bluuebunny/crossref_metadata_2025 huggingface dataset:
Barrier cells: stromal regulation of hematopoiesis and blood cell release in normal and stressed murine bone marrow
Authors: Weiss L , Geduldig U
Published: October 2019

Murine hematopoietic bone marrow is heterogenous in respect to bone- lining cells, hematopoiesis, and release of blood cells. In diaphyseal femoral marrow, bone-lining cells are largely osteoblasts, indifferent endosteum, blood cells, and reticular cells. Hematopoiesis is sustained by rather differentiated progenitors, as myelocytes and polychromatophilic erythroblasts. But in sharply restricted loci within trabeculated bone in the distal medial femoral metaphysis, bone-lining cells are dominated by newly discovered fibroblastic, contractile, stromal barrier cells; activated, multilaminar and branched, enveloping putative stem cells; and extending into the marrow, where they enclose hematopoietic clusters of rather early progenitors, as promyelocytes; and basophilic erythroblasts. Barrier cells may endocytize granules released individually by vicinal differentiating granulocytes, preventing tissue damage. Barrier cells envelope blood vessels and insinuate processes into their wall, augmenting blood-marrow barriers, preventing release of immature cells. Further, extensions of venous sinuses made of extraordinarily thin barrier cell processes receive released blood cells. With heightened hematopoiesis due to mutant hemolytic anemias or after administration of interleukin-1, barrier cells and their associated structures are greatly increased, spreading beyond normally restricted loci. But in microenvironment-deficient mutants, barrier cells are fewer, less activated, and less granulated. Barrier cells thus appear important in hematopoiesis and in the release of blood cells from bone marrow.
In vivo production of interleukin-5, granulocyte-macrophage colony- stimulating factor, macrophages colony-stimulating factor, and interleukin-6 during intravenous administration of high-dose interleukin-2 in cancer patients [see comments]
Authors: Schaafsma MR , Falkenburg JH , Landegent JE , Duinkerken N , Osanto S , Ralph P , Kaushansky K , Wagemaker G , Van Damme J , Willemze R
Published: October 2019

Recombinant human interleukin-2 (IL-2), administered to cancer patients by continuous intravenous (IV) infusion (3 x 10(6) U/m2/d), was found to induce the in vivo production of colony-stimulating factors (CSF). Plasma obtained from patients during IL-2 treatment stimulated in vitro colony formation of normal human bone marrow cells, depleted of mononuclear phagocytes and T lymphocytes. This colony-stimulating activity (CSA) was identified as IL-5, granulocyte-macrophage CSF (GM- CSF), and macrophage CSF (M-CSF), by the ability of specific antibodies against these factors to neutralize their effects. The presence of IL-2- induced GM-CSF and M-CSF was also demonstrated by specific radioimmunoassays. During IL-2 treatment, plasma also contained detectable levels of IL-6, which was measured in a bioassay. Using a cDNA-polymerase chain reaction (PCR) with specific primer sets for the various CSF, we showed that IL-2 treatment induced the expression of mRNA for M-CSF, GM-CSF, IL-3, and IL-5, but not for granulocyte CSF (G- CSF) in peripheral blood mononuclear cells, suggesting differential expression of CSF in vivo in response to IL-2. Furthermore, no negative regulators of hematopoiesis, such as interferon gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha), were found in plasma. These data illustrate that in vivo administration of high-dose IL-2 may result in a stimulatory effect on hematopoiesis. The induction of detectable levels of IL-5 and GM-CSF in the circulation may explain the eosinophilia and neutrophilia observed in these patients.
Effects of in vivo administration of interleukin-2 (IL-2) and IL-4, alone and in combination, on ex vivo human basophil histamine release
Authors: White MV , Igarashi Y , Emery BE , Lotze MT , Kaliner MA
Published: October 2019

Human basophils possess receptors for interleukin-2 (IL-2) and IL-4. The effect of 3 days of intravenous administration of IL-2 and/or IL-4 on basophil histamine release was examined in three groups of patients receiving IL-2, IL-4, or the combination of agents as part of a protocol to treat malignant melanoma or renal cell carcinoma. Because all patients received ranitidine for control of side effects, a control group of patients receiving ranitidine for Zollinger-Ellison's syndrome was also studied. IL-4 significantly inhibited IgE-mediated histamine release, while there was a trend for enhancement of IgE-mediated histamine release by IL-2. Administration of the combination of IL-2 and IL-4 did not alter IgE-mediated basophil histamine release. Both IL- 2 and IL-4, alone and in combination, enhanced basophil histamine release induced by histamine releasing factors in human nasal washings. The effect of IL-2 alone was significantly greater than that of IL-4 alone or the combination of IL-2 plus IL-4. Taken together, the data suggest that when coadministered, IL-4 may inhibit the effects of IL-2 on basophils. Neither cytokine exerted any effect on basophil histamine release induced by the calcium ionophore A23187, nor did ranitidine cause any effects on histamine release induced by any of the stimulants. Thus, human basophil reactivity can be affected by IL-2 and by IL-4. The role that these two cytokines play in basophil function in vivo is likely to be complex.
Chromosomal loss and deletion are the most common mechanisms for loss of heterozygosity from chromosomes 5 and 7 in malignant myeloid disorders
Authors: Neuman WL , Rubin CM , Rios RB , Larson RA , Le Beau MM , Rowley JD , Vardiman JW , Schwartz JL , Farber RA
Published: October 2019

