bluuebunny/crossref_metadata_2025 huggingface dataset:
Transient increase in circulating donor leukocytes after allogeneic transfusions in immunocompetent recipients compatible with donor cell proliferation
Authors: Lee TH , Donegan E , Slichter S , Busch MP
Published: October 2019

Donor leukocytes in therapeutic blood components are implicated in transfusion-related complications ranging from alloimmunization to graft-versus-host disease (GVHD) to viral transmission and reactivation. To further characterize the kinetics of donor leukocyte clearance after allogeneic transfusion, we developed allele-specific polymerase chain reaction (PCR) assays directed at a single-copy Y chromosome gene and HLA class II alleles. These assays enable sensitive detection and quantitation of donor leukocytes at concentrations ranging from one cell to greater than 1,000 cells per 125 microL of recipient blood. When applied to serial samples from five consecutive orthopedic surgery patients who met study criteria, we observed 99.9% clearance of donor leukocytes over the initial 2 days posttransfusion, followed by a transient, 1-log increase in circulating donor leukocytes on days 3 to 5. This phenomenon was reproduced in a canine transfusion model, where the transient donor leukocyte expansion phase was prevented by gamma irradiation of donor blood, and was not observed after transfusions into alloimmunized dogs. We hypothesize that this transient increase in circulating allogeneic donor cells represents one arm of an in vivo mixed lymphocyte reaction, with activated donor T lymphocytes proliferating in an abortive GVHD reaction to HLA- incompatible recipient cells. Further investigation of this phenomenon should provide insight into the mechanisms involved in donor-recipient leukocyte interactions posttransfusion and the relationship of these interactions to leukocyte-induced complications.
High expression of c-kit in K562YO cells due to the prolonged half-life of its mRNA: the effects of modification with serine/threonine kinase signals
Authors: Ogawa K , Takeda Y , Tashima M , Sawai H , Toi T , Okazaki T , Sawada H , Maruyama Y , Okuma M
Published: October 2019

We previously reported that the K562 cell line K562YO expressed a high level of the c-kit gene. In this study, we analyzed the mechanism of this expression and investigated the effects of the serine/threonine kinases such as protein kinase C (PKC) and cyclic adenosine 3′,53′ monophosphate (cAMP)-dependent kinase (PKA) on it. The half-life of the c-kit mRNA in K562YO cells was greater than 10 hours, compared with 2 hours in the original K562 cells, which expressed a very low level of c- kit mRNA. This prolonged half-life can contribute to the high level of c-kit expression in K562YO cells. Cycloheximide (CHX), a protein synthesis inhibitor, caused increases in c-kit mRNA levels in K562YO cells. 12-O-tetradecanoylphorbol-13-acetate (TPA), by which PKC was activated at first and downregulated in a late phase, gradually decreased c-kit mRNA in K562YO cells until 9 hours and then returned to the control level 24 hours after treatment. TPA also rapidly decreased c-kit protein level on the membranes. In whole cells, c-kit protein was also decreased 6 hours after incubation with TPA. Calphostin C, a light- dependent PKC inhibitor, decreased c-kit mRNA levels within 30 minutes in a light-dependent manner. It also decreased c-kit protein in whole cells 2 hours after the addition. However, it increased the amount of c- kit protein on the cell surfaces. Dibutyryl cyclic AMP (dbc-AMP) increased c-kit mRNA as well as c-kit protein on membranes and in whole cells. Run-on transcriptional assay suggested that the agent (dbc-AMP) enhanced the transcription rate of the gene. These results suggest that c-kit protein on the membranes is downregulated by PKC activation and upregulated by PKC inhibition. In the whole cell lysate, c-kit proteins are decreased by PKC inhibition through downregulation of mRNA. On the other hand, the elevation of an intracellular cAMP level causes upregulation of both the mRNA and c-kit protein on membranes and in whole cells through enhanced transcription. Thus, c-kit gene expression is apparently modulated by PKC and PKA.
High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance) [see comments]
Authors: Rosendaal FR , Koster T , Vandenbroucke JP , Reitsma PH
Published: October 2019

Resistance to activated protein C (APC) is a common inherited risk factor for venous thrombosis, which is associated with a mutation in coagulation factor V (factor V Leiden). We investigated the risk of venous thrombosis in individuals homozygous for this abnormality. We determined the factor V Leiden genotype in 471 consecutive patients aged less than 70 years with a first objectively confirmed deep-vein thrombosis and in 474 healthy controls. We found 85 heterozygous and seven homozygous individuals among the cases with thrombosis and 14 heterozygous individuals among the control subjects. The expected number of homozygous individuals among the controls was calculated from Hardy-Weinberg equilibrium and estimated at 0.107 (allele frequency, 1.5%). Whereas the relative risk was increased sevenfold for heterozygous individuals, it was increased 80-fold for homozygous individuals. These patients experienced their thrombosis at a much younger age (31 v 44 years). The homozygous individuals were predominantly women, most likely due to the effect of oral contraceptives. Because of the increased risk of thrombosis with age, the absolute risk becomes most pronounced in older patients, both for heterozygous and homozygous individuals. For the homozygous individuals, the absolute risk may become several percentage points per year. This implies that most individuals homozygous for factor V Leiden will experience at least one thrombotic event in their lifetime.
Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes
Authors: Berthou C , Legros-Maida S , Soulie A , Wargnier A , Guillet J , Rabian C , Gluckman E , Sasportes M
Published: October 2019

