signaling and proliferation signaling in hematopoietic cells. Engagement of particular determinants of CD34 by monoclonal antibodies leads to homotypic adhesiveness of the full-length CD34-transfected BaF3 cells. However, this homotypic adhesiveness is abrogated in BaF3 cells transfected with the truncated CD34 lacking the cytoplasmic domain. Cytoadhesion signaling through the cytoplasmic domain of CD34 cannot be restored through that of erythropoietin receptor (EPOR) or granulocyte colony-stimulating factor receptor (G-CSFR), suggesting that the cytoplasmic domain of CD34 is required for its signal transduction of cellular adhesion. In constrast, we show that replacing the cytoplasmic domain of EPOR or G-CSFR with that of CD34 abolished growth signal transduction in response to EPO or G-CSF in the chimeric receptor-transfected BaF3, 32D, and FDCP1 cells, whereas the wild-type EPOR- or G-CSFR-transfected cells responded to EPO or G-CSF growth signaling well. These results suggest that the cytoplasmic portion of CD34 may not contain the elements necessary to transduce a proliferative signal in hematopoietic cells. Thus, the function of CD34 in hematopoiesis is primarily on hematopoietic cell adhesion.
Risk Factors, Treatment, and Outcome of Central Nervous System Recurrence in Adults With Intermediate-Grade and Immunoblastic Lymphoma
Authors: van Besien Koen , Ha Chul S. , Murphy Sandy , McLaughlin Peter , Rodriguez Alma , Amin Kamal , Forman Arthur , Romaguera Jorge , Hagemeister Fredrick , Younes Anas , Bachier Carlos , Sarris Andreas , Sobocinski Kathleen S. , Cox James D. , Cabanillas Fernando
Published: October 2019

To evaluate the incidence, risk factors, and outcome of central nervous system (CNS) recurrence in adult patients with non-Hodgkin's lymphoma, we evaluated 605 newly diagnosed patients with large-cell and immunoblastic lymphoma who participated in prospective chemotherapy studies. The Kaplan-Meier estimate of probability of CNS recurrence at 1 year after diagnosis was 4.5% (95% confidence interval [CI], 4.4 to 4.6). Twenty-four patients developed CNS recurrence after a median of 6 months from diagnosis (range, 0 to 44 months). In univariate analysis, an increased risk for CNS recurrence was associated with an advanced disease stage (P = .0014), an increased LDH (P = .0000), the presence of B-symptoms (P = .0037), involvement of more than one extranodal site (P = .0000), poor performance status (P = .0005), and B-cell phenotype (P = .008). Bone marrow involvement (P = .005), involvement of parenchymal organs (P = .03), and involvement of skin, subcutaneous tissue, and muscle (P = .002) were also associated with an increased risk for CNS disease. Multivariate logistic regression analysis identified only involvement of more than one extranodal site (P = .0005) and an increased LDH (P = .0008) as independent predictors of CNS recurrence. Established CNS recurrence had a poor prognosis. Only 1 of 24 patients remains alive and the Kaplan-Meier estimate of probability of survival at 1 year after the diagnosis of CNS recurrence is only 25.3% (95% CI, 6.9 to 43.7). Intrathecal treatment provided symptomatic benefit in only 1 of 6 patients. Radiation treatment provided symptomatic improvement in 6 of 9 patients treated. However, remissions were short and followed by systemic or CNS recurrence. Serum LDH and involvement of more than one extranodal site are independent risk factors for CNS recurrence in patients with large-cell lymphoma. The presence of both risk factors identifies a patient group at high risk for CNS recurrence. Established CNS recurrence can be rapidly fatal. Transient responses occur after radiation treatment.
Thrombopoietin, kit Ligand, and flk2/flt3 Ligand Together Induce Increased Numbers of Primitive Hematopoietic Progenitors From Human CD34+Thy-1+Lin− Cells With Preserved Ability to Engraft SCID-hu Bone
Authors: Luens Karin M. , Travis Marilyn A. , Chen Ben P. , Hill Beth L. , Scollay Roland , Murray Lesley J.
Published: October 2019

CD34+Thy-1+Lin− cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34+Thy-1+Lin− cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34+Thy-1+Lin− cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34hiPKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34+Thy-1+Lin− cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34hi PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34hiPKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34+Thy-1+Lin− cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.
Integrin β2 (CD18)-Mediated Cell Proliferation of HEL Cells on a Hematopoietic-Supportive Bone Marrow Stromal Cell Line, HESS-5 Cells
Authors: Tsuji Takashi , Waga Iwao , Tezuka Katsunari , Kamada Masafumi , Yatsunami Kimio , Kodama Hisashi
Published: October 2019

Cellular interactions between hematopoietic cells and stromal cells play important roles in the proliferation and differentiation of hematopoietic cells. The proliferation of a human erythroleukemia cell line, HEL cells, which can differentiate into macrophage- and megakaryocyte-like cells, and erythroid precursors was dramatically induced on coculture with a hematopoietic-supportive stromal cell line, HESS-5 cells, which can support long-term hematopoiesis in vitro without fetal bovine serum. HEL cells proliferated when they were cocultured with but not without direct cell contact. Because the coculture supernatants with direct cell contact and cytokines such as interleukins and growth factors did not exhibit growth-stimulating activity toward HEL cells, it was suggested that some molecule that has growth-stimulating activity exists on the surface of the cells. Extracellular matrix components such as fibronectin, laminin, vitronectin, and collagen did not affect the proliferation of HEL cells. An anti-CD18 monoclonal antibody, which recognizes the common β chain of the β2 integrin subfamily, induced dramatic proliferation of HEL cells. Moreover, the proliferation of HEL cells was inhibited by an antisense oligonucleotide of CD18 mRNA. As judged from these observations, the proliferation of HEL cells was mediated by CD18 molecules expressed on HEL cells. On the contrary, the common counter-receptor of the β2 integrin subfamily, intercellular adhesion molecule-1, which is expressed on CHO-K1 cells, did not stimulate the growth of HEL cells. It is known that other counter molecules of the β2 integrin subfamily, such as complement C3bi and fibrinogen, are not produced by stromal cells. These findings suggest that the proliferation of HEL cells may be induced through an interaction between a novel molecule of the β2 integrin subfamily on HEL cells and the counter-receptor on HESS-5 cells. The β2 integrin subfamily may regulate the growth of hematopoietic cells in hematopoiesis in vivo and/or cause the abnormal growth of leukemia cells.