We have examined a population of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) for loss of heterozygosity of polymorphic markers on chromosomes 5 and 7. The rationale for this study was the observation that the majority of patients with therapy- related leukemia (t-AML or t-MDS), resulting from cytotoxic treatment for prior malignancies, have loss of chromosome 5 and/or 7 or deletions involving the long arms of one or both of these chromosomes. This cytogenetic finding suggested that tumor-suppressor genes, important in the development of AML, may be located in these chromosomal regions. We analyzed a total of 60 patients, 43 with primary MDS/AML de novo and 17 with t-MDS/t-AML. Leukemia cells were evaluated for restriction fragment length polymorphisms (RFLPs). Leukemia cell genotypes were compared with lymphoblastoid cell genotypes from the same patients. Two cases of loss of heterozygosity were identified from chromosomes lacking visible deletions: one involving chromosome 5 in a patient with AML de novo who had a visible deletion of 5q at a later stage of the disease, and one involving chromosome 7 in a patient with t-AML. We conclude that allele loss from loci on chromosomes 5 and 7 in MDS/AML, when it occurs, usually results from major deletion or simple chromosome loss, rather than from mitotic recombination or chromosome loss with duplication of the remaining homologue.
Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559
Authors: Quinn MT , Mullen ML , Jesaitis AJ , Linner JG
Published: October 2019

Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti- cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule- associated Rap1A was translocated to the plasma membrane in PMA- stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide- generating system.
High Rates of Hepatitis G Virus Infection in Multitransfused Patients With Hemophilia
Authors: De Filippi Francesca , Colombo M. , Rumi Maria Grazia , Tradati F. , Prati D. , Zanella A. , Mannucci P.M.
Published: October 2019

The parallel measurement of serum antibodies to the hepatitis G virus (anti-HGV) and of viremia (HGV-RNA) should improve our understanding of HGV transmission by coagulation factor concentrates. The aim of this study was to assess the relationship between HGV, the type of concentrate infused, and liver disease in multitransfused hemophiliacs. To this end, anti-HGV and HGV-RNA were evaluated by an enzyme-linked immunosorbent assay and a nested–polymerase chain reaction assay in patients treated lifelong with nonvirus-inactivated plasma-derived concentrates (n = 128), virus-inactivated concentrates (n = 33), or recombinant factors (n = 7), and in 200 regular blood donors. The prevalence of serum HGV-RNA and anti-HGV was higher in the recipients of nonvirus-inactivated factors than in blood donors (HGV-RNA: 9% v 1.5%, P = .002; anti-HGV: 32% v 5%, P < .0001). In the recipients of virus-inactivated concentrates the prevalences of these markers were similar to those in blood donors (HGV-RNA: 3% v 1.5%; anti-HGV: 15% v 5%). The prevalence of either marker in the recipients of nonvirus-inactivated concentrates was higher than in the recipients of virus-inactivated factors (39% v 18%, P = .04). The former group had serum hepatitis C virus (HCV) RNA or anti-HCV more frequently than the latter group (HCV-RNA: 86% v 15%, P < .0001; anti-HCV: 96% v 18%, P < .0001). Serum alanine aminotransferase was persistently high in 83 (81%) patients with HCV-RNA alone, in 8 (89%) with HCV/HGV coinfection, and in none of the three patients with HGV-RNA only. Thus, HGV infection in hemophiliacs is more common than previous studies of HGV-RNA prevalence have suggested, but it resolved in most cases and caused chronic viremia only in a small number of patients, without biochemical evidence of persistent liver damage.
Expression of Activated Mutants of the Human Interleukin-3/Interleukin-5/Granulocyte-Macrophage Colony-Stimulating Factor Receptor Common β Subunit in Primary Hematopoietic Cells Induces Factor-Independent Proliferation and Differentiation
Authors: McCormack Matthew P. , Gonda Thomas J.
Published: October 2019