Perforin is the cytolytic pore-forming protein, which alone can be responsible for the lethal hit in one of the killing mechanisms used by natural killer (NK) cells or cytotoxic T lymphocytes. In this study, perforin expression was investigated in cord blood (CB) lymphocytes to determine their killing potential in vivo. The majority of CB CD3- NK cells had the protein. Compared with adult perforin-positive NK cells, a significantly lower percentage of cells expressing CD56 and CD57, the related neural cell adhesion molecules, was found (P = .0001). Perforin was also present in a unique immature CB NK-cell subset, characterized by cytoplasmic CD3 antigen (Ag) expression. In CB, very few CD8 perforin-positive T lymphocytes could be detected, but they were in significant numbers in adult peripheral blood (P = .02). A substantial proportion of these cells (70% +/- 23%) lacked the CD28 T-cell coactivation Ag, and they were able to exert NK-like, major histocompatibility complex nonrestricted cytolytic activity. CD4+ and gamma delta-T cells expressing perforin were absent from CB, but low numbers of such cells were detected in adult peripheral blood (P = .0001). Therefore, the spontaneous cytolytic activity of CB lymphocytes appeared to be dependent on well-represented perforin-positive NK cells, which were shown to efficiently lyse NK-sensitive target cells.
High-dose sequential chemoradiotherapy in multiple myeloma: residual tumor cells are detectable in bone marrow and peripheral blood cell harvests and after autografting
Authors: Corradini P , Voena C , Astolfi M , Ladetto M , Tarella C , Boccadoro M , Pileri A
Published: October 2019

Based on preliminary encouraging results in terms of response rate and survival, high-dose chemoradiotherapy has gained considerable interest in the treatment of patients with multiple myeloma (MM). We have evaluated the presence of residual myeloma cells in 15 of 18 patients enrolled in a high-dose sequential (HDS) chemoradiotherapy program followed by autografting. Our analysis has been performed both on bone marrow (BM) and peripheral blood (PB) cell harvests and after autografting. As it has been recently shown that B cells clonally related to malignant plasma cells are detectable in MM patients, we have developed a polymerase chain reaction (PCR)-based strategy to detect both residual B cells and plasma cells using clone-specific sequences derived from the rearrangement of Ig heavy chain (IgH) genes. The complementarity-determining regions (CDR) of IgH genes have been used to generate tumor-specific primers and probes. The constant (C) region usage defined the differentiation stage of residual myeloma cells. We report that plasma cells were detectable in PB and BM cell harvests and after transplantation in all assessable patients, irrespective of disease status. B cells were detectable in a consistent proportion of BM and PB samples at diagnosis, but only in one case at the time of PB and BM cell harvests. These cells became sometimes detectable after transplantation. Whether residual myeloma cells are clonogenic and contribute to relapse is currently unknown, and further investigations are required.
Overexpression of the MDM2 gene by childhood acute lymphoblastic leukemia cells expressing the wild-type p53 gene
Authors: Zhou M , Yeager AM , Smith SD , Findley HW
Published: October 2019

The wild-type (wt) p53 tumor suppressor gene is commonly inactivated in human malignancies, either by mutations or by loss of expression. An additional proposed mechanism for inactivation of wt-p53 is amplification of the murine double minute 2 (MDM2) gene and overexpression of the MDM2 protein, which binds to p53 and eliminates its tumor suppressor function. To investigate a potential role for MDM2 in the inactivation of wt-p53 in pediatric acute lymphoblastic leukemia (ALL), we examined the expression of MDM2 and p53, as well as the occurrence of p53 mutations and possible amplification of the MDM2 gene, in 19 pediatric ALL cell lines and one pediatric acute myelogenous leukemia (AML) line. Although we did not find significant amplification of the MDM2 gene in any of the leukemic lines, we detected overexpression of MDM2 in all 10 lines that expressed wt-p53. Of the 10 lines without overexpression of the MDM2 gene, six (including the AML line) did not express p53, and four expressed mutant p53 with single point mutations in exons 7 and 8. To determine whether primary leukemic cells showed a similar correlation, we analyzed the original cryopreserved leukemic bone marrow cells from seven patients from whom cell lines were established. We obtained similar results from both the primary leukemic cells and the corresponding cell lines: overexpression of MDM2 was present in primary cells that expressed wt-p53 but not in cells that lacked expression of wt-p53. These findings suggest an important role for MDM2 in the pathogenesis of pediatric ALL in which leukemic cells express wt-p53.
Isolation of a novel macrophage-specific gene by differential cDNA analysis
Authors: Spilsbury K , O'Mara MA , Wu WM , Rowe PB , Symonds G , Takayama Y
Published: October 2019

