bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse fetal liver cells lack functional amphotropic retroviral receptors
Authors: Richardson C , Ward M , Podda S , Bank A
Published: October 2019

We have been transducing mouse hematopoietic cells with the human MDR1 (MDR) gene in retroviral vectors to determine the optimal conditions for retroviral gene transfer as a model system for potential human gene therapy. In these studies, we have demonstrated transduction and expression of the human MDR gene using ecotropic and amphotropic MDR- retroviral producer lines. To obtain more mouse hematopoietic cells for detailed study, mouse fetal liver cells (FLC) have been used for MDR transduction and expression, and to reconstitute the ablated marrows of live adult mice. FLC contain hematopoietic cells that have a reconstituting capacity comparable to that of adult mouse bone marrow cells. However, to our surprise, FLC can only be transduced with ecotropic retrovirus and not with amphotropic virus. This restriction of transduction of FLC cannot be overcome by higher titer virus. The resistance to amphotropic transduction by FLC may be part of a changing developmental program that results in a different antigen repertoire on FLC as compared with adult bone marrow cells.
Characterization of recombinant plasminogen activator production by primate endothelial cells transduced with retroviral vectors
Authors: Dichek DA , Lee SW , Nguyen NH
Published: October 2019

Retroviral vector-mediated expression of plasminogen activators (PAs) from endothelial cells (ECs) has been proposed as a potential therapeutic approach for intravascular thrombosis. To define the potential for gene transfer to increase fibrinolytic activity in a primate system, baboon ECs were transduced with retroviral vectors expressing wild-type and glycosylphosphatidylinositol-anchored urokinase, as well as wild-type and serpin-resistant tissue PA (t-PA). Expression of either t-PA or urokinase was increased by one log over baseline levels. There was no specific effect of either t-PA or urokinase overexpression on endogenous t-PA, urokinase, or PA inhibitor 1 (PAI-1) expression. Recombinant urokinase could be anchored to the cell surface at a level eight-fold above that of receptor-bound urokinase. The majority of secreted urokinase accumulated in conditioned medium as a free proenzyme, whereas both wild-type and serpin-resistant t-PA accumulated almost exclusively in complexes with PAI-1. In most but not all of the assays, the urokinase vectors conferred PA activity above that of the t-PA vectors. These data show that PA synthesis and activity are specifically increased subsequent to retroviral vector-mediated gene transfer in primate ECs. However, definition of an optimal PA vector will require in vivo experimentation.
Detection of cytarabine resistance in patients with acute myelogenous leukemia using flow cytometry
Authors: Lacombe F , Belloc F , Dumain P , Puntous M , Makhoul PC , Saux MC , Bernard P , Boisseau MR , Reiffers J
Published: October 2019

Cytarabine (Ara-C) is currently used in the treatment of adult acute myeloid leukemia (AML). To predict the results of induction chemotherapy, it could be useful to detect leukemic cells that are resistant to Ara-C in patients with AML. Using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM), we have developed a cell resistance index to Ara-C (RI). The technique has been applied to 121 bone marrow (BM) samples from patients with de novo AML treated by a regimen containing Ara-C and daunorubicin (DNR). Ninety-seven patients achieved a complete remission (CR), and 24 patients did not and were considered drug-resistant (DR). The BM cells collected at diagnosis were cultured for 48 hours and underwent BrdUrd/DNA analysis. Among 25 patients with no or very low proliferative activity (<3% of cells in S-phase), the proportion of DR patients (nine of 25) was significantly higher than in a second group of 96 patients with detectable proliferative activity (15 of 96) (P < .025). Within this second group, there was a first group of nine patients with high RI values, which included only DR patients; a second group of 63 patients with low RI values, which included 62 CR patients; and a third group of 24 patients with intermediate RI values, which included 19 CR and five DR patients. In view of this series, our results show that it is possible to detect a majority of DR patients treated by Ara-C.
Efficient retrovirus-mediated gene transduction into murine hematopoietic stem cells and long-lasting expression using a Transwell coculture system
Authors: Germeraad WT , Asami N , Fujimoto S , Mazda O , Katsura Y
Published: October 2019

The neomycin phosphotransferase (neo) gene was transduced into murine hematopoietic stem cells by culturing a recombinant retrovirus- producing cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene transduction into stem cells retaining long-term reconstitution ability was successfully performed by using protocols of total 15 to 18 days of culture including establishment of the Dexter culture, transduction, and G418 selection. In the irradiated recipients of these cells, a large majority of the BM, thymus, and spleen cells as well as peripheral blood (PB) leukocytes were of donor origin and the neo gene was present in these organs up to 21 weeks after cell transfer. One third to two thirds of the in vitro colony-forming cells in the BM of the recipient mice were resistant to cultivation with G418. It was further found that the hematopoietic system of secondary recipients given BM cells from a primary recipient mouse was predominated by original donor-type cells. The transduced neo gene was detected in the PB, BM, thymus, and spleen cells of these secondary recipients. These results indicate that our procedure of retroviral vector-mediated gene transfer is highly effective in safely introducing a gene into pluripotent hematopoietic stem cells.
Mobilization of long-term hematopoietic reconstituting cells in mice by the combination of stem cell factor plus granulocyte colony-stimulating factor
Authors: Yan XQ , Briddell R , Hartley C , Stoney G , Samal B , McNiece I
Published: October 2019

