bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergism Between Mycophenolate Mofetil and Cyclosporine in Preventing Graft-Versus-Host Disease Among Lethally Irradiated Dogs Given DLA-Nonidentical Unrelated Marrow Grafts
Authors: Yu Cong , Seidel Kristy , Nash Richard A. , Deeg H. Joachim , Sandmaier Brenda M. , Barsoukov Alexander , Santos Erlinda , Storb Rainer
Published: October 2019

Mycophenolate mofetil (MMF) was evaluated either alone or combined with cyclosporine (CSP) for preventing graft-versus-host disease (GVHD) in dogs given 9.2 Gy total body irradiation and DLA-nonidentical unrelated marrow grafts. Marrow autograft studies showed gut toxicity as limiting MMF side effects. Four groups were studied for GVHD prevention: six dogs in group 1 received MMF 10 mg/kg twice daily subcutaneously (SC) on days 0 to 27. They died between 8 to 28 days from infection or GVHD; survival was better than that of 72 controls given no immunosuppression (P = .04), but not different from 19 dogs given CSP. Four dogs in group 2 received MMF as described, along with CSP at 10 to 15 mg/kg twice daily on days 0 to 27. They died at 6 to 98 days from CSP-associated toxicity, weight loss, or infection. Nine dogs in group 3 received MMF SC twice daily 6 mg/kg/d for 3 days, followed by 10 mg/kg twice daily until day 27, along with CSP as described; four died between 7 to 106 days with intussusception, infection, or GVHD, and five became long-term survivors. Six dogs in group 4 received shortened MMF (21 days) and reduced doses of CSP given through day 100. Three died with GVHD or infection between days 38 to 119, and three became long-term survivors. Results support the notion of synergism between MMF and CSP, as evidenced by stable graft-host tolerance in greater than 50% of dogs.
BCR-ABL Antisense Oligodeoxynucleotide In Vitro Purging and Autologous Bone Marrow Transplantation for Patients With Chronic Myelogenous Leukemia in Advanced Phase
Authors: de Fabritiis Paolo , Petti Maria Concetta , Montefusco Enrico , De Propris Maria Stefania , Sala Roberta , Bellucci Roberto , Mancini Marco , Lisci Alessandro , Bonetto Francesco , Geiser Tim , Calabretta Bruno , Mandelli Franco
Published: October 2019

BCR-ABL antisense oligodeoxynucleotides (ODN) have provided evidence of antileukemia effect when tested in vitro against Philadelphia-positive (Ph-pos) cells and in vivo when injected into leukemic mice. On the basis of the results obtained in vitro at diagnosis, eight patients with chronic myelogenous leukemia (CML) were selected and submitted to autologous bone marrow transplantation (ABMT) with bone marrow (BM) cells purged in vitro with junction-specific (J-sp) BCR-ABL antisense ODN at the time of transformation in accelerated phase or during second chronic phase. Mononuclear BM cells were treated in vitro for 24 or 72 hours with 150 μg/mL of antisense ODN yielding a median recovery of 47.6% mononuclear cells, 48.8% CD34+ cells, and 20.3% clonogenic cells. After a conditioning regimen including busulphan and etoposide, the reinfused treated cells allowed engraftment and hematologic reconstitution in all patients. Evaluation of the antileukemic effect by standard cytogenetic analysis and fluorescence in situ hybridization showed a complete karyotypic response in two cases and a minimal or no response in the other six. The patient autografted in second chronic phase died in blast crisis 7 months after ABMT; of the seven patients autografted in transformation, three developed blast crisis 21 to 39 months after reinfusion, one died from unrelated BMT complications 30 months after ABMT, and three are in persistent second chronic phase 14 to 26 months after autograft. The low toxicity of the protocol and the hemopoietic reconstitution observed in all patients make this approach feasible; the marked karyotypic response observed in some patients and the duration of the second chronic phase show that ODN-mediated BM purging and autograft is a promising treatment for this high-risk group of CML.
Sustained Phenotypic Correction of Murine Hemophilia A by In Vivo Gene Therapy
Authors: Connelly Sheila , Andrews Julie L. , Gallo Angela M. , Kayda Dawn B. , Qian Jiahua , Hoyer Leon , Kadan Michael J. , Gorziglia Mario I. , Trapnell Bruce C. , McClelland Alan , Kaleko Michael
Published: October 2019

Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.
Factor XIIIa Cross-Linking of the Marburg Fibrin: Formation of αm·γn-Heteromultimers and the α-Chain–Linked Albumin·γ Complex, and Disturbed Protofibril Assembly Resulting in Acquisition of Plasmin Resistance Relevant to Thrombophila
Authors: Sugo Teruko , Nakamikawa Chizuko , Takebe Mikihiro , Kohno Isao , Egbring Rudorf , Matsuda Michio
Published: October 2019