To analyze myelomonocytic differentiation we have used the approach of differential cDNA analysis to isolate novel genes that are preferentially expressed in mature macrophages. Differential screening of a macrophage cDNA library led to the identification of a novel cDNA that showed macrophage lineage- and differentiation stage-specific expression. Transcripts from the gene, which we have termed Mpg-1, are found at a high level in mature human and murine macrophages and at a moderate level in certain myelomonocytic cell lines. The expression of Mpg-1 was found to increase when murine fetal liver hematopoietic progenitor cells were induced to differentiate into macrophages. An Mpg- 1-specific transcript was not detected in a wide variety of other tissues and cell lines. The DNA sequence of Mpg-1 (4,214 bp) was obtained from a series of overlapping cDNA, 33′ rapid amplification of cDNA ends (RACE), and genomic clones. Primer extension analysis predicted the existence of multiple transcription start sites, ranging from 26 to 117 bp upstream of the 53′ proximal ATG of the open reading frame. The predicted 669-amino acid, Mpg-1-encoded protein has potential glycosylation and phosphorylation sites in addition to a signal sequence. The core protein is predicted to have a molecular weight of 71 to 74 kD. Computer-assisted local similarity searches indicate that Mpg-1 is a novel gene that may share a distant ancestry to perforin, a lytic protein found in cytotoxic T lymphocytes and natural killer cells.
Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor) [see comments]
Authors: Schmitz N , Dreger P , Suttorp M , Rohwedder EB , Haferlach T , Loffler H , Hunter A , Russell NH
Published: October 2019

Transplantation of allogeneic peripheral blood progenitor cells (PBPCs) may have advantages over bone marrow transplantation (BMT) with regards to the speed of hematopoietic and immunologic recovery, which may then shorten the time spent in hospital and decrease costs. The recipient might also profit by an enhanced graft-versus-leukemia reaction exerted by the high number of natural killer cells contained in such grafts. The donor could be spared the discomfort and risks of general anesthesia and marrow harvesting. Primary transplantation of unmanipulated allogeneic PBPCs has not been reported so far because the vast amount of T cells contained in the collection product was thought to cause severe graft-versus-host disease. We present preliminary data on primary transplantation of allogeneic PBPCs in patients who either suffered from advanced leukemia or had a donor unable to undergo general anesthesia. Eight patients with a median age of 42 years suffering from acute myelogenous leukemia (AML) in first remission (n = 3), AML in third remission, AML in relapse (n = 2), acute lymphoblastic leukemia in second remission, or chronic myelogenous leukemia in accelerated phase received myeloablative therapy followed by transplantation of unmanipulated allogeneic PBPCs mobilized with granulocyte colony-stimulating factor (5 to 10 micrograms/kg of body weight of filgrastim administered for 5 to 6 days) in their HLA- identical donors. Hematopoietic reconstitution was achieved in all patients with a median of 15.5 (16.5) days after transplant needed to surpass an absolute neutrophil count of 0.5 (1.0) x 10(9)/L. The median time to an unsupported platelet count greater than 20 (> 50) x 10(9)/L was 19.5 (41) days after grafting. Three patients did not exhibit signs of acute graft-versus-host disease (GVHD), grade I disease was seen in one patient, and three patients experienced grade II disease limited to the skin. The only patient with severe acute GVHD (grade III) refused to take his oral cyclosporin regularly and had ineffective serum levels for most of the time until relapse. Six of eight patients are currently alive without evidence of disease between 61 and 533 days after grafting; two patients grafted for AML in relapse achieved a complete remission after transplantation but relapsed again and died of leukemia on days +48 and +70, respectively. Primary transplantation of unmanipulated allogeneic PBPCs is feasible and results in long-term engraftment without causing detrimental GVHD.
Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro
Authors: Broudy VC , Lin NL , Kaushansky K
Published: October 2019

Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major regulator of platelet production in vivo. Treatment of mice with purified recombinant Tpo increases platelet count fourfold and expands colony-forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines including interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem cell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examined the effects of recombinant murine Tpo in combination with these cytokines on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% horse serum and beta-mercaptoethanol in the presence of recombinant growth factors, and CFU-Meg colonies were counted on day 5. Megakaryocyte ploidy was analyzed using murine marrow cells cultured for 5 days in IMDM supplemented with 1% nutridoma-SP and recombinant growth factors. Megakaryocytes were identified by labeling with the 4A5 antibody and ploidy was analyzed by flow cytometry. Tpo supported the growth of CFU-Meg in a dose-dependent manner. Although the addition of SCF (50 ng/mL), Epo (2 U/mL), or IL-11 (50 ng/mL) alone exerted only a modest effect on CFU-Meg growth, the combination of SCF plus Tpo, Epo plus Tpo, or IL-11 plus Tpo resulted in a synergistic enhancement of the number of CFU-Meg colonies. IL-3 alone supported CFU- Meg colony growth, and the effects of IL-3 plus Tpo or IL-6 plus Tpo on colony growth appeared to be approximately additive. Fifty percent of megakaryocytes generated in cultures containing IL-3 or Epo displayed < or = 16 N ploidy. In contrast, cultures containing Tpo uniquely generated large numbers (30% to 35% of the total) of megakaryocytes with > or = 64N ploidy. These results show that Tpo stimulates both proliferation of committed megakaryocytic progenitor cells and maturation of megakaryocytes, and that two multipotent cytokines, SCF and IL-11, as well as a late-acting erythroid cytokine, Epo, can synergize with Tpo to stimulate proliferation of CFU-Meg.
Mechanism of shape change in chilled human platelets
Authors: Winokur R , Hartwig JH
Published: October 2019