In this study, we have compared the ability of recombinant human granulocyte colony-stimulating factor (rhG-CSF) alone and the combination of low doses of recombinant rat pegylated stem cell factor (rrSCF-PEG) plus rhG-CSF to mobilize peripheral blood progenitor cells (PBPCs) with long-term engrafting potential. Female recipient irradiated mice were transplanted with PBPCs from male mice that were mobilized with rhG-CSF alone (group A) or rrSCF-PEG plus rhG-CSF (group B). As previously shown, greater short-term survival resulted in group B compared with group A, with 80% and 40% survival at 30 days posttransplant, respectively. Both groups of animals showed long-term donor-derived engraftment in greater than 95% of animals, as determined by quantitative specific polymerase chain reaction amplification of a Y chromosome sequence from whole blood of the mice at 6 to 12 months posttransplantation. Analysis of individual granulocyte-macrophage colonies, picked up from semisolid methylcellulose culture of bone marrow cells from transplanted mice, resulted in detection of donor- derived DNA in 98% of colonies from group B mice compared with 81% from group A mice. These data show that cells with long-term potential are mobilized by rhG-CSF alone and the combination of rrSCF-PEG plus rhG- CSF. Furthermore, an increased number of cells with short-term and long- term engraftment potential was obtained with rrSCF-PEG plus rhG-CSF compared with rhG-CSF alone.
In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells
Authors: Andrews RG , Briddell RA , Knitter GH , Opie T , Bronsden M , Myerson D , Appelbaum FR , McNiece IK
Published: October 2019

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS).
Effects of butyrate on the erythropoietin receptor of cell line IW201
Authors: Hermine O , Dong YJ , Goldwasser E
Published: October 2019

The murine erythroleukemic cell line, IW201, normally expresses only low-affinity erythropoietin receptors. Exposure of these cells for 48 hours to sodium butyrate results in a change in receptor kd from about 600 pmol/L to 100 to 200 pmol/L. This change in affinity is accompanied by downregulation of both receptor number and receptor mRNA. Cells exposed to sodium butyrate for 2 hours show a similar change in kd but no change in receptor number. The butyrate effect on kd at 2 hours is abrogated by either cycloheximide or actinomycin D. These data indicate that an accessory protein induced by sodium butyrate is responsible for high-affinity binding of erythropoietin.
CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits
Authors: Fortenberry JD , Marolda JR , Anderson DC , Smith CW , Mariscalco MM
Published: October 2019

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day- old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.
Prospective comparative trial of autologous versus allogeneic bone marrow transplantation in patients with non-Hodgkin's lymphoma
Authors: Ratanatharathorn V , Uberti J , Karanes C , Abella E , Lum LG , Momin F , Cummings G , Sensenbrenner LL
Published: October 2019

A prospective comparative trial of allogeneic versus autologous bone marrow transplant (BMT) was conducted. Sixty-six consecutive patients (median age, 41; range, 15 to 60; female:male ratio = 21:45) entered this clinical trial. Priority for allogeneic BMT was given to patients who were 55 or younger and had a major histocompatibility complex- matched or 1-antigen-disparate sibling donor. Autologous BMT was offered to all other patients whose age was 60 or younger. Patients who had no sibling donor and who had BM involvement at the time of evaluation were not eligible. Thirty-one patients received an allograft, and 35 patients received an autograft. Thirteen patients received a BM graft purged with 4-hydroperoxycyclophosphamide because of previous BM involvement. Patients who had previous radiation to the thoracic and/or abdominal areas of more than 20 Gy received a preparative regimen consisting of cyclophosphamide (1,800 mg/m2/d for 4 days), VP-16 (200 mg/m2 every 12 hours for 8 doses), and 1,3-bis(2- chloroethyl)-1-nitrosourea (600 mg/m2 as 1 dose). Other patients received cyclophosphamide 1,800 mg/m2/d for 4 days followed by total body irradiation of 12 Gy administered as a single daily fraction over 4 days. With a median follow-up of 14 months, the progression-free survival (PFS) for autograft and allograft recipients was 24% +/- 8% (+/- SE) and 47% +/- 9%, respectively, (P = .21). However, the probability of disease progression was significantly higher in the autologous group (69% +/- 9%) than in the allogeneic group (20% +/- 10%; P = .001). When other confounding prognostic factors were adjusted in the multivariate analysis, chemosensitive disease and allograft were found to have a significant favorable influence on probability of disease progression (P = .03 and .003), but only chemosensitive disease had a significant influence on the PFS (P < .002). Our results suggest the existence of graft-versus-lymphoma effect and also support the rationale of using immunotherapy after autologous BMT. Allogeneic BMT should be preferable to autologous BMT in younger patients with lymphoma.
Differential effects of gangliosides on Ig production and proliferation by human B cells
Authors: Kimata H , Yoshida A
Published: October 2019