To date, several activating mutations have been discovered in the common signal-transducing subunit (hβc) of the receptors for human granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Two of these, FIΔ and I374N, result in a 37 amino acid duplication and a single amino acid substitution in the extracellular domain of hβc, respectively. A third, V449E, results in a single amino acid substitution in the transmembrane domain. Previous studies comparing the activity of these mutants in different hematopoietic cell lines imply that the transmembrane and extracellular mutations act by different mechanisms and suggest the requirement for cell type-specific molecules in signalling. To characterize the ability of these mutant hβc subunits to mediate growth and differentiation of primary cells and hence investigate their oncogenic potential, we have expressed all three mutants in primary murine hematopoietic cells using retroviral transduction. It is shown that, whereas expression of either extracellular hβc mutant confers factor-independent proliferation and differentiation on cells of the neutrophil and monocyte lineages only, expression of the transmembrane mutant does so on these lineages as well as the eosinophil, basophil, megakaryocyte, and erythroid lineages. Factor-independent myeloid precursors expressing the transmembrane mutant display extended proliferation in liquid culture and in some cases yielded immortalized cell lines.
Anti–Sia-lb (Anti-Gd) Cold Agglutinins Bind the Domain NeuNAcα2-3Gal in Sialyl Lewisx, Sialyl Lewisa, and Related Carbohydrates on Nucleated Cells and in Soluble Cancer-Associated Mucins
Authors: Gallart Teresa , Roelcke Dieter , Blay Maite , Pereira Arturo , Martı́nez Antonio , Massó Oriol , Viñas Odette , Cid Mariona , Esparza Jordi , Molina Rafael , Barceló Juanjo
Published: October 2019

Anti–Sia-lb (formerly anti-Gd) cold agglutinins (CAs) recognize sialylated carbohydrates on both adult and neonate red blood cells (RBCs). RBC CA activity inhibition experiments reported here indicate that the domain NeuNAcα2-3Gal, as found in sialyllactose, synthetic sialyl(s) Lewis(Le)x and sLea, sialyllactosamine, sialyl-fucosyllactose, and nonfucosylated sLea, constitutes the minimal epitope for these CAs, implicating that these autoantibodies could be able to bind this domain in sLex and sLea and related carbohydrates expressed on nucleated cells and in soluble cancer-related mucins. The following data obtained with the previously characterized monoclonal IgMk anti-Sia-lb CA, GAS, show that this is the case. GAS epitope expression among leukocytes that lack sLea parallels that of sLex determinant as detected by mouse monoclonal antibodies (MoAbs), especially MoAb KM-93. It is also found on epithelial malignant cells bearing both sLex and sLea. GAS epitope on these nucleated cells, (1) like that present on RBC, is abolished by sialidase, unaffected by proteases, and inhibited by sialyllactose; and (2) is overlapping and/or proximal to that recognized by anti-sLex MoAb, CSLEX-1, and KM-93. Moreover, CAGAS binds soluble cancer-associated mucins bearing sLex and sLea determinants. This binding is inhibited by sialyllactose and these mucins inhibit the RBC CA activity of CAGAS. The possible significance of anti–Sia-lb (anti-Gd) CAs as autoantibodies directed to carbohydrate ligands of host adhesion molecules that might be receptors of microbial adhesins of some CA-inducing pathogens is discussed.
Metalloproteinases in Multiple Myeloma: Production of Matrix Metalloproteinase-9 (MMP-9), Activation of proMMP-2, and Induction of MMP-1 by Myeloma Cells
Authors: Barillé Sophie , Akhoundi Camélia , Collette Madeleine , Mellerin Marie-Paule , Rapp Marie-Josée , Harousseau Jean-Luc , Bataille Régis , Amiot Martine
Published: October 2019

Multiple myeloma is a very devastating cancer with a high capacity to destroy bone matrix. Matrix metalloproteinases (MMPs) play a critical role in bone remodeling and tumor invasion. In this study, we have investigated the involvement of interstitial collagenase (MMP-1) and gelatinases (MMP-2 and MMP-9) in the biology of multiple myeloma. We show (1) that myeloma cells express MMP-9 and (2) that this expression is not subjected to regulation either by interleukin-6 (IL-6), the major myeloma cell growth factor, or by other cytokines involved in the multiple myeloma cytokine network. In the tumoral environment, we show that bone marrow stromal cells express MMP-1 and MMP-2. Whereas MMP-1 is positively regulated by IL-1β, tumor necrosis factor-α, and Oncostatin M, MMP-2 is not modulated by any of these cytokines. To evaluate whether myeloma cells can modify the bone marrow stromal environment, we have examined these MMP activities in coculture. Interestingly, we have observed an upregulation of MMP-1 and a partial conversion of the proMMP-2 into its activated form. We conclude that the increase of MMP activity produced or induced by myeloma cells in these cocultures could favor bone resorption and tumor invasion. Inhibition of such activities could represent a new therapeutical approach in multiple myeloma.
Downregulation of the Proinflammatory Cytokine Response to Endotoxin by Pretreatment With the Nontoxic Lipid A Analog SDZ MRL 953 in Cancer Patients
Authors: Kiani Alexander , Tschiersch Astrid , Gaboriau Elke , Otto Florian , Seiz Alexander , Knopf Hans-Peter , Stütz Peter , Färber Lothar , Haus Ulrike , Galanos Chris , Mertelsmann Roland , Engelhardt Rupert
Published: October 2019