The truncated Aα-chain of fibrinogen Marburg is partly linked with albumin by a disulfide bond. Based on the recovery of the first six amino acid residues assigned to the subunit polypeptides of fibrinogen (the Aα-and γ-chains) and albumin, 0.33 mol of albumin was estimated to be linked to 1 mol of the Marburg fibrinogen. When the Marburg fibrinogen was clotted with thrombin-factor XIIIa-Ca2+, various αmγnheteromultimers were produced, and part of the albumin was cross-linked to the γ-chain. Acid-solubilized Marburg fibrin monomer failed to form large aggregates that could be detected by monitoring turbidity at A350, but it was able to enhance tissue-type plasminogen-activator–catalyzed plasmin generation, though not as avidly as the normal control, indicating that the double-stranded protofibrils had, to some extent, been constructed. This idea seems to be supported by normal factor XIIIa–catalyzed cross-linking of the fibrin γ-chains. However, the cross-linked Marburg fibrin, being apparently fragile and translucent, was highly resistant against plasmin, and its subunit components were considerably retained for 48 hours as noted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the exact mechanisms are still unclear, the albumin-incorporated factor XIIIa–cross-linked Marburg fibrin seems to have undergone a critical structural alteration(s) to acquire resistance against plasmin. This aquisition of plasmin resistance may be contributed to the postoperative pelvic vein thrombosis and recurrent pulmonary embolisms in the patient after caesarian section for her first delivery at the age of 20 years.
gp130, The Cytokine Common Signal-Transducer of Interleukin-6 Cytokine Family, Is Downregulated in T Cells In Vivo by Interleukin-6
Authors: Wang Xue-Jie , Taga Tetsuya , Yoshida Kanji , Saito Mikiyoshi , Kishimoto Tadamitsu , Kikutani Hitoshi
Published: October 2019

gp130 is a common signal-transducing receptor component for the interleukin-6 (IL-6) family of cytokines. To investigate the expression of gp130 in T-cell subsets and its regulation, anti-murine gp130 monoclonal antibody (MoAb) was used for flow cytometric analysis. In normal mice, gp130 was differentially expressed in thymocyte and splenic T-cell subpopulations defined by CD4/CD8 expression. In aged MRL/lpr mice, although gp130 expression was detectable in splenic CD4+ or CD8+ T cells, gp130 expression was significantly downregulated. Because serum levels of IL-6 and soluble IL-6 receptor (sIL-6R) are elevated in these mice, we examined the possibility that the downregulation of gp130 expression on splenic T cells might be produced in response to continuous activation of gp130 by high levels of serum IL-6. In transgenic mice overexpressing IL-6, gp130 expression in the splenic T cells was significantly decreased. After stimulation with IL-6 in vitro, the level of gp130 on CD4+ or CD8+ splenic T cells from normal mice was significantly decreased. These results suggest that the expression of gp130 in splenic T cells could be downregulated by the IL-6 stimulation under physiological or pathological circumstances.
Interleukin-12 Inhibits Graft-Versus-Host Disease Through an Fas-Mediated Mechanism Associated With Alterations in Donor T-Cell Activation and Expansion
Authors: Dey Bimalangshu R. , Yang Yong-Guang , Szot Gregory L. , Pearson Denise A. , Sykes Megan
Published: October 2019

We have recently made the paradoxical observation that a single injection of recombinant murine interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) inhibits graft-versus-host disease (GVHD) in lethally irradiated mice receiving fully major histocompatability complex (MHC)-mismatched bone marrow and spleen cells. We have now examined the mechanism of this effect of IL-12 on acute GVHD. By day 4 post-BMT, IL-12–treated mice showed marked reductions in splenic donor CD4+ and CD8+ T cells compared with GVHD controls. Expression of the early activation markers IL-2R alpha chain (CD25) and CD69 on splenic donor CD4+ cells was considerably higher at early time points (36 and 72 hours post-BMT) in IL-12–treated mice compared with GVHD controls. However, the later, GVHD-associated increase in CD25 and very late antigen-4 (VLA-4) expression on donor T cells was greatly depressed in IL-12–protected mice compared with GVHD controls. The marked GVHD-associated expansion of host-reactive T helper cells by day 4 was also completely inhibited in the IL-12–treated group. Expression of Fas was increased on donor CD4 cells of IL-12–treated mice compared with those of controls on days 3 through 7 post-BMT. Furthermore, the ability of IL-12 to protect against GVHD was at least partially dependent on the ability of donor cells to express functional Fas molecules. We conclude that IL-12 treatment at the time of BMT markedly perturbs the activation of alloreactive donor CD4+ T cells that play a critical role in the pathogenesis of acute GVHD. We hypothesize that these perturbations culminate in Fas-dependent apoptosis of donor T cells, thus impeding their expansion and their GVHD-promoting activity.
High CD30 Ligand Expression by Epithelial Cells and Hassal's Corpuscles in the Medulla of Human Thymus
Authors: Romagnani Paola , Annunziato Francesco , Manetti Roberto , Mavilia Carmelo , Lasagni Laura , Manuelli Cinzia , Vannelli Gabriella B. , Vanini Vittorio , Maggi Enrico , Pupilli Cinzia , Romagnani Sergio
Published: October 2019