The effects of gangliosides on human B-cell responses were studied. Of various gangliosides tested, only GM2 and GM3 inhibited production of IgG subclasses and IgM, but not IgA subclasses, and thymidine uptake by human B cells stimulated with SAC plus interleukin-2 (IL-2). In contrast, GM1, GD1a, GD1b, GD3, GT1b, and GQ1b were without effects. GM2- and GM3-induced inhibition were specific, because each was blocked by a corresponding antibody. Of various cytokines tested, tumor necrosis factor-alpha (TNF-alpha) alone counteracted GM2- and GM3- induced inhibitions of Ig production and thymidine uptake, whereas other cytokines including IL-1 beta, IL-3, IL-5, IL-6, and interferon- gamma each failed to do so. Moreover, anti-TNF-alpha antibody, but not control IgG, blocked the counteraction of inhibition by TNF-alpha. GM2 and GM3 each inhibited Ig production, thymidine uptake, and TNF-alpha production by surface IgG1+ (slG1+), sIgG2+, sIgG3+, sIgG4+, and sIgM+ B cells without affecting IL-2 binding or TNF-alpha binding to B cells, but had no such inhibitory effects on sIgA1+ or sIgA2+ B cells. These findings indicate that GM2 and GM3 inhibit Ig production and thymidine uptake by human sIgG1+, sIgG2+, sIgG3+, sIgG4+, and sIgM+ B cells by inhibiting endogenous TNF-alpha production.
Differential expression of bcl-2 and susceptibility to anti-Fas- mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils
Authors: Iwai K , Miyawaki T , Takizawa T , Konno A , Ohta K , Yachie A , Seki H , Taniguchi N
Published: October 2019

The recently identified Fas antigen (Ag) is a cell surface molecule that can mediate apoptosis. The cytoplasmic product of proto-oncogene bcl-2 has been shown to prolong the cellular survival by inhibiting apoptosis. To elucidate the physiologic significance of expression of both molecules, we examined the expression of Fas Ag and bcl-2 on blood leukocyte populations and evaluated their sensitivity to the cytolytic action of anti-Fas antibody. Although Fas Ag was expressed on a fraction of lymphocytes, both neutrophils and monocytes expressed Fas Ag constitutively. In contrast, there was marked difference among these leukocytes regarding bcl-2 expression. Lymphocytes expressed bcl-2 intensely, but monocytes showed weaker bcl-2 expression, and neutrophils were essentially absent for bcl-2 expression. Seemingly reflecting this lack of bcl-2-expression, neutrophils more easily underwent apoptotic cell death in vitro as compared with monocytes and lymphocytes. We showed that anti-Fas antibody affectively accelerated apoptotic cell death in neutrophils. However, the apoptosis-inducing effect of anti-Fas antibody was minimal on monocytes, and lymphocytes were resistant to this antibody. These results suggest that anti-Fas- mediated cell death may, in part, be determined by bcl-2 expression status in Fas+ lymphoid and hematopoietic cells.
Crystallization of recombinant hemoglobins with basic amino acid substitutions (Lys and Arg) at the beta 6 position
Authors: Adachi K , Lai CH , Konitzer P , Donahee M , Campbell A , Surrey S
Published: October 2019

We have produced recombinant hemoglobins (rHbs) alpha 2 beta 2(6Glu-- >Lys) (rHb beta E6K) and alpha 2 beta 2(6Glu-->Arg) (rHb beta E6R) using a yeast expression system coupled with a polymerase chain reaction (PCR)-based mutagenesis strategy for studies focused on defining determinants that facilitate crystallization of Hb C (alpha 2 beta 2(6Lys)). rHb beta E6K had the same electrophoretic mobility as native human Hb C, whereas rHb beta E6R migrated slightly slower than Hb C on cellulose acetate electrophoresis. The carbonmonoxy (CO) forms of rHb beta E6K and rHb beta E6R formed tetrahedral crystals in vitro in 2.3 mol/L phosphate buffer just like native Hb C. The Hb concentration required for crystallization of CO-rHb beta E6R was lower than that of CO-rHb beta E6K, suggesting that stronger basic amino acids at the beta 6 position accelerate crystallization of Hb. However, the size of rHb beta E6R crystals was smaller than that of rHb beta E6K. Crystallization of native Hb C and both rHbs was inhibited by Hb F. These results suggest that alpha 2 beta gamma-heterohybrids that have basic amino acids at the beta 6 position behave similarly and are unable to crystallize like Hb C.
Activation of the coagulation cascade after infusion of a factor XI concentrate in congenitally deficient patients
Authors: Mannucci PM , Bauer KA , Santagostino E , Faioni E , Barzegar S , Coppola R , Rosenberg RD
Published: October 2019