Interfering with the endotoxin-mediated cytokine cascade is thought to be a promising approach to prevent septic complications in gram-negative infections. The synthetic lipid A analog SDZ MRL 953 has been shown to be protective against endotoxic shock and bacterial infection in preclinical in vivo models. As part of a trial of unspecific immunostimulation in cancer patients, we conducted a double-blind, randomized, vehicle-controlled phase I trial of SDZ MRL 953 to investigate, first, its biologic effects and safety of administration in humans and, second, its influence on reactions to a subsequent challenge of endotoxin (Salmonella abortus equi). Twenty patients were treated intravenously with escalating doses of SDZ MRL 953 or vehicle control, followed by an intravenous application of endotoxin (2 ng/kg of body weight [BW]). Administration of SDZ MRL 953 was safe and well-tolerated. SDZ MRL 953 itself increased granulocyte counts and serum levels of granulocyte colony-stimulating factor (G-CSF ) and interleukin-6 (IL-6), but not of the proinflammatory cytokines tumor necrosis factor-α (TNF-α), IL-1β, and IL-8. Compared with vehicle control, pretreatment with SDZ MRL 953 markedly reduced the release of TNF-α, IL-1β, IL-8, IL-6, and G-CSF, but augmented the increase in granulocyte counts to endotoxin. Induction of tolerance to the endotoxin-mediated cascade of proinflammatory cytokines by pretreatment with SDZ MRL 953 in patients at risk may help to prevent complications of gram-negative sepsis.
Normal Immunologic Response to a Neoantigen, Bacteriophage ΦX-174, in Baboons With Long-Term Lymphohematopoietic Reconstitution From Highly Purified CD34+ Lin− Allogeneic Marrow Cells
Authors: Andrews Robert G. , Winkler Aaron , Potter Jennifer , Bryant Eileen , Knitter Glenn H. , Bernstein Irwin D. , Ochs Hans D.
Published: October 2019

The CD34 antigen is thought to be expressed by hematopoietic stem cells in adult humans and nonhuman primates. We present data that baboons transplanted with highly purified allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes and myeloid cells, isolated from sex-mismatched mixed lymphocyte culture (MLC) nonreactive siblings, have maintained stable lymphohematopoietic engraftment with donor cells for greater than 4.9, greater than 6.0, and 5.0 years. Cytogenetic analysis of unfractionated marrow and peripheral blood cells at multiple time points after transplantation show virtually all donor cells in two animals and stable mixed chimerism in the third. We used polymerase chain reaction to show that colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, and high proliferative potential colony-forming cells (HPP-CFC) were virtually all of donor origin in two animals and present at lower levels in the stable mixed chimera. CD20+ B-lymphoblastoid cell lines derived by Herpesvirus Papio transformation of peripheral blood cells were virtually all donor in two animals and 50% donor in the mixed chimera. CD4+ and CD8+ T cells and neutrophils purified from the peripheral blood of the two female animals also were all donor-derived. To assess immunologic function after transplantation, we immunized the three long-term chimeric animals and two normal control animals with bacteriophage ΦX-174, a neoantigen that requires the interaction of antigen-presenting cells, T lymphocytes, and B lymphocytes to mount a normal antibody response. Experimental and control animals, when immunized with bacteriophage, had similar serum Ig levels. The experimental and control animals generated similar titers of antibacteriophage antibodies after primary and secondary immunizations with evidence of amplification and class switching. These findings further support the hypothesis that the CD34+ antigen is expressed on hematopoietic stem cells that can mediate stable long-term lymphohematopoiesis in vivo and, importantly, that normal immunologic function can be reconstituted in vivo after transplantation of the highly purified CD34+ Lin− cells alone.
An In Vitro Model for Toxin-Mediated Vascular Leak Syndrome: Ricin Toxin A Chain Increases the Permeability of Human Endothelial Cell Monolayers
Authors: Lindstrom Alan L. , Erlandsen Stanley L. , Kersey John H. , Pennell Christopher A.
Published: October 2019

Vascular leak syndrome (VLS) is the dose-limiting toxicity observed in clinical trials of immunotoxins containing ricin toxin A chain (RTA). RTA itself is thought to cause VLS by damaging vascular endothelial cells, but the exact mechanism remains unclear. This is partially due to the paucity of appropriate models. To study VLS, we developed an in vitro model in which human umbilical vein-derived endothelial cells were first grown to confluence on microporous supports and then cultured under low pressure in the presence or absence of RTA. Endothelial cell barrier function was assessed by measuring the volume of fluid that passed through each monolayer per unit time. We found that RTA significantly increased monolayer permeability at times and concentrations consistent with the onset of VLS in patients treated with RTA-based immunotoxins. Scanning electron microscopy showed that intercellular gaps formed in endothelial monolayers exposed to RTA. Intercellular gap formation followed endothelial cell death caused by the enzymatic activity of RTA. We conclude that RTA is directly toxic to endothelial cells in vitro and speculate that this contributes to VLS in vivo.
Conjugation of Blocked Ricin to an Anti-CD19 Monoclonal Antibody Increases Antibody-Induced Cell Calcium Mobilization and CD19 Internalization
Authors: Goulet Anne-Christine , Goldmacher Victor S. , Lambert John M. , Baron Chantal , Roy Denis-Claude , Kouassi Edouard
Published: October 2019

