bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of humoral immune tolerance to major histocompatibility complex antigens by transfusions of UVB-irradiated leukocytes
Authors: Kao KJ
Published: October 2019

To determine whether immune tolerance to donor major histocompatibility complex (MHC) antigens can be induced by transfusions of UVB-irradiated leukocytes, studies were conducted in inbred strains of mice with well-characterized MHC antigens. CBA mice with H-2k phenotype and BALB/c mice with H-2d phenotype were used as recipients and donors, respectively. Humoral immune tolerance is defined as absence of antibody response after challenge with transfusions of untreated donor leukocytes. It was found that transfusions of purified peripheral blood mononuclear leukocytes irradiated with 1,200 mJ/cm2 UVB not only prevented allo-immunization but also induced humoral immune tolerance to donor class-I and -II MHC antigens in all recipient mice. Donor plasma and platelets interfered with the induction of this tolerance. The tolerance induction by UVB-irradiated leukocytes is dose-dependent. Four weekly transfusions of 2 x 10(5) leukocytes were required to ensure tolerance induction in all mice. The results of a long-term follow-up study showed that the induced tolerance is long-lasting and can withstand repeated challenges by untreated donor leukocytes. Recipient mice tolerant to H-2d antigens also became tolerant to H-2b, H-2r, and H-2n MHC antigens but did not have impaired antibody responses to antigens unrelated to donor leukocytes. Adoptive transfer experiments showed the development of negative regulatory cells in spleens of the tolerized mice. In view of recent feasibility of using UVB-irradiated human platelet concentrates for prevention of HLA alloimmunization, the findings of this study support that UVB- irradiated donor leukocytes could have potential, important clinical application in transplantation medicine.
Hereditary spherocytosis with band 3 deficiency. Association with a nonsense mutation of the band 3 gene (allele Lyon), and aggravation by a low-expression allele occurring in trans (allele Genas)
Authors: Alloisio N , Maillet P , Carre G , Texier P , Vallier A , Baklouti F , Philippe N , Delaunay J
Published: October 2019

We describe an 18-year-old with moderate hereditary spherocytosis. The condition was associated with a 35% decrease in band 3. The underlying mutation was Arg to stop at codon 150 (CGA-->TGA) and was designated R150X, which defined allele Lyon of the EPB3 gene. The inheritance pattern was dominant. However, the mother, who also carried the allele Lyon, had a milder clinical presentation and only a 16% decrease of band 3. We suggested that the father had transmitted a modifying mutation that remained silent in the heterozygous state. Nucleotide sequencing after single strand conformation polymorphism analysis of the band 3 cDNA and promoter region revealed a G-->A substitution at position 89 from the cap site in the 5′-untranslated region, designated 89G-->A, which defined allele Genas. A ribonuclease protection assay showed that (1) the allele Genas (father) resulted in a 33% decrease in the amount of band 3 mRNA, (2) the reduction caused by the allele Lyon (mother) was 42%, and (3) the compound heterozygous state for both alleles (proband) resulted in a 58% decrease. These results suggest that some mildly deleterious alleles of the EPB3 gene are compensated for by the normal allele in the heterozygous state. They are shown through the aggravation of the clinical picture, based on more obvious molecular alterations when they occur in trans to an allele causing a manifest reduction of band 3 membrane protein concentration.
In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation-and chemotherapy- induced bone marrow suppression
Authors: Ohwada A , Rafii S , Moore MA , Crystal RG
Published: October 2019

Thrombopoietin (TPO, c-mpl ligand) has emerged as a major hematopoietic cytokine stimulating megakaryocyte proliferation, endomitosis, and platelet production. This study shows that a single administration of an adenovirus (Ad) vector encoding TPO (AdCMV.TPO) abrogates thrombocytopenia induced in mice by carboplatin and irradiation. Normal Balb/c mice receiving the vector had increased platelet counts peaking at 7 days and returning to baseline by day 15. Mice rendered pancytopenic with 500 rads and 1.2 mg of carboplatin had a nadir platelet count of five percent of the baseline. Mice receiving AdCMV.TPO 3 days before receiving irradiation and chemotherapy achieved a platelet nadir fourfold higher, and had significant reduction in duration of thrombocytopenia, than mice receiving the control Ad vector. Introduction of AdCMV.TPO the same day of chemotherapy and irradiation was equally effective in acceleration of platelet recovery, but administration of AdCMV.TPO 3 days after chemotherapy-radiation had little effect on platelet recovery. At 30 days after therapy bone marrow and spleen of mice treated with AdCMV.TPO were populated with a large number of polyploid megakaryocytes, but there was no evidence of circulating megakaryocytes in the liver or lungs and no pathologic bone abnormalities such as osteosclerosis or myelofibrosis. These observations suggest that an Ad vector may be an excellent delivery system to provide adequate TPO production to maintain platelet levels in circumstances associated with life-threatening thrombocytopenia.
Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines
Authors: Weber-Nordt RM , Egen C , Wehinger J , Ludwig W , Gouilleux-Gruart V , Mertelsmann R , Finke J
Published: October 2019

Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta- casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1–6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus- positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.
Direct synergistic effects of leukemia inhibitory factor on hematopoietic progenitor cell growth: comparison with other hematopoietins that use the gp130 receptor subunit
Authors: Keller JR , Gooya JM , Ruscetti FW
Published: October 2019

Because leukemia inhibitory factor (LIF) has little or no effect on murine hematopoietic progenitor cell growth yet enhances hematopoiesis in vivo, we sought to determine whether the effects of LIF were directly or indirectly mediated, or a combination of both. Although LIF alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) has no effect on colony formation of unfractionated bone marrow cells (BMCs), it enhances M-CSF- induced colony formation. In comparison, LIF synergizes with IL-3, GM- CSF, M-CSF, and Steel Factor (SLF) to promote the colony formation of partially purified lineage-negative (Lin-) BM progenitors without altering their differentiation. These effects were directly mediated since identical results were observed in single-cell assays. Comparing the effect of LIF with other members of this subclass of hematopoietins (IL-6, oncostatin M [OSM], and ciliary neurotrophic factor [CNTF]), we found that while LIF and IL-6 equally synergize with M-CSF and SLF to promote the colony formation of Lin- BMCs, OSM, and CNTF have no effect. In agreement with OSMs ability to directly bind gp130, preincubation of BMCs with OSM inhibits progenitor cell growth stimulated by the combination of LIF or IL-6 plus SLF. LIF can also directly enhance the growth of further purified more primitive Lin- c- kit+ progenitor cells in the presence of IL-3, GM-CSF, or SLF. Thus, LIF can directly synergize with growth factors to promote the proliferation of purified hematopoietic progenitors, suggesting that the direct effects of LIF on hematopoietic cell growth can, in part, explain the observed hematopoietic effects in vivo. This is a US government work. There are no restrictions on its use.
Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen
Authors: Lijnen HR , Carmeliet P , Bouche A , Moons L , Ploplis VA , Plow EF , Collen D
Published: October 2019

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.
Factor VIIa and antithrombin III activity during severe sepsis and septic shock in neutropenic patients
Authors: Mesters RM , Mannucci PM , Coppola R , Keller T , Ostermann H , Kienast J
Published: October 2019

Septic shock and multiple organ failure may be associated with coagulation activation, disseminated fibrin formation, and consumption of coagulation inhibitors such as antithrombin III. We have evaluated prospectively coagulation measurements in patients with severe chemotherapy-induced neutropenia. This group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing before and during evolving sepsis or septic shock. Sixty-two patients with febrile infectious events were accrued to the study. Of these, 13 patients progressed to severe sepsis and 13 additional patients to septic shock as defined according to standard diagnostic criteria. At the onset of fever, factor (F) VIIa activity, FVII antigen and antithrombin III (AT III) activity decreased from normal baseline levels and were significantly lower in the group of patients who progressed to septic shock compared with those that developed severe sepsis (medians: 0.3 v 1.4 ng/mL, 21 v 86 U/dL and 45% v 95%; P < .001). The decrease of these measurements in septic shock was accompanied by an increase in prothrombin fragment 1+2 (median: 3.6 v 1.4 nmol/L; P = .05), a marker of thrombin generation. These differences were sustained throughout the septic episode (P < .0001). FVIIa and AT III levels of < 0.8 ng/mL and < 70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100% and 85%, and a specificity of 75% and 85%, respectively. In contrast, FXIIa-alpha antigen levels were not different between groups at onset of fever but increased modestly during the course of septic shock (P = .001). Thus, septic shock in neutropenic patients is associated with increased thrombin generation. Furthermore, both FVIIa and AT III measurements are sensitive markers of an unfavorable prognosis.
Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets
Authors: Wagner CL , Mascelli MA , Neblock DS , Weisman HF , Coller BS , Jordan RE
Published: October 2019