Virally inactivated, high-purity factor XI concentrates are available for treatment of patients with factor XI deficiency. However, preliminary experience indicates that some preparations may be thrombogenic. We evaluated whether a highly purified concentrate produced signs of activation of the coagulation cascade in two patients with severe factor XI deficiency infused before and after surgery. Signs of heightened enzymatic activity of the common pathway of coagulation (elevated plasma levels of prothrombin fragment 1 + 2 and fibrinopeptide A) developed in the early post-infusion period, accompanied by more delayed signs of fibrin formation with secondary hyperfibrinolysis (elevated D-dimer and plasmin-antiplasmin complex). These changes occurred in both patients, but were more severe in the older patient with breast cancer when she underwent surgery, being accompanied by fibrinogen and platelet consumption. There were no concomitant signs of heightened activity of the factor VII-tissue factor mechanism on the factor Xase complex (plasma levels of activated factor VII and of factor IX and X activation peptides did not increase). The observed changes in biochemical markers of coagulation activation indicate that concentrate infusions increased thrombin generation and activity and that such changes were magnified by malignancy and surgery. Because some factor XI concentrates may be thrombogenic, they should be used with caution, especially in patients with other risk factors for thrombosis.
Modeling human lymphoid precursor cell gene therapy in the SCID-hu mouse
Authors: Akkina RK , Rosenblatt JD , Campbell AG , Chen IS , Zack JA
Published: October 2019

Gene therapy of human T-lymphocyte disorders, including acquired immunodeficiency syndrome (AIDS), would be greatly facilitated by the development of an in vivo system in which transduced human hematopoietic stem cells can be used to reconstitute the T-lymphoid compartment. Here we use the SCID-hu mouse as a recipient for human CD34+ hematopoietic progenitor cells transduced in vitro with a retroviral vector carrying the neomycin resistance gene (neoR). The transduced cells engraft and reconstitute the lymphoid compartments of the human thymus implant with as few as 5 x 10(4) CD34+ cells. The neoR gene was expressed at low levels in human thymocytes and there was no apparent effect on thymocyte differentiation as a result of vector transduction. Thus, this SCID-hu mouse system is the first in vivo model showing human thymopoiesis after transduction of exogenous vectors, and should allow preclinical testing of gene therapeutic reagents designed to function in human cells of the T-lymphoid lineage. Because human immunodeficiency virus type 1 infection induces depletion of human thymocytes in SCID-hu mice, this system may be particularly valuable in evaluating efficacy of gene therapies to combat AIDS.
The B7-2 (B70) costimulatory molecule expressed by monocytes and activated B lymphocytes is the CD86 differentiation antigen
Authors: Engel P , Gribben JG , Freeman GJ , Zhou LJ , Nozawa Y , Abe M , Nadler LM , Wakasa H , Tedder TF
Published: October 2019

T-cell activation is initiated after T-cell receptor binding to antigen, but also requires interactions between costimulatory molecules expressed on antigen-presenting cells. An important costimulatory molecule expressed by monocytes and activated B lymphocytes has been recently identified and termed B7–2 or B70. Independently, a new Cluster of Differentiation was defined in the Fifth International Leukocyte Differentiation Antigen Workshop as CD86, a molecule predominantly expressed by monocytes and activated B lymphocytes. In this study, the two monoclonal antibodies that defined CD86, FUN-1 and BU-63, were shown to bind to cDNA transfected cells expressing B7– 2/B70. The FUN-1 monoclonal antibody also completely blocked the costimulatory activity of B7–2/B70 in functional assays. Therefore, the serologically defined CD86 differentiation antigen is the B7–2/B70 molecule.
Positively selected autologous blood CD34+ cells and unseparated peripheral blood progenitor cells mediate identical hematopoietic engraftment after high-dose VP16, ifosfamide, carboplatin, and epirubicin
Authors: Brugger W , Henschler R , Heimfeld S , Berenson RJ , Mertelsmann R , Kanz L
Published: October 2019