CD19 (B4) is a signal transduction molecule restricted to the B-cell lineage and the target of the immunotoxin anti-B4–blocked ricin (anti-B4–bR), which is composed of the monoclonal antibody (MoAb) anti-B4 and the modified plant toxin blocked ricin. To explore the influence of conjugation of blocked ricin to anti-B4 on functional activation of CD19, we investigated the effects of anti-B4–bR, and that of unconjugated anti-B4, on intracellular calcium mobilization and ligand/receptor internalization. The data showed that anti-B4–bR was more potent than anti-B4 in triggering cell calcium mobilization. Two other immunotoxins that bind to the B-cell surface, anti-CD20–bR and anti-CD38–bR, were devoid of the calcium increasing effect of anti-B4–bR. Furthermore, anti-B4 conjugated to ricin A-chain was also without effect in Namalwa cells, indicating that the ricin B-chain component was required for anti-B4–bR effect. Anti-B4–bR-induced calcium mobilization was inhibited in the presence of lactose, yet the calcium response induced by cross-linking anti-B4–bR with a second step antibody was not affected. The extent of CD19 modulation induced by anti-B4–bR was higher than that induced by anti-B4, and lactose dampened the effect of the immunotoxin down to that of the MoAb. Moreover, the number of internalized immunotoxin molecules was higher than that of unconjugated MoAb. Although a mechanism involving dimerization of the immunotoxin cannot be excluded, our findings suggest that the residual binding activity of the blocked ricin B-chain to cell surface molecules plays an important role in the greater calcium fluxes and greater internalization rate of anti-B4–bR, and is of functional significance in the mechanism of intoxication of cells by the immunotoxin.
CD84 Leukocyte Antigen Is a New Member of the Ig Superfamily
Authors: de la Fuente Miguel Angel , Pizcueta Pilar , Nadal Marga , Bosch Jaime , Engel Pablo
Published: October 2019

cDNA isolated from a human B-cell line Raji library was analyzed and shown to encode the full-length cDNA sequence of a novel cell-surface glycoprotein, initially termed HLy9-β. The predicted mature 307-amino acid protein was composed of two extracellular Ig-like domains, a hydrophobic transmembrane region, and an 83-amino acid cytoplasmic domain. The extracellular Ig-like domains presented structural and sequence homology with a group of members of the Ig superfamily that included CD2, CD48, CD58, and Ly9. Northern blot analysis showed that the expression of HLy9-β was predominantly restricted to hematopoietic tissues. Chromosome localization studies mapped the HLy9-β gene to chromosome 1q24, where other members of this Ig superfamily (CD48 and HumLy9) have been mapped. CD84 monoclonal antibodies (MoAbs) were shown to react with cells transfected with the cloned cDNA. These MoAbs were further used to show that CD84 is expressed as a single chain cell-surface glycoprotein of Mr 64,000 to 82,000, which was highly glycosylated. CD84 had a unique pattern of expression, being found predominantly on lymphocytes and monocytes. Thus, the glycoprotein HLy9-β is recognized by MoAbs previously clustered as CD84 and represents a newly identified member of the Ig superfamily that may play a significant role in leukocyte activation.
CD7+ and CD56+ Myeloid/Natural Killer Cell Precursor Acute Leukemia: A Distinct Hematolymphoid Disease Entity
Authors: Suzuki Ritsuro , Yamamoto Kazuhito , Seto Masao , Kagami Yoshitoyo , Ogura Michinori , Yatabe Yasushi , Suchi Taizan , Kodera Yoshihisa , Morishima Yasuo , Takahashi Toshitada , Saito Hidehiko , Ueda Ryuzo , Nakamura Shigeo
Published: October 2019