CD30 is a member of tumor necrosis factor (TNF) receptor superfamily that is expressed by activated T cells in the presence of interleukin-4 (IL-4). Although CD30 can mediate a variety of signals, CD30-deficient mice have impaired negative selection of T cells, suggesting that at least in the context of murine thymus, CD30 is a cell death–mediating molecule. The ligand for CD30 (CD30L) is a membrane-associated glycoprotein related to TNF, which is known to be expressed mainly by activated T cells and other leukocytes. However, the nature of CD30L-expressing cells involved in the interaction with CD30+ thymocytes is unclear. We report here that in postnatal human thymus the great majority of CD30+ cells are double positive (CD4+CD8+), activated, IL-4 receptor–expressing T cells which selectively localize in the medullary areas. Moreover, many medullary epithelial cells and Hassal's corpuscles in the same thymus specimens showed unusually high expression of CD30L in comparison with other lymphoid or nonlymphoid tissues. These findings provide additional information on the nature and localization of CD30+ thymocytes and show that epithelial cells are the major holder of CD30L in the thymic medulla.
Highly Sensitive Fluorescence In Situ Hybridization Method to Detect Double BCR/ABL Fusion and Monitor Response to Therapy in Chronic Myeloid Leukemia
Authors: Dewald Gordon W. , Wyatt William A. , Juneau Amy L. , Carlson Richard O. , Zinsmeister Alan R. , Jalal Syed M. , Spurbeck Jack L. , Silver Richard T.
Published: October 2019

We investigated a new method using fluorescence in situ hybridization and DNA probes that span the common breakpoints of t(9;22)(q34;q11.2) and that detect double BCR/ABL fusion (D-FISH) in bone marrow cells with this translocation, one on the abnormal chromosome 9 and one on the Philadelphia chromosome (Ph chromosome). D-FISH patterns were abnormal in 30 of 30 specimens with classic, simple, complex, and masked Ph chromosomes. Based on 200 nuclei from each of 30 normal specimens, the mean percentage of false-positive cells was 0.25 ± 0.39. Thirty-seven specimens from 10 patients were studied before treatment and two or more times at 4-month intervals after treatment with interferon-α2b (IFN-α2b) with or without ara-C. Based on 200 nuclei, the results of D-FISH in these specimens correlated closely with quantitative cytogenetics and accurately quantified disease within a few percent. We studied 6,000 nuclei for each of six specimens, three normal and three from patients with chronic myeloid leukemia (CML) in cytogenetic remission. The normal cutoff for 6,000 nuclei was 0.079% and patients in cytogenetic remission had residual disease ranging from 7 (0.117%) to 53 (0.883%) Ph-positive nuclei. We conclude that D-FISH can detect the Ph chromosome and its variant translocations and accurately quantify disease in CML at diagnosis and at all times after treatment, including cytogenetic remission.
Prognostic Value of Numerical Chromosome Aberrations in Multiple Myeloma: A FISH Analysis of 15 Different Chromosomes
Authors: Pérez-Simón J.A. , Garcı́a-Sanz R. , Tabernero M.D. , Almeida J. , González M. , Fernández-Calvo J. , Moro M.J. , Hernández J.M. , Miguel J.F. San , Orfão A.
Published: October 2019

Recent observations indicate that chromosome aberrations are important prognostic factors in patients with multiple myeloma (MM) treated with high-dose chemotherapy. Nevertheless, the inherent problems of conventional cytogenetics have hampered the systematic evaluation of this parameter in series of patients treated with conventional chemotherapy. Fluorescence in situ hybridization (FISH) analysis is an attractive alternative for evaluation of numerical chromosomal changes. In the present study, we analyze the relationship between aneuploidies of 15 different chromosomes assessed by FISH and prognosis in a series of 63 patients with MM treated with conventional chemotherapy. After a median follow-up of 61 months (range, 6 to 109), 49% of patients are still alive with a median survival of 33 months. The overall incidence of numerical chromosome abnormalities was 70%. This incidence significantly increased when seven or more chromosomes were analyzed (53 patients), reaching 81%. Trisomies of chromosomes 6, 9, and 17 were associated with prolonged survival (P = .033, P = .035, and P = .026, respectively); by contrast, overall survival (OS) was lower in cases with monosomy 13 (as assessed by deletion of Rb gene,P = .0012). From the clinical point of view, loss of Rb gene was associated with a poor performance status; low hemoglobin levels; high creatinine, C-reactive protein, and lactic dehydrogenase serum levels; high percentage of bone marrow plasma cells (BMPC); extensive bone lytic lesions; and advanced clinical stage. Other chromosome abnormalities such as trisomy of chromosome 9 and 17 were associated with good prognostic features including high hemoglobin levels, early clinical stage, β2microglobulin less than 6 μg/mL, and low percentage of BMPC. A multivariate analysis for OS showed that S-phase PC greater than 3% (P = .010) and β2microglobulin serum levels greater than 6 μg/mL (P = .024), together with monosomy of chromosome 13 (P = .031) and nontrisomy of chromosome 6 (P = .048) was the best combination of independent parameters for predicting survival in patients with MM. According to these results, chromosomal analysis is of great use in patients with MM at diagnosis to have a correct prognostic evaluation for clinical decision making.
Neutropenia Associated With T-Cell Large Granular Lymphocyte Leukemia: Long-Term Response to Cyclosporine Therapy Despite Persistence of Abnormal Cells
Authors: Sood Raman , Stewart Carleton C. , Aplan Peter D. , Murai Hiroyuki , Ward Pamela , Barcos Maurice , Baer Maria R.
Published: October 2019