The so-called cold activation of platelets that precludes refrigeration of platelets for storage has long been recognized, but its mechanism has remained a mystery. Cooling of discoid resting platelets to temperatures below 15 degrees C causes shape distortions, and the chilled cells rewarmed to above 25 degrees C are spheres rather than discs. As platelet shape change responsive to receptor activation at normal temperatures requires the remodeling of an actin scaffolding (Hartwig JH, 1992, J Cell Biol 118:1421–1442), we examined the role of actin in the morphologic changes induced by cooling. The addition of actin monomers onto the fast-exchanging (barbed) ends of actin filaments accompanies the initial physiologic platelet shape changes, and a key control point in this growth is the removal of proteins (caps) from the filament ends. This uncapping of actin filament ends is mediated by polyphosphoinositide aggregates in vitro, suggesting that cold-induced phase changes in membrane lipids might uncap actin filaments and thereby account for actin assembly-mediated shape alterations during cooling. Consistent with this hypothesis, reversible inhibition of actin assembly with cytochalasin B prevented the distortions in shape, although cooled platelets had increased actin nucleation sites and became spherical. Another step in normal platelet shape changes requires the severing of actin filaments that maintain the resting platelet. The proteins that sever initially bind to the broken filament ends, and uncapping of these fragmented filaments provides numerous nucleation sites for growth of actin filaments to fill in spreading filopodia and lamellae. Actin filament fragmentation requires a rise in intracellular calcium, and we showed that chilling platelets from 37 degrees C to 4 degrees C increases free cytosolic calcium levels from 80 nmol/L to approximately 200 nmol/L in minutes, thus providing an explanation for the spherical shape of cooled, rewarmed platelets. Blocking the calcium transient with nanomolar concentrations of the permeant calcium chelators Quin-2 and Fura-2 prevented the increase in nucleation sites and the sphering, but not the other shape changes of chilled and rewarmed platelets. However, a combination of micromolar cytochalasin B and millimolar intracellular calcium chelators preserved the discoid shapes of chilled and rewarmed platelets. After removal of cytochalasin B and addition of sufficient extracellular calcium, these platelets responded with normal morphologic alterations to glass and thrombin activation.
Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11;19)(q23;p13.1) translocation
Authors: Mitani K , Kanda Y , Ogawa S , Tanaka T , Inazawa J , Yazaki Y , Hirai H
Published: October 2019

The t(11;19)(q23;p13.1) translocation is thought to play an important role in pathogenesis of myeloid leukemias in older patients. The MLL gene involved in other 11q23 abnormalities was also rearranged by this translocation. Screening of cDNA libraries of the t(11;19)(q23;p13.1)-carrying leukemic cells resulted in the isolation of several species of fusion cDNAs between the MLL gene and an unknown gene on 19p13.1, named MEN (myeloid eleven-nineteen translocation), which is ubiquitously expressed. Although the MLL gene was alternatively spliced, the fusion protein should contain an N-terminal half of the MLL, including AT hook motifs, that is fused to the MEN protein with a lysine-rich sequence, suggesting that the MLL/MEN fusion protein could be a chimeric transcription factor. The MLL/MEN fusion transcripts of 8.0 kb were detected in leukemic cells of two cases with the translocation. The MLL/MEN fusion was consistent in all three cases of the t(11;19)(q23;p13.1)-carrying leukemia examined by RNA-based polymerase chain reaction. These findings strongly suggest that the t(11;19)(q23;p13.1) results in the fusion formation encoding a new class of potential chimeric transcription factor that contributes to leukemogenesis of myeloid lineage.
Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis
Authors: Traycoff CM , Kosak ST , Grigsby S , Srour EF
Published: October 2019

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
The Apple 1 and Apple 4 domains of factor XI act synergistically to promote the surface-mediated activation of factor XI by factor XIIa
Authors: Baglia FA , Seaman FS , Walsh PN
Published: October 2019