The disease spectrum of natural killer (NK) cell leukemias and lymphomas has recently been expanding with the continuing evolution in diagnostic concepts. We describe here seven cases of acute leukemia of conceivable myeloid and NK cell precursor phenotype in six men and one woman varying from 19 to 59 years of age (median, 46 years). Striking extramedullary involvement was evident at initial presentation, with peripheral lymphadenopathy and/or mediastinal masses. Two lacked any leukemic cells in the bone marrow at diagnosis. Using cytochemical myeloperoxidase staining, less than 3% of the leukemic cells showed positive reactivity. However, expression of CD7, CD33, CD34, CD56, and frequently HLA-DR, but not other NK, T-cell, and B-cell markers was observed. Cytoplasmic CD3 was detected in three of the cases by flow cytometry and in six by Northern blotting, suggesting an origin from common progenitors between the NK cell and myeloid lineages. All but one presented germline configurations of the T-cell receptor β and γ chain genes and Ig heavy chain gene. With regard to morphology, the cells were generally L2-shaped, with variation in cell size, round to moderately irregular nuclei and prominent nucleoli, pale cytoplasm, and a lack of azurophilic granules. Histopathologic examination of biopsied specimens of extramedullary tumors showed a lymphoblast-like morphology, implying the differential diagnostic problem from lymphoblastic lymphomas, especially in cases lacking bone marrow involvement. Three patients were successfully treated with chemotherapy for acute myeloid leukemia (AML), whereas three other patients proved refractory to chemotherapeutic regimens for lymphoid malignancies, although two responded to subsequent AML chemotherapy. However, despite intensive chemotherapy, including allogeneic bone marrow transplantation, most persued fatal courses within 41 months. These data suggested that the CD7+ and CD56+ myeloid/NK cell precursor acute leukemia might constitute a distinct biologic and clinical disease entity. Its recognition appears to be particularly important for the clinicopathologic evaluation of CD56+ hematolymphoid malignancies and the development of therapeutic approaches to such disease.
Apoptosis Resistance of Blood Cells From Patients With Paroxysmal Nocturnal Hemoglobinuria, Aplastic Anemia, and Myelodysplastic Syndrome
Authors: Horikawa Kentaro , Nakakuma Hideki , Kawaguchi Tatsuya , Iwamoto Norihiro , Nagakura Shoichi , Kagimoto Tadashi , Takatsuki Kiyoshi
Published: October 2019

Bone marrow (BM) hypoplasia is a major cause of death in paroxysmal nocturnal hemoglobinuria (PNH). However, little is known about the molecular events leading to the hypoplasia. Considering the close pathologic association between PNH and aplastic anemia (AA), it is suggested that a similar mechanism operates in the development of their BM failure. Recent reports have indicated apoptosis-mediated BM suppression in AA. It is thus conceivable that apoptosis also operates to cause BM hypoplasia in PNH. If this is the case, PNH clones need to survive apoptosis and show considerable expansion leading to clinical manifestations. We report here that granulocytes obtained from 11 patients with PNH were apparently less susceptible than those from 20 healthy individuals to both spontaneous apoptosis without any ligands and that induced by anti-FAS (CD95) antibody in vitro. The patients' BM CD34+ cells were also resistant to apoptosis induced by treatment with tumor necrosis factor-α, interferon-γ, and subsequently with anti-FAS antibody. In lymphocytes, the pathologic resistance was not discriminated from inherent resistance to apoptosis. Granulocytes from 13 patients with AA and 12 patients with myelodysplastic syndrome (MDS) exhibited similar resistance to apoptosis. CD34+ cells from MDS-BM also showed similar tendency. Thus, the comparative resistance to apoptosis supports the pathogenic implication of apoptosis in marrow injury of PNH and related stem cell disorders.
Regulation of Neutrophil Apoptosis by Tumor Necrosis Factor-α: Requirement for TNFR55 and TNFR75 for Induction of Apoptosis In Vitro
Authors: Murray Joanna , Barbara Jeffrey A.J. , Dunkley Sarah A. , Lopez Angel F. , Van Ostade Xaveer , Condliffe Alison M. , Dransfield Ian , Haslett Christopher , Chilvers Edwin R.
Published: October 2019

Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-α (TNF-α), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-α inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4 , and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-α was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-α neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-α to accelerate apoptosis was lost if neutrophils were primed with 1 μmol/L PAF or aged for 6 hours before TNF-α addition. The TNFR55-selective TNF-α mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-α. Although the TNFR75-selective mutant (D143F ) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-α–stimulated apoptosis. Hence, TNF-α has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.
Interleukin-15 (IL-15) Induces IL-8 and Monocyte Chemotactic Protein 1 Production in Human Monocytes
Authors: Badolato Raffaele , Ponzi Alessandro Negro , Millesimo Maura , Notarangelo Luigi D. , Musso Tiziana
Published: October 2019

Interleukin-15 (IL-15) is a recently characterized cytokine that shares many biological activities with IL-2 and interacts with the β and γ components of the IL-2 receptor. Unlike IL-2, which is secreted only by T cells, IL-15 is expressed preferentially by nonlymphoid tissues, epithelial, and fibroblast cell lines and by activated monocytes/macrophages. High concentrations of IL-15 have been shown in inflamed joints of rheumatoid arthritis patients, suggesting a role for IL-15 in inflammatory diseases where there is recruitment of leukocytes. Although monocytes have been shown to bind IL-15, its effects on these cells are not defined. In this report we show that supernatants of monocytes treated with IL-15–contained chemotactic activity for neutrophils and monocytes which was neutralized by anti-IL-8 or by anti-monocyte chemotactic protein 1 (MCP-1) antibodies, respectively. Secretion of IL-8 and MCP-1 proteins is detectable by enzyme-linked immunosorbent assay as early as 6 hours after stimulation with IL-15. Production of the two chemokines is correlated with induction by IL-15 of mRNA expression in monocytes. In addition, IL-8 and MCP-1 induction by IL-15 is differently regulated by interferon-γ (IFN-γ) and IL-4. IFN-γ inhibited IL-15–induced IL-8 secretion, but synergized with IL-15 in MCP-1 induction; whereas IL-4 inhibited both IL-8 and MCP-1 induction by IL-15. These results show that IL-15 can stimulate monocytes to produce chemokines that cause inflammatory cell accumulation. Thus, IL-15 locally produced at sites of inflammation may play a pivotal role in the regulation of the leukocyte infiltrate.
Treatment of Thrombocytopenia in Chimpanzees Infected With Human Immunodeficiency Virus by Pegylated Recombinant Human Megakaryocyte Growth and Development Factor
Authors: Harker Laurence A. , Marzec Ulla M. , Novembre Francis , Sundell I. Birgitta , Waller Edmund K. , Karpatkin Simon , McClure Harold M. , Kelly Andrew B. , Stead Richard B.
Published: October 2019