A large number of glycoprotein (GP) IIb/IIIa receptors are present on the surface of platelets. Studies to define precisely the number of GPIIb/IIIa receptors using specific monoclonal antibodies (MoAbs) or fibrinogen binding have, however, yielded varying estimates of receptor number. To refine the quantitative estimation of GPIIb/IIIa receptors on resting platelets, we have used the MoAb 7E3, which has high affinity for GPIIb/IIIa. Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG, as well as fragments and derivatives of 7E3. For platelets obtained from any single individual, the numbers of 7E3 F(ab′)2 and IgG molecules bound per platelet were equivalent (approximately 40,000), whereas the number of Fab molecules bound per platelet was consistently approximately twofold higher (approximately 80,000). To investigate the basis of the quantitative disparity in binding of intact 7E3 and 7E3 F(ab′)2 versus 7E3 Fab, we studied the binding of a newly constructed, bispecific (Fab′)2 molecule containing only a single 7E3 combining site. Because this construct bound to the same extent as the Fab species, the larger size of the intact 7E3 and 7E3 F(ab′)2 molecules could not explain the reduced number of molecules that bound per platelet compared to the Fab fragment. Rather, it appears that the valency of the antibody is the critical factor determining the number of antibody molecules bound per platelet. Thus, we conclude that the binding of 7E3 Fab corresponds most closely with surface GPIIb/IIIa number and that the number of GPIIb/IIIa receptors is approximately 80,000 per platelet.
Molecular characterization of a glycosylphosphatidylinositol-linked ADP- ribosyltransferase from lymphocytes
Authors: Okazaki IJ , Kim HJ , McElvaney NG , Lesma E , Moss J
Published: October 2019

Mono ADP-ribosyltransferases catalyze the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) to proteins. It was reported by Wang et al (J Immunol 153:4048, 1994) that incubation of mouse cytotoxic T lymphocytes (CTL) with NAD resulted in the ADP- ribosylation of membrane proteins and inhibition of cell proliferation and cytotoxicity. Treatment of CTL with phosphatidylinositol-specific phospholipase C (PI-PLC) before incubation with NAD prevented the inhibitory effects of NAD on the cells, consistent with the removal of a glycosylphosphatidylinositol (GPI)-anchored ADP-ribosyltransferase on the lymphocyte surface. We have identified and cloned a GPI-linked ADP- ribosyltransferase from Yac-1 mouse T-cell lymphoma cells. The deduced amino acid sequence of the Yac-1 transferase was 70% and 41% identical to those of the rabbit skeletal muscle and chicken heterophil, respectively. It contained three noncontiguous sequences similar to those found in several of the bacterial toxin and vertebrate ADP- ribosyltransferases. Based on crystallography of the bacterial toxins, these regions are believed to form, in part, the catalytic site consistent with a common mechanism for the ADP-ribose transfer reaction. In rat mammary adenocarcinoma (NMU) cells transformed with the Yac-1 transferase cDNA, transferase activity was present on the cell surface and was released into the medium by treatment of cells with PI-PLC. Thus, we have cloned a novel gene that has properties identical to the transferase detected in CTL, and may be involved in the NAD-dependent regulation of proliferation and cytotoxicity.
Cystamine inhibits human immunodeficiency virus-1 replication in cord blood-derived mononuclear phagocytes and lymphocytes
Authors: Ho WZ , Kaufman D , Song L , Cutillii JR , Douglas SD
Published: October 2019