T-cell large granular lymphocyte (T-LGL) leukemia is clinically indolent, but is associated with severe neutropenia in approximately 50% of cases. The pathogenesis of the neutropenia is unclear. We report reversal of severe neutropenia associated with T-LGL leukemia in five patients treated with cyclosporine (CSA). All five had persistent neutrophil counts below 0.5 × 109/L, two had agranulocytosis, and four had recurrent infections. Increased populations of LGL were present in blood and marrow, with a T-LGL immunophenotype (CD3+CD8+CD16±CD56±CD57+) shown by multiparameter flow cytometry, and clonal T-cell receptor (TCR) gene rearrangements in two of two pretreatment blood samples studied. CSA was initiated at doses of 1 to 1.5 mg/kg orally every 12 hours, with subsequent dose adjustments based on trough serum levels. Four patients attained normal neutrophil counts with CSA alone; one required addition of low-dose granulocyte-macrophage colony-stimulating factor. Time to attainment of 1.5 × 109/L neutrophils ranged from 21 to 75 days. Attempts to taper and withdraw CSA resulted in recurrent neutropenia. Three patients have maintained normal neutrophil counts on continued CSA therapy for 2, 8, and 8.5 years. Two patients died 1.7 and 4.6 years after initiation of CSA despite normal neutrophil counts—one of metastatic melanoma and one of complications after aortofemoral bypass surgery. Despite resolution of neutropenia, increased populations of T-LGL cells have persisted in all patients during CSA therapy, as shown by morphology and flow cytometry and by the presence of clonal TCR gene rearrangements in four patients' posttreatment blood samples. We conclude that CSA is an effective therapy for neutropenia associated with T-LGL leukemia, and that resolution of neutropenia despite persistence of abnormal cells implies that CSA may inhibit T-LGL secretion of yet unidentified mediators of neutropenia.
Inhibition of BCR-ABL Expression With Antisense Oligodeoxynucleotides Restores β1 Integrin-Mediated Adhesion and Proliferation Inhibition in Chronic Myelogenous Leukemia Hematopoietic Progenitors
Authors: Bhatia Ravi , Verfaillie Catherine M.
Published: October 2019

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to β1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210BCR-ABL, interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210BCR-ABL in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34+HLA-DR+(DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated β1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210BCR-ABL levels in CML CD34+ cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors.
Erythroid Maturation and Globin Gene Expression in Mice With Combined Deficiency of NF-E2 and Nrf-2
Authors: Martin Florence , van Deursen Jan M. , Shivdasani Ramesh A. , Jackson Carl W. , Troutman Amber G. , Ney Paul A.
Published: October 2019

NF-E2 binding sites, located in distant regulatory sequences, may be important for high level α- and β-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2–related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2–deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of α- and β-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2–deficient mice is not the result of compensation by Nrf-2.
Comparative Oxidation of Hemoglobins A and S
Authors: Sheng Kuan , Shariff Michelle , Hebbel Robert P.
Published: October 2019

The mutant hemoglobin S (HbS) previously was reported to undergo accelerated autooxidation during incubation in vitro. However, subsequent observations have raised the possibility that this might be explained by adventitious association of molecular iron with HbS, rather than reflecting an inherent property of HbS. Using purified HbA and HbS obtained from genotypic HbAS donors, we found that the observed oxidation rate of HbS, but not of HbA, is indeed exaggerated by adventitious iron. This result suggests a preferential partitioning of molecular iron to HbS over HbA, which was further supported by experimentation. However, after elimination of this effect, there still remains a significant increase in inherent autooxidation rate for HbS. Physiologic oxidants (superoxide, peroxide, hydroxyl radical) and various Fe(III) chelates all stimulate oxidation of oxyHb, but they do so equivalently for HbA and HbS. Nevertheless, these mechanisms also would contribute to excessive biologic oxidation of HbS because the cytoplasm of sickle red blood cells, unlike that of normal cells, would be exposed to abnormal amounts of oxidants and low–molecular-weight iron compounds.
Identification of Patients Who May Benefit From Prophylactic Immunotherapy After Bone Marrow Transplantation for Acute Myeloid Leukemia on the Basis of Lymphocyte Recovery Early After Transplantation
Authors: Powles Ray , Singhal Seema , Treleaven Jennifer , Kulkarni Samar , Horton Clive , Mehta Jayesh
Published: October 2019