Binding sites for high molecular weight kininogen (HK) and for factor XIIa are present in the Apple 1 (A1) and the A4 domains of factor XI, respectively. To define the roles of these two sites in surface-mediated factor-XI activation we prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) and determined their effects on the activation of factor XI by factor XIIa in the presence of HK and either kaolin or dextran sulfate. Surface-mediated factor-XI activation by factor XIIa was inhibited by a conformationally constrained A4 peptide (Ala317-Gly350), by an A1 peptide (Phe56-Ser86), and by rA1 (Glu1-Ser90). When used in combination at equimolar concentrations, rA1 and A4 peptide were 10-fold more effective than either one alone in inhibiting surface-mediated activation of factor XI by factor XIIa. The A4 peptide was a competitive inhibitor of factor XIIa amidolytic activity and a noncompetitive inhibitor of factor-XI activation by factor XIIa, whereas rA1 and the A1 peptide did not inhibit factor XIIa. The rA1 domain inhibited factor XI binding to HK, whereas the A4 peptide did not. We conclude that specific sequences exposed on the surfaces of the A1 (Val59-Lys83) and A4 (Ala317-Gly350) domains of factor XI act synergistically to promote surface-mediated factor-XI activation by factor XIIa in the presence of HK by binding factor XI to surface-bound HK (A1 domain) and by binding factor XIIa near the cleavage site (Arg369-Ile370) of factor XI (A4 domain).
Differential effects of ifosfamide on the capacity of cytotoxic T lymphocytes and natural killer cells to lyse their target cells correlate with intracellular glutathione levels
Authors: Multhoff G , Meier T , Botzler C , Wiesnet M , Allenbacher A , Wilmanns W , Issels RD
Published: October 2019

We established an in vitro model to study the influence of ifosfamide treatment on intracellular glutathione (GSH) levels in activated human effector cells with specific phenotypes and immunologic functions. Besides its role as the major intracellular reductant, GSH has been shown to affect the initiation and progression of lymphocyte activation after stimulation with lectins. An incubation of activated human peripheral blood lymphocytes (PBL) with 4-hydroxyifosfamide, the activated form of ifosfamide (4-OH-IF), resulted in a depletion of the intracellular GSH levels and a significant inhibition of the proliferative capacity in a dose-dependent manner. The cytotoxic activity of separated CD3-natural killer (NK) cells and CD3+ allospecific, cytotoxic T lymphocytes (CTL), either untreated or treated with 4-OH-IF at different concentrations, was compared in a standard 51chromium release assay (CML). There were three major findings. (1) The capacity of CD3+ major histocompatibility complex (MHC)-restricted CTL to lyse their specific allogeneic target cells was substantially reduced by preincubation of the effector cells with 4-OH-IF. This inhibition of the lytic activity in CD3+ CTL correlated with a substantial depletion of the intracellular GSH levels in this population. Rapid reconstitution of depleted GSH levels and restoration of cytotoxic activity of CTL was achieved by incubation of the effector cells with thiols, eg, glutathione ester (GSH-ester) or 2-mercaptoethanesulfonate (mesna). (2) In contrast, the lytic activity in CD3-NK cells was not substantially affected (up to 100 mumol/L 4-OH-IF). This result correlates with the capacity of NK cells to maintain their intracellular GSH levels after an ifosfamide treatment. (3) In comparison with CD3+ CTL, CD3-NK cells are more resistant to an ifosfamide treatment because they have higher initial GSH levels and a more than fourfold higher relative rate of GSH synthesis.
Predictive value for treatment outcome in acute myeloid leukemia of cellular daunorubicin accumulation and P-glycoprotein expression simultaneously determined by flow cytometry
Authors: Guerci A , Merlin JL , Missoum N , Feldmann L , Marchal S , Witz F , Rose C , Guerci O
Published: October 2019

To evaluate the clinical relevance of multidrug resistance (MDR) phenotype, the intracellular daunorubicin accumulation (IDA) and P-glycoprotein (P-gp) expression were investigated in 87 adult patients with acute leukemia: 69 patients with de novo acute myeloid leukemia (AML), 10 with AML at relapse, and eight with secondary leukemia to myelodysplastic syndromes (MDS-AML). IDA and P-gp expression were determined by double-labeling flow cytometry analysis. Of 87 patients, 36 expressed P-gp (41%). P-gp expression was more frequently observed in AML at relapse and MDS-AML as compared with de novo AML (P = .0001). P-gp expression was significantly associated with CD34 expression (P = .0003) and chromosome 7 abnormalities (P = .027). A significantly reduced IDA was observed in P-gp+ as compared with P-gp- patients (P = .0007). Of the 87 patients, 51 achieved complete remission (CR). A reduced IDA was observed in patients in failure as compared with patients in CR (22% +/- 17% v 42% +/- 21%; P = 10(-4). Twelve of 36 P-gp+ patients as compared with 40 of 51 P-gp- patients achieved CR (33% v 78%; P = 10(-4). The prognostic value of IDA and P-gp expression was confirmed in multivariate analysis. These data suggest that the determination of IDA and P-gp expression may be useful in designing therapy for patients with AML.
Highly purified primitive hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia
Authors: Turhan AG , Lemoine FM , Debert C , Bonnet ML , Baillou C , Picard F , Macintyre EA , Varet B
Published: October 2019