The effects of cystamine on the human immunodeficiency virus (HIV-1) expression in cord blood monocytes-derived macrophages (CBMDM) and lymphocytes were investigated. Cystamine suppressed HIV-1 expression in CBMDM and lymphocytes in a concentration-dependent fashion as determined by HIV-1 reverse transcriptase (RT) activity. This inhibitory effect of cystamine occurred with all five HIV-1 strains (both laboratory adopted and fresh isolates) tested in the study. The addition of cystamine to cultures of HIV-1 chronically infected CBMDM also suppressed 80% to 90% of RT activity in comparison with untreated controls. Cystamine also decreased HIV-1 protein expression in CBMDM as determined by indirect immunofluorescence assay. The inhibitory effects of cystamine on HIV-1 did not appear to be caused by toxicity to CBMDM or lymphocytes because there was no change in cell viability or cellular DNA synthesis as evaluated by trypan blue dye exclusion and [3H]-thymidine incorporation at doses of cystamine that inhibit the virus. HIV-1 infected CBMDM or lymphocyte cultures (without cystamine treatment) demonstrated giant syncytium formation or cytopathic effect (CPE), respectively, whereas cystamine-treated cultures lacked the giant syncytia or CPE induced by HIV-1 infection. Thus, these observations indicate that cystamine may have the potential to limit HIV-1 replication in monocytes/macrophages and lymphocytes in vivo and may represent a potentially useful compound in the treatment of pediatric HIV-1 infection and acquired immunodeficiency syndrome.
Application of long-distance polymerase chain reaction to detection of junctional sequences created by chromosomal translocation in mature B- cell neoplasms
Authors: Akasaka T , Muramatsu M , Ohno H , Miura I , Tatsumi E , Fukuhara S , Mori T , Okuma M
Published: October 2019

Junctional sequences created by chromosomal translocations in mature B- cell neoplasms, which involve immunoglobulin gene loci (IG) and putative proto-oncogenes on reciprocal partner chromosomes, are unique to neoplastic cells characterized by particular histological and immunological phenotypes. To establish a rapid and sensitive method to detect neoplastic cells carrying a specific chromosomal translocation, we have developed a novel strategy based on long-distance polymerase chain reaction (LD-PCR) amplification. Genomic DNA was extracted from tumor cells carrying t(14;19)(q32;q13), t(8;14)(q24;q32), t(3;22)(q27;q11), t(2;3)(p12;q27), or t(3;14)(q27;q32). Thirty-two to 35-mer oligonucleotide primer pairs were designed to be complementary to exons or flanking sequences of the BCL3, c-MYC and BCL6 oncogenes, and to IG constant region genes. LD-PCR with a newly available Taq polymerase for longer product synthesis successfully amplified fragments representing BCL3/C alpha junctional sequences for t(14;19); c-MYC/C mu, c-MYC/C gamma, and c-MYC/C alpha for t(8;14); BCL6/C lambda for t(3;22); BCL6/C kappa for t(2;3); 5′-BCL6/C mu, and 5′-BCL6/C gamma for t(3;14). In Burkitt's lymphoma/leukemia, all materials in which c- MYC rearrangements were detectable by conventional Southern blot hybridization showed positive LD-PCR amplification. The sizes of the amplified fragments varied from 1.8 kb to 12 kb, and these were specific to each material. Serial dilution of tumor cells or DNA in negative materials demonstrated a single band on agarose gel electrophoresis stained with ethidium bromide at a level of sensitivity of 10(-3), and hybridization with radioactive probe improved the level by one order of magnitude (1 cell in 10(4)), indicating that this LD- PCR approach is a sensitive technique capable of detecting minimal residual disease. Thus, the present study provided a useful tool for diagnosis and subsequent management of B-cell neoplasms characterized by specific chromosomal translocations.
Shiga-like toxin purges human lymphoma from bone marrow of severe combined immunodeficient mice
Authors: LaCasse EC , Saleh MT , Patterson B , Minden MD , Gariepy J
Published: October 2019

Shiga-like toxin-1 (SLT-1) is a bacterial toxin that kills cells by inhibiting protein synthesis. SLT-1 is composed of one cytotoxic A- subunit and five B-subunits that bind to CD77, a cell-surface glycolipid. In the human hematopoietic system, CD77 expression is restricted to a subset of activated B cells and derived cancers. Here we report that SLT-1 treatment of murine bone marrow ex vivo effectively cures severe combined immunodeficient mice of a human B- cell lymphoma xenograft while sparing normal hematopoietic precursor cells. Flow cytometry results using fluorescein isothiocyanate-labeled SLT-1 B-subunit show the high prevalence of expression of SLT-1 receptors (CD77) in human non-Hodgkin's lymphomas, especially follicular lymphomas. These results suggest the use of SLT-1 for the purging of human bone marrow before autologous bone marrow transplant in the case of CD77+ B-cell lymphomas as just one of many possible uses.
Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2
Authors: Silva M , Grillot D , Benito A , Richard C , Nunez G , Fernandez-Luna JL
Published: October 2019

Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.
Prolonged administration of granulocyte colony-stimulating factor (filgrastim) to patients with Fanconi anemia: a pilot study
Authors: Rackoff WR , Orazi A , Robinson CA , Cooper RJ , Alter BP , Freedman MH , Harris RE , Williams DA
Published: October 2019

This report examines the effect of filgrastim (granulocyte colony- stimulating factor, [G-CSF] in 12 patients with neutropenia [absolute neutrophil count [ANC] < 1,000/mm3]) caused by Fanconi anemia (FA). Two of 14 patients who were evaluated for study entry were ineligible because of unsuspected cytogenetic abnormalities in their bone marrow (BM). G-CSF was started at 5 micrograms/kg/d. All patients had an increase in their ANC at week 8 (mean increase = 15,664/mm3). The median ANC during therapy was 5,030/mm3. Eight of 10 patients who completed 40 weeks on study maintained an ANC > 1,500/mm3 on G-CSF given every-otherday. Four patients had an increase in their platelet count by week 8 without transfusion (maximum increase = 23,000 to 45,000/mm3); however, platelet counts fell toward baseline levels as the G-CSF dose was reduced. BM CFU-MK were increased at week 8 in three of four evaluable patients. Four patients who did not receive red blood cell transfusions had an increase in their hemoglobin level of at least 2.0 g/dL. A fifth patient had a red blood cell transfusion in week 2 and then had a similar increase in hemoglobin level without subsequent transfusion. Eight of 10 patients who completed 40 weeks of treatment showed increases in the percentage of BM CD34+ cells measured by flow cytometry. The same proportion showed increases in peripheral blood CD34+ cells. Increased BM cellularity and myeloid hyperplasia were constant findings and were associated with increased expression of the proliferating cell nuclear antigen. Adverse experiences were mild fever (1 patient) and a new BM cytogenetic abnormality at week 40 (1 patient). This study shows that prolonged administration of G-CSF exerts a stimulatory effect on the BM of FA patients and may be used to maintain a clinically adequate ANC in these patients. G-CSF has beneficial effects on multiple hematopoietic lineages in some patients and may be a good candidate for use in combination cytokine protocols for FA patients with progressive aplastic anemia. G-CSF use results in an increase in circulating CD34+ cells, a finding with important implications for future gene transfer protocols.
Expression and function of receptors for stem cell factor and erythropoietin during lineage commitment of human hematopoietic progenitor cells
Authors: Olweus J , Terstappen LW , Thompson PA , Lund-Johansen F
Published: October 2019

The aim of the present study was to determine whether stem cell factor (SCF) and erythropoietin (EPO) act differently on defined subsets of progenitor cells, and if potential differences correlate with the receptor density on each subset. To investigate this possibility directly, we optimized conditions for the identification and purification of homogeneous progenitor cell subpopulations from human bone marrow. Populations containing 40% and 44% colony forming cells (CFCs) with 99% and 95% purity for the granulomonocytic and erythroid lineage, respectively, were sorted on the basis of differential expression of CD34, CD64, and CD71. In addition, a population containing 67% CFCs, of which 29–43% were CFU-MIX, was sorted from CD34hi CD38loCD50+ cells. Purified progenitor cell subsets were compared directly for responsiveness to SCF and EPO using a short-term proliferation assay. Expression of the receptors for SCF and EPO were then examined on each subset using a flow cytometer modified for high- sensitivity fluorescence measurements. The results show that EPO induces extensive proliferation of erythroid progenitor cells, but has no effect on the proliferation or survival of primitive or granulomonocytic progenitors, even when used in combination with other cytokines. The majority of erythroid progenitor cells furthermore stained positively with anti-EPO receptor (EPO-R) monoclonal antibodies, whereas other progenitor cells were negative. SCF alone induced extensive proliferation of erythroid progenitor cells, and had a stronger synergistic effect on primitive than on granulo-monocytic progenitors. In spite of these differences in SCF activity, there were no significant differences in SCF-R expression between the progenitor subsets. These results suggest that the selective action of EPO on erythropoiesis is determined by lineage-restricted receptor expression, whereas there are additional cell-type specific factors that influence progenitor cell responses to SCF.
Effect of thrombopoietin on the development of megakaryocytes and platelets: an ultrastructural analysis
Authors: Zucker-Franklin D , Kaushansky K
Published: October 2019