Two hundred and one patients (median age, 29 years) with acute myeloid leukemia (AML) underwent bone marrow transplantation (BMT) from HLA-identical sibling donors after conditioning with melphalan-total-body irradiation (TBI) (57%), cyclophosphamide-TBI (35%), or chemotherapy alone (8%). Graft-versus-host disease (GVHD) prophylaxis included cyclosporine alone (68%), cyclosporine-methotrexate (26%), or T-cell depletion (6%). The probability of relapse was calculated as a function of the absolute lymphocyte count (109/L) on days 27 to 30 posttransplant (<0.1 v ≥0.1, <0.2 v ≥0.2, and <0.3 v≥0.3). In each of these 12 comparisons, the probability of relapse was higher for the group with the lower lymphocyte count. Because the difference was most significant (P = .004) for an absolute lymphocyte count of <0.2 on day 29 (3-year relapse probability, 42%) versus ≥ 0.2 (16%), this variable was included in a Cox model to determine factors independently affecting relapse. Multivariate analysis showed that conditioning regimens other than melphalan-TBI, a low lymphocyte count on day 29, French-American-British (FAB) subtypes M4-7, and a nucleated cell dose of > 2.42 × 108/kg was associated with a higher risk of relapse. We conclude that slow lymphocyte recovery after allogeneic BMT, to < 0.2 × 109/L 29 days in this analysis, appears to be associated with a higher risk of relapse in patients with AML. This group of patients may benefit from posttransplant immune manipulations such as abbreviated GVHD prophylaxis, or donor cell or cytokine administration to enhance graft-versus-leukemia reactions to reduce relapse.
Efficient Retroviral-Mediated Gene Transfer to Human Cord Blood Stem Cells With In Vivo Repopulating Potential
Authors: Conneally E. , Eaves C.J. , Humphries R.K.
Published: October 2019

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34+ cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34+ (myeloid) CFC and human CD19+ (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% ± 3% and 17% ± 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% ± 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit–granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a ≥ 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.
Specific Signals Generated by the Cytoplasmic Domain of the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Not Required for G-CSF–Dependent Granulocytic Differentiation
Authors: Jacob Jason , Haug Jeffery S. , Raptis Sofia , Link Daniel C.
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) is the principal growth factor regulating the production of neutrophils, yet its role in lineage commitment and terminal differentiation of hematopoietic progenitor cells is controversial. In this study, we describe a system to study the role of G-CSF receptor (G-CSFR) signals in granulocytic differentiation using retroviral transduction of G-CSFR–deficient, primary hematopoietic progenitor cells. We show that ectopic expression of wild-type G-CSFR in hematopoietic progenitor cells supports G-CSF–dependent differentiation of these cells into mature granulocytes, macrophages, megakaryocytes, and erythroid cells. Furthermore, we show that two mutant G-CSFR proteins, a truncation mutant that deletes the carboxy-terminal 96 amino acids and a chimeric receptor containing the extracellular and transmembrane domains of the G-CSFR fused to the cytoplasmic domain of the erythropoietin receptor, are able to support the production of morphologically mature, chloroacetate esterase-positive, Gr-1/Mac-1–positive neutrophils in response to G-CSF. These results demonstrate that ectopic expression of the G-CSFR in hematopoietic progenitor cells allows for multilineage differentiation and suggest that unique signals generated by the cytoplasmic domain of the G-CSFR are not required for G-CSF–dependent granulocytic differentiation.
A Novel Function of Stat1α and Stat3 Proteins in Erythropoietin-Induced Erythroid Differentiation of a Human Leukemia Cell Line
Authors: Kirito Keita , Uchida Mie , Takatoku Masaaki , Nakajima Koichi , Hirano Toshio , Miura Yasusada , Komatsu Norio
Published: October 2019