The hierarchical level of stem cell involvement in acute promyelocytic leukemia (APL) characterized by the pathognomonic PML-RARA fusion gene is unknown. To determine if the cells of the primitive hematopoietic stem cell compartment are involved in the leukemic process, we have used molecular and cell sorting techniques in peripheral blood and bone marrow (BM) cells at diagnosis from three patients with APL and t(15; 17). In two of them, clonality analysis was also possible using the BstXI polymorphic site of the PGK gene. The PML-RARA fusion gene was readily identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of BM cells obtained at diagnosis in all three patients. These same samples were then used to sort CD34+ cells and their CD38+ and CD38-subsets by fluorescence-activated cell sorting. In both female patients, CD34+/CD38+ and CD34+/CD38- cell fractions were polyclonal using PCR, whereas a monoclonal pattern was identified at the BM sample obtained at diagnosis either by Southern blotting or by PCR. Because of the high sensitivity of the PCR analysis, the polyclonal pattern of these cell populations could mask the presence of a minor clone. To detect this clone, we preformed RT-PCR analysis for t(15; 17). In one female patient, the abnormal PML-RAR fusion gene was found only in the more mature CD34+/CD38+ cell fraction using a nested PCR approach, whereas the polyclonal CD34+/CD38- fraction was PML-RARA negative. These findings were confirmed in a third patient with APL in whom the PML-RARA transcripts were absent in the CD34+/CD38- cell fraction. To study the clonality at the level of clonogenic progenitors, we used in one patient PGK analysis by PCR of individual burst-forming units-erythroid and colony-forming units-granulocyte- macrophage obtained from the CD34+/CD38- and CD34+/CD38+ cell populations at diagnosis and from the BM sample obtained during remission. The two highly purified cell populations gave rise to morphologically normal colonies clonal for both the BstXI site containing (A) and the BstXI site lacking (B) PGK allelles, indicating their polyclonal content, a pattern that was also found in clonogenic progenitors obtained at remission. These findings strongly suggest that the primitive hematopoietic stem cells as defined by the CD34+/CD38- antigens are not involved by the neoplastic process in APL. These results may have important implications for autografting strategies of retinoic acid/chemotherapy-resistant or relapsed patients.
Comparative evaluation of diepoxybutane sensitivity and cell cycle blockage in the diagnosis of Fanconi anemia
Authors: Seyschab H , Friedl R , Sun Y , Schindler D , Hoehn H , Hentze S , Schroeder-Kurth T
Published: October 2019

Fanconi anemia (FA) is a clinically and genetically heterogenous disease that is usually diagnosed on the basis of chromosomal instability reflecting the hypersensitivity towards the DNA cross-linking agents diepoxybutane (DEB) and/or mitomycin C. A less well-known cellular feature that characterizes FA patients is an intrinsic cell cycle disturbance consisting of prolonged progression through, and arrest within, the G2 phase compartment of the cell cycle. In a collaborative blind study, we have evaluated 72-hour lymphocyte cultures from 66 patients with clinical suspicion of FA both for DEB sensitivity and cell cycle disturbance. A concordant result was obtained in 63 of 66 cases. Each of the 3 discordant, but only 1 of the concordant cases presented with overt leukemia. Seventeen cases were identified as classical FA because of their increased DEB sensitivity and G2 phase blockage. Five cases showed a cell cycle disturbance but only borderline DEB sensitivity. These cases might represent atypical or nonclassical forms of FA. They would have been missed by cell cycle studies without concomitant DEB testing. Used in conjunction, cytogenetic and flow cytometric testing provide for the currently optimal diagnosis of FA in nonleukemic patients.
Interleukin-3 (IL-3) poor-responsive inbred mouse strains carry the identical deletion of a branch point in the IL-3 receptor alpha subunit gene
Authors: Hara T , Ichihara M , Takagi M , Miyajima A
Published: October 2019

Interleukin-3 (IL-3) stimulates colony formation of multiple lineages of hematopoietic cells. Bone marrow cells of A/J mice are nonresponsive to IL-3, and this observation has recently been correlated with aberrant mRNA splicing and impaired expression of the IL-3 receptor alpha subunit (IL-3R alpha), a binding component of the high-affinity receptors. We examined the IL-3R alpha gene in 27 inbred mouse strains and found the identical mutation, a 5-bp deletion at the branch point of intron 7, in 10 of these mouse strains. Bone marrow cells isolated from these 10 mouse strains did not express IL-3R alpha on the cell surface and did not form colonies in response to IL-3. Because the defective IL-3R alpha gene was found in several distantly related mouse strains, it appears to be a recessive allele rather than a sporadic mutation. In contrast, only 1 of 21 wild-derived mouse strains carried the 5-bp deletion in the IL-3R alpha gene. This study suggests that IL-3 function is not required for normal hematopoiesis in mice, but the retention of the IL-3 and IL-3R system may be of some selective advantage in wild populations.
Analysis of hematopoiesis in max 41 transgenic mice that exhibit sustained elevations of blood granulocytes and monocytes
Authors: Metcalf D , Lindeman GJ , Nicola NA
Published: October 2019