Megakaryocytopoiesis and platelet production can be assessed with reasonable accuracy by quantitative and functional analyses of circulating platelets. The evaluation of megakaryocytopoiesis in culture has remained unsatisfactory, particularly because platelet production is rarely observed. In mouse culture systems, megakaryocytes have been identified almost entirely by measurements of acetyl cholinesterase, size, and ploidy without concomitant assessment of maturation based on such criteria as the formation of granules, demarcation membranes, and cytoplasmic fragmentation. The availability of various thrombopoietic cytokines, in particular thrombopoietin (TPO), and their imminent clinical use has made a more detailed understanding of their effect on differentiation and maturation of the MK lineage more urgent. Therefore, ultrastructural analyses were performed on megakaryocyte-depleted serum-free mouse bone marrow cultures in the presence of TPO alone, TPO plus other cytokines, or under conditions in which TPO and/or other cytokines were blocked with neutralizing agents. These studies show that, while cytokines that use the gp130 receptor subunit may function synergistically with TPO, in the absence of TPO, such cultures do not yield morphologically recognizable MK. On the other hand, TPO alone is able to drive MK to full maturation as evidenced by the generation of granules, demarcation membranes, and cytoplasmic fragmentation into platelets.
Human CD34+ hematopoietic progenitors have low, cytokine-unresponsive O6-alkylguanine-DNA alkyltransferase and are sensitive to O6- benzylguanine plus BCNU
Authors: Gerson SL , Phillips W , Kastan M , Dumenco LL , Donovan C
Published: October 2019

Human bone marrow (BM) cells contain low levels of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, which may explain their susceptibility to nitrosourea-induced cytotoxicity and the development of secondary leukemia after nitrosourea treatment. Isolated CD34+ myeloid progenitors were also found to have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells or in mononuclear BM cells did not increase after incubation with granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor, the combination, or 5637 conditioned medium. BCNU sensitivity remained unchanged as well. In addition, O6-benzylguanine depleted alkyltransferase activity in BM cells at concentrations as low as 1.5 mumol/L after a 1-hour exposure. O6-benzylguanine pretreatment markedly sensitized hematopoietic progenitor colony-forming cells to BCNU, resulting in a reduction in the dose of drug (termed the dose- modification factor) required to inhibit 50% of the colony formation (IC50) of threefold to fivefold. Since, unlike many other cell types, proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase, myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials.
Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function
Authors: Lee SB , Rao AK , Lee KH , Yang X , Bae YS , Rhee SG
Published: October 2019

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate, mobilization of intracellular Ca2+, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation (see accompanying report). A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, 7 out of 10 known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC- gamma 2 > PLC-beta 2 > PLC-beta 3 > PLC-beta 1 > PLC-gamma 1 > PLC- delta 1 > PLC-beta 4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-beta 2, whereas PLC-beta 4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-beta 2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.
Clearance of normal and type 2A von Willebrand factor in the rat
Authors: Stoddart JH Jr , Andersen J , Lynch DC
Published: October 2019

A model for the in vivo clearance of normal and mutant forms of human von Willebrand factor (vWF) has been established using catheterized rats. vWF clearance rates in rat plasma were determined by quantitation of reduced vWF subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and multimeric vWF was analyzed using nondenaturing SDS-agarose gels. Normal vWF derived from human umbilical vein endothelial cells displayed a biphasic pattern of clearance, with half times of 35 minutes (T 1/2 a; SD 15. min.) and 245 minutes (T 1/2 b; SD 76. min.); metabolic clearance rate = 0.65%/minute. High molecular weight multimers of vWF were cleared more rapidly than dimeric vWF. vWF containing the S1613P mutation found in some type 2A von Willebrand disease (vWD) patients was observed to undergo proteolysis in vivo resulting in a reduction of high molecular weight vWF and concomitant appearance of rapidly-migrating satellite species, although the overall clearance rate of vWF antigen was similar to wild type vWF. These results provide direct in vivo evidence that the S1613P mutation causes the characteristic type 2A vWD phenotype. Full-length recombinant vWF produced from transfected Chinese hamster ovary cells was cleared at a similar rate to endothelial cell-derived vWF, and recombinant vWF devoid of O-linked carbohydrates was cleared significantly faster. vWF devoid of sulfate was cleared at a similar rate as wild type vWF, indicating the sulfate moiety of vWF does not regulate in vivo clearance. This animal model should prove useful in subsequent in vivo analysis of additional forms of vWD and in the development of protease inhibitor therapy for 2A vWD.
Nitric oxide interactions with cobalamins: biochemical and functional consequences
Authors: Brouwer M , Chamulitrat W , Ferruzzi G , Sauls DL , Weinberg JB
Published: October 2019

Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.
A unique gene encodes spliceoforms of the B-cell adhesion molecule cell surface glycoprotein of epithelial cancer and of the Lutheran blood group glycoprotein
Authors: Rahuel C , Le Van Kim C , Mattei MG , Cartron JP , Colin Y
Published: October 2019

Two new members of the Ig superfamily, the Lutheran (Lu) blood group glycoprotein and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen, have been recently cloned from human placenta and colon cancer HT29 cell line, respectively. Although amino acid sequences deduced from cDNA analysis suggested that B-CAM should represent an abridged form of the Lu glycoprotein lacking the last 40 amino acids of the putative cytoplasmic tail, the relationship between the genes encoding these polypeptides has not been determined. In the present report, we showed by Southern blot analysis that the Lu and B-CAM cDNAs derived from a unique LU gene which exhibited an HindIII RFLP associated with the Lua/Lub blood group polymorphism. Accordingly, in situ hybridization of the Lu cDNA probe confirmed the localization of the Lutheran blood group locus to chromosome 19 q13.2–13.3, as previously shown for a B-CAM DNA probe. Sequence comparison between cDNA and genomic PCR fragments indicated that the Lu and B-CAM transcripts previously isolated are generated through the alternative use of internal splice donor and acceptor sites within an exon located at the 3′ end of the LU gene. These spliceoforms corresponded to 2.5 kb and 4.0 kb mRNA species detectable by Northern blot in all tissues and cell lines in which the LU gene is expressed; their primary structures are consistent with the presence of both the Lu and B-CAM antigens on two glycoprotein isoforms. However, the 4.0 kb transcript was very poorly expressed as compared to the 2.5 kb species except in the colon carcinoma HT29 cell line, suggesting a differential regulation of the Lu/B-CAM messenger RNA in some tumor tissues.
A prospective study of transfusion-transmitted GB virus C infection: similar frequency but different clinical presentation compared with hepatitis C virus
Authors: Wang JT , Tsai FC , Lee CZ , Chen PJ , Sheu JC , Wang TH , Chen DS
Published: October 2019

To study the incidence and outcome of GB virus C (GBV-C) infection in blood recipients. Serum samples collected in a prospective study were examined for GBV-C RNA by a nested polymerase chain reaction assay. Among the 400 adults who underwent cardiac surgery, 40 were positive for GBV-C RNA, including six whose pretransfusion sera were already positive and seven coinfected with hepatitis C virus (HCV) during transfusion. The risk of transmission was estimated to be approximately 0.46% per donor. GBV-C viremia was detectable 1 week after transfusion and could persist for 8 years. However, no evident symptoms or signs were noted in the 25 patients infected by GBV-C alone, and the average peak serum alanine aminotransferase activity was 31 IU/L only (range, 12 to 123), with persistently normal levels in 20 patients. In the seven patients coinfected with HCV, the clinical courses of posttransfusion hepatitis were similar to those infected by HCV alone. In eight patients with posttransfusion non-A approximately E hepatitis, only one was positive for GBV-C RNA. Sixty samples were chosen to test hepatitis G virus (HGV) sequences, 26 of the 30 GBV-C positives were positive for HGV RNA in contrast to none of the 30 GBV-C negative samples. In conclusion, GBV-C can be transmitted by transfusion in approximately 9% of patients who underwent cardiac surgery. Nevertheless, this virus does not seem to cause classic hepatitis in most instances.
Identification of a Serpin Specifically Expressed in Multipotent and Bipotent Hematopoietic Progenitor Cells and in Activated T Cells
Authors: Hampson I.N. , Hampson L. , Pinkoski M. , Cross M. , Heyworth C.M. , Bleackley R.C. , Atkinson E. , Dexter T.M.
Published: October 2019