We recently determined that erythropoietin (EPO) activates 3 members of the signal transducer and activator of transcription (STAT) family, Stat1α, Stat3, and Stat5, in the human EPO-dependent cell lines, UT-7 and UT-7/EPO (Kirito et al, J Biol Chem 272:16507, 1997). In addition, we have shown that Stat1α, but not Stat3, is involved in EPO-induced cellular proliferation. In this study, we examined the roles of Stat1α and Stat3 in EPO-induced erythroid differentiation. UT-7/GM was used as a model system, because this cell line can differentiate into erythroid-lineage cells with EPO treatment (Komatsu et al, Blood 89:4021, 1997). We found that EPO did not activate Stat1α or Stat3 in UT-7/GM cells. Transfection experiments showed that both Stat1α and Stat3 inhibited the induction by EPO of γ-globin and erythroid-specific 5-aminolevulinate synthetase transcripts, resulting in a reduction of the percentage of hemoglobin-positive cells. Dominant negative forms of Stat1α or Stat3 promoted the EPO-induced erythroid differentiation of UT-7/GM cells, even in the presence of granulocyte-macrophage colony-stimulating factor, although this cytokine never induced erythroid differentiation of the parent UT-7/GM cells with or without EPO. A cell cycle analysis showed that the constitutive activation of Stat1α, but not Stat3, shortened the period of G0/G1 prolongation caused by EPO stimulation. Taken together, our data suggest that Stat1α and Stat3 act as negative regulators in EPO-induced erythroid differentiation. Specifically, Stat1α may activate a cell cycle-associated gene(s), leading to the entry of cells into the cell cycle.
In Vivo Expression of Murine Platelet Glycoprotein Ibα
Authors: Fujita Hiroyuki , Hashimoto Yoshimi , Russell Susan , Zieger Barbara , Ware Jerry
Published: October 2019

We have performed a systematic in vivo evaluation of gene expression for the glycoprotein (GP) Ibα subunit of the murine platelet adhesion receptor, GP Ib-IX-V. This study is warranted by in vitro observations of human GP Ibα expression in cells of nonhematopoietic lineage and reports of regulation of the GP Ibα gene by cytokines. However, an in vivo role for a GP Ib-IX-V receptor has not been established beyond that described for normal megakaryocyte/platelet physiology and hemostasis. Our Northern analysis of mouse organs showed high levels of GP Ibα mRNA in bone marrow with a similar expression pattern recapitulated in mice containing a luciferase transgene under the control of the murine GP Ibα promoter. Consistently high levels of luciferase activity were observed in the two hematopoietic organs of mice, bone marrow (1,400 relative light units/μg of protein [RLUs]) and spleen (500 RLUs). Reproducible, but low-levels of luciferase activity were observed in heart, aorta, and lung (30 to 60 RLUs). Among circulating blood cells, the luciferase activity was exclusively localized in platelets. No increase in GP Ibα mRNA or luciferase activity was observed after treatment of mice with lipopolysaccharides (LPS) or tumor necrosis factor-α (TNF-α). We conclude the murine GP Ibα promoter supports a high level of gene expression in megakaryocytes and can express heterologous proteins allowing an in vivo manipulation of platelet-specific proteins in the unique environment of a blood platelet.
Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers
Authors: Bendetowicz Ana Victoria , Morris Jill A. , Wise Robert J. , Gilbert Gary E. , Kaufman Randal J.
Published: October 2019

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.
Shiga Toxin Type 1 Activates Tumor Necrosis Factor-α Gene Transcription and Nuclear Translocation of the Transcriptional Activators Nuclear Factor-κB and Activator Protein-1
Authors: Sakiri Ramesh , Ramegowda Belakere , Tesh Vernon L.
Published: October 2019

Shiga toxins (Stxs) produced by Shigella dysenteriae 1 andEscherichia coli have been implicated in the pathogenesis of bloody diarrhea, acute renal failure, and neurologic abnormalities. The pathologic hallmark of Stx-mediated tissue damage is the development of vascular lesions in which endothelial cells are swollen and detached from underlying basement membranes. However, in vitro studies using human vascular endothelial cells demonstrated minimal Stx-induced cytopathic effects, unless the target cells were also incubated with the proinflammatory cytokines tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). These cytokines have been shown to upregulate the expression of the Stx-binding membrane glycolipid globotriaosylceramide (Gb3). We show here that purified Stx1 induces TNF secretion by a human monocytic cell line, THP-1, in a dose- and time-dependent manner. Treatment of cells with both lipopolysaccharides (LPS) and Stx1 results in augmented TNF production. Treatment with the nontoxic Gb3-binding subunit of Stx1 or with an anti-Gb3 monoclonal antibody did not trigger TNF production. Northern blot analyses show that Stx1 causes increased TNF-α production through transcriptional activation. Increased levels of TNF-α mRNA are preceded by the nuclear translocation of the transcriptional activators NF-κB and AP-1 and the loss of cytoplasmic IκB-α. These data are the first to show that, in addition to direct cytotoxicity, Stxs possess cellular signaling capabilities sufficient to induce the synthesis of cytokines that may be necessary for target cell sensitization and the development of vascular lesions.
Lack of Clinically Significant Contact System Activation During Platelet Concentrate Filtration by Leukocyte Removal Filters
Authors: Scott Cheryl F. , Brandwein Harvey , Whitbread John , Colman Robert W.
Published: October 2019