To investigate the feasibility of peripheral blood CD34+ cell selection and to analyze CD34+ cell-mediated engraftment after high-dose chemotherapy, we performed a phase I/II trial in 21 patients with advanced malignancies. The rationale for the selection of CD34+ cells from peripheral blood progenitor cell (PBPC) collections is based on the observation that contaminating tumor cells can be depleted approximately 3 logs using this procedure. CD34+ cells from chemotherapy+granulocyte colony-stimulating factor-mobilized PBPCs were positively selected with an avidin-biotin immunoadsorption column (CEPRATE SC system). One leukapheresis product with a median number of 2.8 x 10(6) CD34+ cells/kg was labeled with a biotinylated anti-CD34 monoclonal antibody and subsequently processed over the column. The yield of selected CD34+ cells was 73% +/- 24.6%. The purity of the CD34+ cell fraction was 61.4% +/- 19.7%. CD34+ cells were shown to represent predominantly committed progenitors coexpressing CD33, CD38, and HLA-DR molecules (lin+). They gave rise to myeloid as well as erythroid and multilineage colonies in vitro. In addition, positively selected CD34+ cells also comprised early hematopoietic progenitor cells, as shown by the presence of CD34+/lin- cells. Transfusion of positively selected CD34+ cells (2.5 x 10(6) CD34+/kg; range, 0.45 to 5.1) after high-dose VP16 (1,500 mg/m2), ifosfamide (12 g/m2), carboplatin (750 mg/m2), and epirubicin (150 mg/m2) (VIC-E) in 15 patients resulted in a rapid and stable engraftment of hematopoiesis without any adverse events. As compared with 13 historical control patients reconstituted with a comparable number of unseparated PBPCs, time to neutrophil and platelet recovery was identical in both groups (absolute neutrophil count > 500/microL, day + 12; platelet count > 50,000/microL, day + 15). These data indicate that autologous peripheral blood CD34+ cells and unseparated PBPCs mediate identical reconstitution of hematopoiesis after high-dose VIC-E chemotherapy. Because positive selection of CD34+ cells from mobilized blood results in a median 403-fold depletion of T cells, allogeneic CD34+ cells from mobilized blood should be investigated as an alternative to bone marrow cells for allotransplantation.
Stem cell factor and interleukin-7 synergize to enhance early myelopoiesis in vitro
Authors: Fahlman C , Blomhoff HK , Veiby OP , McNiece IK , Jacobsen SE
Published: October 2019

Interleukin-7 (IL-7) has been shown to be a critical factor in murine lymphoid development. It stimulates pre-B cells to divide in the absence of stroma cells and it is an important growth regulator of immature and mature T cells. IL-7 has been shown to synergize with stem cell factor (SCF) to provide a potent growth stimulus for pre-B cells. However, the combined effects of IL-7 and SCF on murine primitive hematopoietic cells in vitro have not been established. In the present study, the effects of recombinant rat (rr) SCF and recombinant human (rh) IL-7 on primitive murine bone marrow progenitors (Lin-Sca1+) were investigated in single-cell cloning experiments. rhIL-7 alone had no proliferative effect on Lin-Sca1+ cells, but in a dose-dependent manner directly enhanced rrSCF-induced colony formation, with an average increase in colony numbers of 2.7-fold. Interestingly, the cells formed in response to SCF and IL-7 were predominantly mature granulocytes. Thus, SCF and IL-7 synergize to stimulate early myelopoiesis in vitro.
Corticosteroid modulation of interleukin-1 hematopoietic effects and toxicity in a murine system
Authors: Rinehart J , Delamater EW , Keville L , Measel J
Published: October 2019

Interleukin-1 (IL-1) has been shown to ameliorate the hematopoietic toxicities of antitumor chemotherapeutic agents in both mice and humans. However, IL-1 toxicity in humans is considerable and is similar to the systemic inflammatory toxicities induced by IL-3, IL-6, and other cytokines with pleiotropic biologic activities, eg, fever, nausea, malaise, and hypotension. We hypothesized that corticosteroids may reduce IL-1 toxicity without reducing IL-1 hematopoietic effects in vivo. C3H/HeJ mice (female, 6 weeks) were treated for 7 days subcutaneously with cortisone acetate (CA), (0.1, 0.25, or 0.5 mg/d/mouse), intraperitoneally with IL-1 (1 or 2 micrograms/d/mouse), or both. As expected, IL-1 increased white blood cell counts, splenic granulocyte-macrophage colony-forming units, and spleen cell number, and protected mice from lethal doses of carboplatin (200 mg/kg; Paraplatin, Bristol Laboratories, Evansville, IN) administered the day after completion of the 7 days of IL-1 administration. CA did not significantly block the hematopoietic effects of IL-1 or the ability of IL-1 to protect mice from the hematopoietic toxicity of carboplatin. IL- 1 administered to mice at 8 micrograms/d/mouse for 5 days induced decreased activity, roughening of hair, diarrhea, pancytopenia, multiple metabolic abnormalities, and death in 60% of mice. IL-1 at the therapeutic doses (0.5 to 2 micrograms/d) was not toxic. CA in a dose- dependent manner blocked all of the above mentioned toxicities when administered 24 hours and 30 minutes before each IL-1 injection. CA also decreased IL-1-induced increase in plasma tumor necrosis factor levels at the time point examined.
Erythropoietin induces association of the JAK2 protein tyrosine kinase with the erythropoietin receptor in vivo
Authors: Miura O , Nakamura N , Quelle FW , Witthuhn BA , Ihle JN , Aoki N
Published: October 2019