An unusual mouse line (max 41) that carries an inserted transgene encoding the nuclear transcription factor, Max, exhibits a 50- to 60-fold elevation of blood neutrophils and a 10-fold elevation of blood monocytes. Analysis showed that these elevated levels of blood cells were sustained incrementally by a sevenfold increase in mature neutrophils in the bone marrow and spleen and a fourfold increase in granulocyte-committed progenitor cells with normal turnover times for mature neutrophils and monocytes in the marrow and blood. the progenitor cells were not autonomous and exhibited a normal quantitative responsiveness to stimulation in vitro by the four colony-stimulating factors. A consistent anomaly noted was that, when stimulated by macrophage colony-stimulating factor, max 41 progenitor cells formed mainly granulocyte-containing colonies, rather than the usual macrophage colonies. The blast colony-forming cells from max 41 mice did not generate abnormal numbers or types of progenitor cells. The transgenic max 41 model requires further analysis to establish the regulatory anomaly responsible for the excessive production of granulocytes and monocytes, but has emphasized that most neutrophils generated in the marrow normally never leave the organ.
Recombinant human interleukin-1 receptor antagonist protects early myeloid progenitors in a murine model of cyclophosphamide-induced myelotoxicity
Authors: Chudgar UH , Rundus CH , Peterson VM
Published: October 2019

Expression of hematoregulatory cytokines such as interleukin-1 (IL-1) in response to cytotoxic chemotherapy hastens hematopoietic recovery, but may also potentiate myelotoxicity if myeloid progenitors enter cell cycle before drug clearance. In the present study, the ability of recombinant human IL-1 receptor antagonist (IL-1ra) to protect hematopoietic progenitors was studied in a murine model of cyclophosphamide (CPA)-induced myelotoxicity. CF-1 female mice received 200 mg/kg CPA and either 10 mg/kg IL-1ra or an equal volume of 0.05% human serum albumin (HSA) intraperitoneally (i.p.), followed 12 hours later by IL-1ra or HSA. CPA and IL-1ra increased absolute neutrophil counts (ANCs) at days 2 (P = .001) and 14 (P = .0025) after CPA. In IL-1ra-treated mice, colony-forming units granulocyte-macrophage (CFU-GM)/tibia were increased twofold and threefold at days 2 (P = .0047) and 7 (P = .023), respectively, whereas high proliferative potential colony-forming cells (HPP-CFC)/tibia were decreased twofold to threefold at 8 hours (P = .039) and 24 hours (P = .0033), but were approximately threefold higher than HSA-treated mice at day 7 after CPA. Coadministration of CPA and IL-1 enhanced myelotoxicity compared with mice injected with CPA and IL-1ra or HSA. In vivo, IL-1ra protected HPP-CFC, but not CFU-GM, from hydroxyurea suicide after a single dose of CPA, suggesting that IL-1ra inhibited cycling of HPP-CFC. In vitro, IL-1ra did not alter proliferation of CFU-GM, but inhibited IL-1-enhanced proliferation of HPP-CFC. These data suggest that IL-1ra acts as an indirect negative regulator of hematopoiesis and protects HPP-CFC from CPA, possibly by inhibiting IL-1-enhanced proliferation of early myeloid progenitors.
Nonrandom inactivation of the X chromosome in early lineage hematopoietic cells in carriers of Wiskott-Aldrich syndrome
Authors: Wengler G , Gorlin JB , Williamson JM , Rosen FS , Bing DH
Published: October 2019

The Wiskott-Aldrich syndrome (WAS) is an X-linked (Xp11.22) recessive immunodeficiency syndrome characterized by susceptibility to opportunistic and pyogenic infections, thrombocytopenia, and eczema. Previous studies of obligate carriers of WAS documented that nonrandom inactivation of the X chromosome carrying the defective gene is observed in all peripheral blood cells. The existence of both abnormal platelets and lymphocytes is consistent with a defect that affects early hematopoietic precursors. We isolated CD34+ hematopoietic progenitor cells collected from obligate carriers of WAS by apheresis and used polymerase chain reaction analysis of a polymorphic variable number of repeats (VNTR) within the X-linked androgen receptor to document nonrandom inactivation. These data show that nonrandom inactivation of the X-chromosome in WAS-obligate carriers occurs early during hematopoietic differentiation.
Role of B7-1 in mediating an immune response to myeloid leukemia cells
Authors: Matulonis UA , Dosiou C , Lamont C , Freeman GJ , Mauch P , Nadler LM , Griffin JD
Published: October 2019