When blood (plasma) contacts certain foreign surfaces, factor XII can activate and trigger a series of reactions leading to cleavage of kininogens with subsequent release of bradykinin. In this study, we investigated two different widely used leukocyte removal filters, Pall PXL8K (A) and Asahi PLS-5A (B), to test whether clinically significant contact activation occurred during leukodepletion of platelet-rich plasma (PRP). Kininogens were measured by particle concentration fluorescence immunoassay (PCFIA), which can detect cleavage of high and low molecular weight kininogens (HK and LK), the parent molecules of bradykinin, to determine if contact activation had occurred. A slight, nonsignificant decrease in HK and LK was observed with filter A after the first 5 mL was filtered that returned to prefiltration levels by the end of the filtration. Specific TotK (the combined measurement of HK and LK heavy chains divided by plasma protein concentration) showed a small, significant decrease with filter A after the first 5 mL of platelet concentrates was filtered that returned to prefiltration levels by the end of the filtration. There were no significant increases or decreases in the cleaved kininogen index (CKI), an index of HK proteolytic activation or HK and LK destruction (with release of bradykinin). These data suggest that small amounts of both HK and LK initially adsorb to filter A and then desorb, primarily intact. These data also indicate that no significant contact activation, as measured by PCFIA, occurs during leukodepletion of platelet concentrates with either filter A or B.
The Iron Chelator L1 Potentiates Oxidative DNA Damage in Iron-Loaded Liver Cells
Authors: Cragg Louise , Hebbel Robert P. , Miller Wesley , Solovey Alex , Selby Scott , Enright Helen
Published: October 2019

Iron-mediated carcinogenesis is thought to occur through the generation of oxygen radicals. Iron chelators are used in attempts to prevent the long term consequences of iron overload. In particular, 1,2-dimethyl-3-hydroxypyrid-4-one (L1), has shown promise as an effective chelator. Using an established hepatocellular model of iron overload, we studied the generation of iron-catalyzed oxidative DNA damage and the influence of iron chelators, including L1, on such damage. Iron loading of HepG2 cells was found to greatly exacerbate hydrogen peroxide–mediated DNA damage. Desferrithiocin was protective against iron/hydrogen peroxide–induced DNA damage; deferoxamine had no effect. In contrast, L1 exposure markedly potentiated hydrogen peroxide–mediated oxidative DNA damage in iron-loaded liver cells. However, when exposure to L1 was maintained during incubation with hydrogen peroxide, L1 exerted a protective effect. We interpret this as indicating that L1's potential toxicity is highly dependent on the L1:iron ratio. In vitro studies examining iron-mediated ascorbate oxidation in the presence of L1 showed that an L1:iron ratio must be at least 3 to 1 for L1 to inhibit the generation of free radicals; at lower concentrations of L1 increased oxygen radical generation occurs. In the clinical setting, such potentiation of iron-catalyzed oxidative DNA damage at low L1:iron ratios may lead to long-term toxicities that might preclude administration of L1 as an iron chelator. Whether this implication in fact extends to the in vivo situation will have to be verified in animal studies.
Six Previously Undescribed Pyruvate Kinase Mutations Causing Enzyme Deficiency
Authors: Demina Anna , Varughese Kottayil I. , Barbot José , Forman Linda , Beutler Ernest
Published: October 2019

Erythrocyte pyruvate kinase deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. We present 6 previously undescribed mutations of the PKLR gene associated with enzyme deficiency located at cDNA nt 476 G→T (159Gly→Val), 884 C→T (295Ala→Val), 943 G→A (315Glu→Lys), 1022 G→A (341Gly→Asp), 1511 G→T (504Arg→Leu), and 1528 C→T (510Arg→Ter). Two of these mutations are near the substrate binding site: the 315Glu→Lys (943A) mutation may be involved in Mg2+ binding and159Gly→Val (476T) mutation has a possible effect on ADP binding. Four of six mutations produce deduced changes in the shape of the molecule. Two of these mutations,504Arg→Leu (1511T) and510Arg→Ter (1528T), are located at the interface of domains A and C. One of them (510Arg→Ter) is a deletion of the C-terminal residues affecting the integrity of the protein. The 504Arg→Leu mutation eliminates a stabilizing interaction between domains A and C. Changes in amino acid 341(nt 1022) from Gly to Asp cause local perturbations. The mutation295Ala→Val (884T) might affect the way pyruvate kinase interacts with other molecules. We review previously described mutations and conclude that there is not yet sufficient data to allow us to draw conclusions regarding genotype/phenotype relationship.
Stem Cell Factor Influences Mast Cell Mediator Release in Response to Eosinophil-Derived Granule Major Basic Protein
Authors: Furuta Glenn T. , Ackerman Steven J. , Lu Lei , Williams Rachel E. , Wershil Barry K.
Published: October 2019