Protein tyrosine phosphorylation has been hypothesized to play a key role in the growth signaling induced by erythropoietin (Epo), although the Epo receptor (EpoR), a member of the cytokine receptor superfamily, lacks a tyrosine kinase domain. Recently, the JAK2 tyrosine kinase was shown to be activated on Epo stimulation and to bind to the cytoplasmic domain of EpoR in vitro. To further explore the mechanisms of activation of JAK2 in EpoR-mediated signal transduction, we assessed the conditions for association of JAK2 with EpoR in vivo. Epo stimulation rapidly induced association of JAK2 with the EpoR in an interleukin 3 (IL-3)-dependent cell line transfected with the wild-type EpoR. On Epo stimulation JAK2 also associated with a truncated mutant EpoR (H-mutant), which is mitogenetically active but not tyrosine phosphorylated, indicating that association does not require receptor phosphorylation and occurs in the membrane proximal region. However, association was not detected with mutant receptors inactivated by an internal deletion or a point mutation, Trp282 to Arg, in a membrane- proximal cytoplasmic region (PB or PM4 mutant, respectively). Immune complex kinase assays of anti-EpoR immunoprecipitates also revealed that activated JAK2 associates with the EpoR in Epo-stimulated cells. By this approach, association also occurred with the mitogenically active H mutant but not with the mitogenically inactive PB or PM4 mutants. In the immune complex kinases assays, EpoR, JAK2, and a 150-kD protein were phosphorylated on tyrosine. Taken together, the results further support the hypothesis that, on Epo stimulation, JAK2 associates with the membrane-proximal cytoplasmic region of the EpoR to be activated and induces tyrosine phosphorylation of cellular substrates, including the EpoR, to transduce a growth signal.
Interleukin-4 inhibits the expression of Kit and tryptase during stem cell factor-dependent development of human mast cells from fetal liver cells
Authors: Nilsson G , Miettinen U , Ishizaka T , Ashman LK , Irani AM , Schwartz LB
Published: October 2019

Although interleukin-4 (IL-4) in mice is known to augment the proliferation of mast cells and to modulate the expression of certain mast cell protease transcripts, its effect on human mast cells is less well understood. The current study examined the effects of recombinant human IL-4 (rhuIL-4) on stem cell factor (SCF)-dependent fetal liver- derived human mast cells in liquid culture. In no case did rhuIL-4 augment proliferation of mast cells. rhuIL-4 selectively inhibited certain aspects of the development of mast cells in cultures of fetal liver cells with rhuSCF. These include lower numbers and percentages of cells expressing tryptase and surface Kit, smaller cells, and lower contents of cells for tryptase, histamine, and Kit. Development of metachromasia was not attenuated. The downregulation of Kit, the surface receptor for SCF, is probably a critical factor, because cells lacking this molecule would not be able to respond to SCF. In contrast to mast cell progenitors, mast cells already developed in vitro from fetal liver cells are relatively resistant to rhuIL-4, but are still dependent for survival on the presence of rhuSCF.
Structural elements influencing von Willebrand factor (vWF) binding affinity for platelet glycoprotein Ib within a dispase-digested vWF fragment
Authors: Miura S , Fujimura Y , Sugimoto M , Kawasaki T , Ikeda Y , Titani K , Yoshioka A
Published: October 2019

We investigated the structural elements in human von Willebrand factor (vWF) that influence binding affinity for platelet glycoprotein (GP) Ib using a dispase-digested vWF fragment as a prototype (residues Leu480/Val481-Gly718 of the vWF subunit; Andrews et al, Biochemistry 28:8326, 1989). The major structural features of this fragment are a large A1-loop formed by an intrachain disulfide bond between Cys509 and Cys695 and six O-linked sugar chains. The fragment was chemically modified by (1) reduction and S-carboxyamido-methylation (R/A), (2) desialylation (DS), or (3) a combination of both (R/A-DS). The GPIb binding affinity of these fragments was basically evaluated by competitive binding assay with anti-GPIb monoclonal antibody (LJ-Ib1), a receptor blocker for vWF (Sugimoto et al, Biochemistry 30:5202, 1991). Both the prototype and the R/A fragments were also assessed for their function in shear-induced platelet aggregation. Results unambiguously demonstrated that the presence of a disulfide bridge (Cys509-Cys695) within this domain downregulates the affinity of vWF to GPIb. In addition, it was also demonstrated that the terminal sialic acids attached to six o-linked sugar chains within this domain contribute to optimal functional modulation by the antibiotic ristocetin, but not by snake venom botrocetin.
Immunophenotype of adult acute lymphoblastic leukemia, clinical parameters, and outcome: an analysis of a prospective trial including 562 tested patients (LALA87). French Group on Therapy for Adult Acute Lymphoblastic Leukemia
Authors: Boucheix C , David B , Sebban C , Racadot E , Bene MC , Bernard A , Campos L , Jouault H , Sigaux F , Lepage E
Published: October 2019