Three chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 ± 19 × 103/μL (P = .004 compared with 228 ± 92 × 103/μL in 44 normal control animals), mean platelet volumes of 11.2 ± 1.8 fL (P > .5 compared with 10.9 ± 0.7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 ± 364 pg/mL (P < .001 compared with 324 ± 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 × 103 RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 μg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 ± 19 to 599 ± 260 × 103 platelets/μL;P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 ± 6.5 × 106/kg to 353 ± 255 × 106/kg;P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 ± 0.6 to 14.1 × 103 CFU-Meg/1,000 CD34+ marrow cells); and (4) serum levels of Mpl ligand from 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of 1,840 ± 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/μL to 9.9 ± 5.0 × 103/μL (P= .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P > .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P < .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 × 103 RNA viral copies/mL; P > .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.
Treatment of Autoimmune Disease by Intense Immunosuppressive Conditioning and Autologous Hematopoietic Stem Cell Transplantation
Authors: Burt Richard K. , Traynor Ann E. , Pope Richard , Schroeder James , Cohen Bruce , Karlin Karyn H. , Lobeck Lorri , Goolsby Charles , Rowlings Philip , Davis Floyd A. , Stefoski Dusan , Terry Cass , Keever-Taylor Carolyn , Rosen Steve , Vesole David , Fishman Maryanne , Brush Mary , Mujias Salim , Villa Marcelo , Burns William H.
Published: October 2019

Multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis are immune-mediated diseases that are responsive to suppression or modulation of the immune system. For patients with severe disease, immunosuppression may be intensified to the point of myelosuppression or hematopoietic ablation. Hematopoiesis and immunity may then be rapidly reconstituted by reinfusion of CD34+progenitor cells. In 10 patients with these autoimmune diseases, autologous hematopoietic stem cells were collected from bone marrow or mobilized from peripheral blood with either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and G-CSF. Stem cells were enriched ex vivo using CD34+ selection and reinfused after either myelosuppressive conditioning with cyclophosphamide (200 mg/kg), methylprednisolone (4 g) and antithymocyte globulin (ATG; 90 mg/kg) or myeloablative conditioning with total body irradiation (1,200 cGy), methylprednisolone (4 g), and cyclophosphamide (120 mg/kg). Six patients with multiple sclerosis, 2 with systemic lupus erythematosus, and 2 with rheumatoid arthritis have undergone hematopoietic stem cell transplantation. Mean time to engraftment of an absolute neutrophil count greater than 500/μL (0.5 × 109/L) and a nontransfused platelet count greater than 20,000/μL (20 × 109/L) occurred on day 10 and 14, respectively. Regimen-related nonhematopoietic toxicity was minimal. All patients improved and/or had stabilization of disease with a follow-up of 5 to 17 months (median, 11 months). We conclude that intense immunosuppressive conditioning and autologous T-cell–depleted hematopoietic transplantation was safely used to treat these 10 patients with severe autoimmune disease. Although durability of response is as yet unknown, all patients have demonstrated stabilization or improvement.
Gene Delivery to Human B-Precursor Acute Lymphoblastic Leukemia Cells
Authors: Mascarenhas Leo , Stripecke Renata , Case Scott S. , Xu Dakun , Weinberg Kenneth I. , Kohn Donald B.
Published: October 2019

Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus–type 1 (HIV-1)–based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1–based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.
Pharmacokinetics and Pharmacodynamics of Oral Methotrexate and Mercaptopurine in Children With Lower Risk Acute Lymphoblastic Leukemia: A Joint Children’s Cancer Group and Pediatric Oncology Branch Study
Authors: Balis Frank M. , Holcenberg John S. , Poplack David G. , Ge Jeffrey , Sather Harland N. , Murphy Robert F. , Ames Matthew M. , Waskerwitz Mary J. , Tubergen David G. , Zimm Solomon , Gilchrist Gerald S. , Bleyer W. Archie
Published: October 2019