We have identified a gene that has a high level of mRNA expression in undifferentiated, multipotential hematopoietic cells (FDCP-Mix) and that downregulates both transcript and protein, as these cells are induced to differentiate into mature myeloid cells. Sequence analysis of this gene has identified it as a serine protease inhibitor EB22/3 (serpin 2A). Constitutive expression of serpin 2A in FDCP-Mix cells was associated with an increase in the clonogenic potential of the cells and with a delay in the appearance of fully mature cells in cultures undergoing granulocyte macrophage differentiation when compared with control cells. Serpin 2A was also found to be expressed in bone marrow-derived bipotent granulocyte macrophage progenitor cells (GM-colony forming cell [CFC]), but not in erythrocyte progenitor cells from day 15 fetal liver. Expression of serpin 2A also showed a marked up regulation during the activation of cytotoxic suppressor CD8+ T cells, with a clear lag between the appearance of transcript and detection of protein.
A Novel Herpes Vector for the High-Efficiency Transduction of Normal and Malignant Human Hematopoietic Cells
Authors: Dilloo Dagmar , Rill Donna , Entwistle Claire , Boursnell Michael , Zhong Wanyun , Holden William , Holladay Martha , Inglis Stephen , Brenner Malcolm
Published: October 2019

Herpes simplex viruses (HSVs) would offer numerous advantages as vectors for gene transfer, but as yet they have not proved capable of transducing hematopoietic cells. Using a genetically inactivated form of HSV that is restricted to a single cycle of replication (disabled single-cycle virus, [DISC-HSV]), we have transduced normal human hematopoietic progenitor cells and primary leukemia blasts with efficiencies ranging from 80% to 100%, in the absence of growth factors or stromal support. Toxicity was low, with 70% to 100% of cells surviving the transduction process. Peak expression of transferred genes occurred at 24 to 48 hours after transduction with the DISC-HSV vector, declining to near background levels by 14 days. Despite this limitation, sufficient protein is produced by the inserted gene to permit consideration of the vector for applications in which transient expression is adequate. One example is the transfer of immunostimulatory genes, to generate leukemia immunogens. Thus, murine A20 leukemia cells transduced with a DISC-HSV vector encoding granulocyte-macrophage colony-stimulating factor were able to stimulate a potent antitumor response in mice, even against pre-existing leukemia. The exceptional transducing ability of the DISC-HSV vector should therefore facilitate genetic manipulation of normal and malignant human hematopoietic cells for biological and clinical investigation.
Cytokine Prestimulation as a Gene Therapy Strategy: Implications for Using the MDR1 Gene as a Dominant Selectable Marker
Authors: Blau C. Anthony , Neff Tobias , Papayannopoulou Thalia
Published: October 2019

A major obstacle to stem cell gene therapy is the extremely low efficiency of stem cell transduction. In vivo selection is a strategy for enriching a minor population of genetically modified bone marrow cells through the introduction of a drug resistance gene, followed by subsequent administration of the corresponding cytotoxic drug in vivo. Achieving persistent effects from in vivo selection is expected to require selection at the level of stem cells or, minimally, selection at the level of progenitors. Major limitations to in vivo selection are the nonhematologic toxicities of the cytotoxic drugs used and the resistance of stem cells and progenitors to killing by most cytotoxic agents. Experiments were performed in mice to evaluate whether the drugs used for selection in combination with multiple drug resistance gene 1 (MDR1) could have an enhanced effect on clonogenic progenitors if preceded by administration of the cytokine, stem cell factor (SCF). Single doses of taxol, navelbine, or vinblastine produced 10-fold reductions in the total number of mononuclear cells per femur, indicating a significant depletion of nonclonogenic precursor cells. However, for each of these agents, clonogenic progenitors, assayed as colony-forming unit cells and day-12 spleen colony-forming units, were relatively spared. Administration of SCF before taxol, navelbine, or vinblastine completely abrogated the progenitor-sparing phenomenon, because clonogenic progenitors were depleted as effectively as nonclonogenic precursor cells. Furthermore, the administration of SCF before drug administration allowed the dosages of taxol and vinblastine to be reduced by more than half, while retaining reductions in progenitor numbers that were unachievable using very high doses of the cytotoxic drug alone. Doxorubicin administration resulted in a 30- to 40-fold depletion in progenitors that was not significantly altered by preceding SCF administration. These results suggest that previous observations of in vivo selection using MDR1 gene transfer followed by taxol administration may have resulted from selection at the level of relatively mature, nonclonogenic precursor cells. Furthermore, these data suggest that cytokine prestimulation may be a useful strategy for improving the selection of drug-resistant clonogenic progenitors and, possibly, stem cells in vivo.