The aim of the multicentric trial LALA87 was to test the efficacy of different postremission therapies in adults (15 to 60 year olds) with acute lymphoblastic leukemia (ALL). An immunologic subclassification based on surface marker expression was proposed. Among the 562 tested patients, 511 were assigned either to the B lineage (361 cases, 63%) or to the T lineage (150 cases, 26%). T-ALL were significantly associated with male sex, age less than 35 years, mediastinal mass, central nervous system involvement, high white blood cell count, and low anemia. In a univariate and multivariate analysis, T-cell leukemia had a more favorable outcome than B-cell leukemia with respective median disease-free survivals (DFSs) of 28 and 14 months (P < .005). However, the type of postremission therapy modifies the value of the immunophenotype prognostic factor. In the chemotherapy arm, T-ALL patients (26 patients) had a more favorable outcome than B-ALL patients (57 patients) (P < .003). In the autologous bone marrow transplantation (ABMT) arm, the apparent better outcome of T-ALL patients (35 T/50 B) did not reach statistical significance (P = .2) and there was no difference in the allogeneic bone marrow transplantation (alloBMT) arm (37 T/71 B: P = .9). In the B-cell-lineage leukemias, subclassification by stages and myeloid antigen coexpression (10%) were not associated with different prognosis. CD10+ T-ALL (31 patients) were associated with a better DFS compared with the CD10- T-ALL (73 patients) with respective median DFS, not reached and 18.5 months (P = .04).
Activation of Ras and formation of GAP complex during TPA-induced monocytic differentiation of HL-60 cells
Authors: Katagiri K , Hattori S , Nakamura S , Yamamoto T , Yoshida T , Katagiri T
Published: October 2019

In this study, it was shown that the proportion of guanosine triphosphate (GTP)-bound active Ras increased in TPA (12-o- tetradecanoyl phorbol-13-acetate)-induced monocytic differentiation of HL-60 cells. The increase of active Ras was observed at 24 hours after TPA stimulation and attained to threefold (15%) over the proportion in nontreated HL-60 cells. Herbimycin A, an inhibitor of tyrosine kinase, prevented the activation of Ras, as well as the induction of monocytic differentiation. In parallel with the activation of Ras, the proteins with molecular weights of 52, 56, 62, and 190 kD were tyrosine- phosphorylated and formed a complex with GTPase-activating protein (GAP) for Ras. In addition to the 116-kD GAP (type I GAP), the 100-kD GAP (type II GAP) molecule was markedly induced at 24 hours after TPA stimulation of HL-60 cells. These phenomena sustained for a further 24 hours during monocytic differentiation. However, they were not observed during retinoic acid-induced granulocytic differentiation of the cells. The HL-60 transfectants, which expressed a dominant inhibitory Ha-ras Asn17, showed a low level of tyrosine-phosphorylated GAP-associated proteins and did not undergo full differentiation in response to TPA. Taken together, these data indicate that the activation of Ras and GAP complex formation mutually correlate and function downstream of protein- tyrosine kinases in the signaling pathway for monocytic differentiation of HL-60 cells.
Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: cooperation between the integrin alpha 4 beta 1 and the CD44 adhesion receptor
Authors: Verfaillie CM , Benis A , Iida J , McGlave PB , McCarthy JB
Published: October 2019