We prospectively assessed the pharmacokinetics of methotrexate, mercaptopurine, and erythrocyte thioguanine nucleotide levels in a homogenous population of children with lower risk acute lymphoblastic leukemia and correlated pharmacokinetic parameters with disease outcome. The maintenance therapy regimen included daily oral mercaptopurine (75 mg/m2) and weekly oral methotrexate (20 mg/m2). One hundred ninety-one methotrexate doses and 190 mercaptopurine doses were monitored in 89 patients. Plasma drug concentrations of both agents were highly variable. The area under the plasma concentration-time curve (AUC) of methotrexate ranged from 0.63 to 12 μmol•h/L, and the AUC of mercaptopurine ranged from 0.11 to 8 μmol•h/L. Drug dose, patient age, and duration of therapy did not account for the variability. Methotrexate AUC was significantly higher in girls than boys (P = .007). There was considerable intrapatient variability for both agents. Erythrocyte thioguanine nucleotide levels were also highly variable (range, 0 to 10 pmol/g Hgb) and did not correlate with mercaptopurine dose or AUC. A Cox regression analysis showed that mercaptopurine AUC was a marginally significant (P = .043) predictor of outcome, but a direct comparison of mercaptopurine AUC in the remission and relapsed patient groups failed to show a significant difference. Methotrexate and mercaptopurine plasma concentrations and erythrocyte thioguanine nucleotide levels were highly variable, but measurement of these pharmacokinetic parameters at the start of maintenance will not distinguish patients who are more likely to relapse.
An In Vivo Topoisomerase II Cleavage Site and a DNase I Hypersensitive Site Colocalize Near Exon 9 in the MLLBreakpoint Cluster Region
Authors: Strissel Pamela L. , Strick Reiner , Rowley Janet D. , Zeleznik-Le Nancy J.
Published: October 2019

The human myeloid-lymphoid leukemia gene, MLL (also calledALL-1, Htrx, or HRX ), maps to chromosomal band 11q23. MLL is involved in translocations that result in de novo acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), mixed lineage leukemia, and also in therapy AML (t-AML) and therapy ALL (t-ALL) resulting from treatment with DNA topoisomerase II (topo II) targeting drugs. MLL can recombine with more than 30 other chromosomal bands, of which 16 of the partner genes have been cloned. Breaks in MLL occur in an 8.3-kb breakpoint cluster region (BCR) encompassing exons 5 through 11. We recently demonstrated that 75% of de novo patient breakpoints in MLL mapped in the centromeric half of the BCR between two scaffold-associated regions (SAR), whereas 75% of the t-AML patient breakpoints mapped to the telomeric half of the BCR within a strong SAR. We have mapped additional structural elements in the BCR. An in vivo DNA topo II cleavage site (induced with several different drugs that target topo II) mapped near exon 9 in three leukemia cell lines. A strong DNase I hypersensitive site (HS) also mapped near exon 9 in four leukemia cell lines, including two in which MLL was rearranged [a t(6;11) and a t(9;11)], and in two lymphoblastoid cell lines with normalMLL. Two of the leukemia cell lines also showed an in vivo topo II cleavage site. Our results suggest that the chromatin structure of the MLL BCR may influence the location of DNA breaks in both de novo and therapy-related leukemias. We propose that topo II is enriched in the MLL telomeric SAR and that it cleaves the DNase I HS site after treatment with topo II inhibitors. These events may be involved in recombination associated with t-AML/t-ALL breakpoints mapping in the MLL SAR.
Development of a Model for Evaluating the Interaction Between Human Pre-B Acute Lymphoblastic Leukemic Cells and the Bone Marrow Stromal Cell Microenvironment
Authors: Shah Nisha , Oseth LeAnn , LeBien Tucker W.
Published: October 2019

Clonal expansion of B-cell precursor acute lymphoblastic leukemia (ALL) is potentially regulated by survival, growth, and death signals transduced by the bone marrow (BM) microenvironment. Using a human BM stromal cell culture that supports the growth of normal human B-cell precursors, we established a pre-B ALL cell line designated BLIN-2. BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, a dic(9;20) chromosomal abnormality, and a bi-allelic deletion of thep16INK4a and p19ARF genes. The most interesting feature of BLIN-2 is an absolute dependence on adherent human BM stromal cells for sustained survival and growth. BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis, and direct contact with viable BM stromal cells is essential for optimal growth. BLIN-2 cells also grow on vascular cell adhesion molecule-1 (VCAM-1)–negative human skin fibroblasts, making it unlikely that a very late antigen-4 (VLA-4)/VCAM-1 interaction is required for BLIN-2 growth. Western blot analysis of BLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16INK4ap19ARF but does not require BM stromal cells for growth, does not undergo Rb phosphorylation after BM stromal cell contact. The BLIN-2 cell line will facilitate identification of ligand/receptor interactions at the B-cell precursor/BM stromal cell interface and may provide new insight into microenvironmental regulation of leukemic cell survival and growth.