A costimulatory signal from B7–1 (CD80) to its counter-receptor CD28 is required for T-cell activation. Many tumors, including most human leukemias, lack expression of B7–1, and this has been suggested to contribute to the failure of immune recognition of these diseases. A murine leukemia model system was developed to assess the potential role of B7–1 in the induction immunity to leukemia cells. The nonleukemic 32Dc13 myeloid cell line was transformed by transfection of the BCR/ABL gene, generating a subline (32Dp210/clone 26) that was leukemic and rapidly lethal to syngeneic, immunocompetent C3H/HeJ mice or T-cell-deficient nude mice. B7–1-modified leukemic cells remained lethal in nude mice, but caused only a transient, nonlethal leukemia in C3H/HeJ mice. After a single exposure to live, nonirradiated B7–1-modified leukemic cells, C3H/HeJ mice developed protective immunity against subsequent challenge with B7–1(-) leukemic cells. Further, hyperimmunization with B7–1(+) leukemic cells prolonged the survival of mice previously injected with a lethal number of B7–1(-) leukemic cells. These results indicate that myeloid leukemic cells may be attractive candidates for B7–1 gene transfer.
Identification of a major positive regulatory element located 5' to the human zeta-globin gene
Authors: Sabath DE , Koehler KM , Yang WQ , Patton K , Stamatoyannopoulos G
Published: October 2019

The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5′ to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5′ to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.
Interleukin-11 stimulates multilineage progenitors, but not stem cells, in murine and human long-term marrow cultures
Authors: Du XX , Scott D , Yang ZX , Cooper R , Xiao XL , Williams DA
Published: October 2019

Interleukin-11 (IL-11) is a bone marrow microenvironment-derived growth factor with pleiotropic effects on a variety of hematopoietic cells. To more accurately assess the effects of IL-11 on stem and progenitor compartments within the hematopoietic microenvironment (HM), we added recombinant human (rh) IL-11 to human and murine long-term bone marrow cultures (LTMC) and analyzed primitive (high proliferative potential- colony forming cells [HPP-CFC], long-term culture-initiating cells [LTC- IC], and long-term reconstituting stem cells) and progenitor (day 12 colony forming unit-spleen [CFU-S12], colony forming unit-megakaryocyte [CFU-Mk] and colony forming unit-granulocyte/macrophage [CFU-GM]) compartments throughout the duration of the cultures. rhIL-11 (100 ng/mL) added twice weekly resulted in significantly increased nonadherent (NA) cellularity, CFU-GM, and CFU-Mk production in human LTMC. Addition of rhIL-11 to murine LTMC was associated with a 5- to 40- fold increase in CFU-GM and a four- to 20-fold increase in day 12 CFU-S in NA cells. However, IL-11 had no significant effect on total HPP-CFC concentration and decreased the size of the more primitive stem/progenitor compartment as evidenced by both decreased LTC-IC frequency in human LTMC and decreased frequency of long-term reconstituting stem cells in murine LTMC. These data suggest that IL-11 may increase commitment of stem cells into a multipotential progenitor compartment.
Retinoic acid reduces induction of monocyte tissue factor and tissue factor/factor VIIa-dependent arterial thrombus formation
Authors: Barstad RM , Hamers MJ , Stephens RW , Sakariassen KS
Published: October 2019

Agents that downregulate the induction of monocyte/macrophage tissue factor (TF) activity may attenuate the thrombotic risk associated with mechanical restoration of vessel patency or artificial arterial grafting. In such events, procoagulant macrophages in the atherosclerotic plaque and procoagulant monocytes adherent to artificial materials may be exposed to the blood stream. Ishii et al (Blood 80:2556, 1992) reported that induction of endothelial TF is downregulated by all-trans retinoic acid (ATRA), and Conese et al (Thromb Haemost 66:662, 1991) reported that retinoids downregulate monocyte procoagulant activity (PCA). These findings led us to investigate the effect of ATRA on monocyte TF expression, and to study the effect of ATRA on monocyte-induced thrombus formation in a model system of human arterial thrombogenesis. Induction of PCA in human peripheral blood monocytes by 0.5 microgram/mL lipopolysaccharide (LPS) was dose dependently reduced by ATRA, reaching a reduction of 56% at 10(-5) mol/L ATRA (P < .0001). A 38% reduction (P < .0007) in LPS- induced TF antigen expression was observed at an ATRA concentration of 10(-6) mol/L. Adherence of monocytes to plastic cover slips (Thermanox, Miles Laboratories, Naperville, IL) also triggered induction of cellular PCA, which was inhibited by more than 80% by an anti-TF monoclonal antibody (MoAb) (P < .002). Inclusion of ATRA (10(-6) mol/L) reduced this PCA by 40% (P < .03), and the TF antigen expression by 30% (P < .0001). Exposure of Thermanox adherent monocytes to flowing nonanticoagulated human blood in a parallel-plate perfusion chamber device at an arterial wall shear rate of 650 s-1 elicited significant fibrin deposition and platelet thrombus formation. Partial interruption of this thrombus formation was achieved by 10(-6) mol/L ATRA, which reduced the fibrin deposition by 80% (P < .02) and platelet thrombus formation by 50% (P < .05). In comparison, incubation of adherent monocytes with the anti-TF MoAb before the blood exposure, reduced the fibrin deposition by 83% (P < .02) and platelet thrombus volume by 75% (P < .0008). Thus, ATRA is an effective down-regulator of monocyte TF- PCA, and may reduce thrombotic complications at sites of plaque rupture, at plaque disruption after percutaneous transluminal angioplasty procedures, or on surfaces introduced by artificial arterial grafting.