Close interaction of human hematopoietic progenitors with the bone marrow microenvironment is important for the ordered progression of human hematopoiesis. Progenitor cell adhesion to stroma has a complex molecular basis, involving various cell-extracellular matrix and cell- cell interactions. We have previously shown that adhesion of colony- forming cells (CFC) to fibronectin, present in stromal extracellular matrix, involves multiple sites, including two heparin-binding synthetic peptides (FN-C/H I and FN-C/H II) and the alpha 4 beta 1 integrin-binding peptide CS1. These synthetic peptides are located in close proximity in the type III repeat 14 and the immediately adjacent type IIIcs region of fibronectin. In the current study, we evaluate receptors expressed by CFC responsible for their adhesion to fibronectin. We show that the alpha 4 beta 1 integrin mediates adhesion to CFC to the peptides FN-C/H I and CS1. Adhesion of CFC to fibronectin is also mediated by proteoglycans, because removal of cell surface chondroitin-sulfate proteoglycans resulted in decreased adhesion of CFC to FN-C/ I and FN-C/H II. The core protein of this proteoglycan was identified by immunoprecipitation as a 90-kD member of the CD44 group of adhesion molecules. Interestingly, although the proteoglycan core protein failed to adhere to FN-C/H II affinity columns, anti-CD44 monoclonal antibodies blocked CFC adhesion to FN-C/H II, indicating that these monoclonal antibodies may interfere with core protein- mediated intracellular signalling. Finally, we show that CD44 and alpha 4 beta 1 may cooperate in establishing progenitor adhesion, because anti-CD44 antibodies potentiated the adhesion-inhibitory effects of suboptimal concentrations of anti-alpha 4 or anti-beta 1 monoclonal antibodies. These results provide a working model for progenitor cell recognition of fibronectin (and possibly the marrow micro-environment) in which the coordinated action of integrins and cell surface proteoglycans is necessary for cell adhesion. This model can now be used to study the complex relationship between progenitor cell adhesion and the regulation of their proliferation and differentiation.
Characterization of a factor IX variant with a glycine207 to glutamic acid mutation
Authors: Lin SW , Lin CN , Hamaguchi N , Smith KJ , Shen MC
Published: October 2019

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.
A subset of human CD34+ hematopoietic progenitors express low levels of CD4, the high-affinity receptor for human immunodeficiency virus-type 1
Authors: Zauli G , Furlini G , Vitale M , Re MC , Gibellini D , Zamai L , Visani G , Borgatti P , Capitani S , La Placa M
Published: October 2019

We investigated the expression of CD4 antigen in normal bone marrow (BM) samples, enriched in CD34+ hematopoietic progenitor cells. At flow cytometry, a significant fraction (ranging from 25% to 65%) of CD34+ cells also showed low levels of CD4 antigen on their surface. The CD4 receptor densities on the surface of hematopoietic progenitors was approximately 50% that of peripheral blood monocytes and 5% of peripheral blood T lymphocytes. In immunoprecipitation experiments, the CD4 antigen expressed by BM hematopoietic progenitors appeared to be the same form expressed by mature peripheral blood CD4+ cells and appeared to be a potentially functional receptor for human immunodeficiency virus-type 1, because it specifically bound recombinant envelope gp120. Moreover, BM samples, highly enriched in CD34+ cells, showed the presence of CD4 mRNA at reverse transcription- polymerase chain reaction examination. In experiments of complement- mediated cytotoxicity with Leu3-a+Leu3b anti-CD4 monoclonal antibody, a significant reduction in the number of both classes of megakaryocyte (burst-forming unit-meg [BFU-meg] and colony-forming unit-meg [CFU- meg]) and granulocyte/macrophage (CFU-GM) progenitors was observed, whereas erythroid (BFU-E) progenitors were only slightly affected. Moreover, purified CD4+ BM cells obtained by immunomagnetic selection, using high concentrations of Leu3a+Leu3b, showed a colony-forming ability of megakaryocyte and granulocyte/macrophage progenitors comparable with that of CD4- BM cells. In conclusion, the present data show that immature hematopoietic progenitor cells express low levels of CD4, the high-affinity receptor of human immunodeficiency virus-type 1.
Marrow transplantation for chronic myeloid leukemia: a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide
Authors: Clift RA , Buckner CD , Thomas ED , Bensinger WI , Bowden R , Bryant E , Deeg HJ , Doney KC , Fisher LD , Hansen JA
Published: October 2019

A prospective randomized study was conducted comparing two conditioning regimens for the treatment of patients with chronic myeloid leukemia in chronic phase by marrow transplantation from HLA identical siblings. Sixty-nine patients received 60 mg/kg of cyclophosphamide on each of 2 successive days followed by 6 fractions of total body irradiation each of 2.0 Gy (CY-TBI), and 73 patients received 16 mg/kg of busulfan delivered over 4 days followed by 60 mg/kg CY on each of 2 successive days (BU-CY). There was no significant difference between the CY-TBI and the BU-CY groups in the 3-year probabilities of survival (0.80 for both), relapse (0.13 for both), or event-free survival (CY-TBI, 0.68; BU-CY, 0.71) or in speed of engraftment or incidence of venocclusive disease of the liver. The 4-year probabilities of survival and event- free survival for patients transplanted within 1 year of diagnosis were 0.86 and 0.72, respectively, for each group. Significantly more patients in the CY-TBI group experienced major creatinine elevations. There was significantly more acute graft-versus-host disease in the CY- TBI group. Fever days, positive blood cultures, hospitalizations, and inpatient hospital days were significantly more common in the CY-TBI group than in the BU-CY group. In conclusion, the BU-CY regimen was better tolerated than, and associated with survival and relapse probabilities that compare favorably with, the CY-TBI regimen.