Stem cell factor (SCF) is an important mast cell growth, differentiation, and survival factor. We investigated whether SCF influenced the response of mouse mast cells to an IgE-independent stimulus, eosinophil-derived granule major basic protein (MBP). Mouse bone marrow cultured mast cells (BMCMC) were derived in either concanavalin-stimulated mouse spleen conditioned medium (CM) or SCF. The cloned growth, factor-independent mast cell line Cl.MC/C57.1 was also studied. BMCMC in SCF exhibited cytochemical staining properties, protease and histamine content, and increased serotonin uptake consistent with more mature differentiated mast cells as compared with BMCMC in CM or Cl.MC/ C57.1 cells. BMCMC in SCF released serotonin,14C-labeled arachidonic acid metabolites and tumor necrosis factor-α (TNF-α) on stimulation with MBP, while no response was seen from either BMCMC in CM or Cl.MC/C57.1 cells. All three mast cell populations released mediators on stimulation with the cationic MBP analog, poly-L-arginine, indicating that the cationic charge did not explain the selective response of BMCMC in SCF to eosinophil-derived granule MBP. These findings show that SCF significantly influences mast cell differentiation and the responsiveness of mast cells to eosinophil-derived granule MBP.

© 1998 by The American Society of Hematology.
Characterization of the Role of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor α Subunit in the Activation of JAK2 and STAT5
Authors: Doyle Sean E. , Gasson Judith C.
Published: October 2019

The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) consists of an alpha (GMRα) and a common beta (βc) subunit. The intracellular domain of βc has been extensively characterized and has been shown to be critical for the activation of both the JAK/STAT and MAP kinase pathways. The function of the intracellular domain of GMRα, however, is not as well characterized. To determine the role of this domain in GMR signaling, an extensive structure-function analysis was performed. Truncation mutants α362, α371, and α375 were generated, as well as the site-directed mutants αVQVQ and αVVVV. Although α375β, αVQNQβ, and αVVVVβ stimulated proliferation in response to human GM-CSF, the truncation mutants α362β and α371β were incapable of transducing a proliferative signal. In addition, both α371 and αVVVV were expressed at markedly reduced levels, indicating the importance of residues 372 to 374 for proper protein expression. More importantly, we show that GMRα plays a direct role in the activation of the JAK/STAT pathway, and electrophoretic mobility shift assays (EMSA) indicate that both GMRα and βc play a role in determining the STAT5 DNA binding complex activated by the GMR. Thus, the intracellular domain of the human GMRα is important for activation of the JAK/STAT pathway and protein stabilization.

© 1998 by The American Society of Hematology.
Use of the Microculture Kinetic Assay of Apoptosis to Determine Chemosensitivities of Leukemias
Authors: Kravtsov Vladimir D. , Greer John P. , Whitlock James A. , Koury Mark J.
Published: October 2019

Chemotherapeutic agents exert their antitumor effects by inducing apoptosis. The microculture kinetic (MiCK) assay provides an automated, continuous means of monitoring apoptosis in a cell population. We used the MiCK assay to determine the chemosensitivities of the human promyelocytic HL-60 and lymphoblastic CEM cell lines and leukemia cells freshly isolated from patients with acute nonlymphocytic (ANLL) or acute lymphocytic (ALL) leukemias. Continuous monitoring of apoptosis in the MiCK assay permits determination of the time to the maximum apoptosis (Tm) and its two components which are initiation time (Ti) and development time (Td). Duration of the three timing components of apoptosis varies from hours to days depending on the drug, drug concentration, and type of target cells. In the MiCK assay, the extent of apoptosis is reported in kinetic units of apoptosis. Kinetic units are determined by the slope of the curve created when optical density caused by cell blebbing is plotted as a function of time. Using the leukemia cell lines, we define the relationship between kinetic units determined by the MiCK assay and the percentage of morphologically apoptotic cells in the culture. Flow cytometry analysis of apoptosis in Annexin-V-fluorescein isothiocyanate–labeled preparations of HL-60 and CEM cells was also used to compare with data obtained by the MiCK assay. The feasibility of the MiCK assay of apoptosis as a chemosensitivity test was confirmed by its comparison with a 3H-thymidine incorporation assay. We show that samples from 10 ANLL and ALL patients patients tested for sensitivity to various doses of idarubicin (IDR), daunorubicin (DNR), or mitoxantrone (MTA) gave the same percentages of apoptotic cells when calculated by the MiCK assay as when determined by morphological analysis. The MiCK assay was used for dose-response analyses of the sensitivities to IDR, DNR, and MTA of leukemia cells from 4 other patients (2 ANLL and 2 ALL). The results from both cell lines and patient samples indicate that ANLL cells are more sensitive than ALL cells to all three of these chemotherapeutic agents. However, for individual patients the chemosensitivities varied significantly among the three chemotherapeutic agents. These varying responses to IDR, DNR, and MTA indicate that the MiCK assay results can be of potential use in designing a treatment regimen for a specific patient with acute leukemia. Among several drugs of presumed similar efficacy, the MiCK assay can permit the selection of the specific chemotherapeutic agent that causes the most apoptosis in the patient's leukemic cells.

© 1998 by The American Society of Hematology.