bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil- treated murine marrow
Authors: Szilvassy SJ , Cory S
Published: October 2019

Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the “lineage-specific” markers CD45R (B220), Gr- 1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with “compromised” marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.
Echicetin: a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib
Authors: Peng M , Lu W , Beviglia L , Niewiarowski S , Kirby EP
Published: October 2019

Echicetin, a new protein isolated from Echis carinatus venom by reverse phase and ion exchange chromatography specifically inhibited agglutination of fixed platelets induced by several platelet glycoprotein Ib (GPIb) agonists, such as bovine von Willebrand factor (vWF), alboaggregins, and human vWF in the presence of botrocetin. Unlike alboaggregins, echicetin bound to GPIb but did not induce agglutination of washed or fixed platelets. In contrast to disintegrins, it did not block adenosine 5'-diphosphate (ADP)-induced platelet aggregation in the presence of fibrinogen. The apparent molecular weight of echicetin measured on sodium dodecyl sulfate (SDS) gel electrophoresis was 26 Kd under nonreducing conditions. On reduction, echicetin showed 16 and 14-Kd subunits suggesting that the molecule is a dimer. Reduced echicetin retained its binding activity and its inhibitory effect on the agglutination of fixed platelets induced by bovine vWF. 125I-echicetin bound to fixed platelets with high affinity (kd = 30 +/- 1.8 nmol/L) at 45,000 +/- 2,400 binding sites per platelet. The binding was selectively inhibited by a monoclonal antibody to the 45-Kd N-terminal domain of platelet GPIb, but not by monoclonal antibodies to other regions on GPIb. Binding of 125I-bovine vWF to fixed platelets was strongly inhibited by echicetin. In contrast, bovine vWF showed a much weaker inhibitory activity on binding of 125I-echicetin to platelets. The half life of echicetin in blood was approximately 170 minutes with no detectable degradation. Echicetin significantly prolonged the bleeding time of mice, suggesting that it may inhibit vWF binding to GPIb in vivo as well as in vitro.
Retrovirus-mediated transfer and expression of an allogeneic major histocompatibility complex class II DRB cDNA in swine bone marrow cultures
Authors: Emery DW , Shafer GE , Karson EM , Sachs DH , LeGuern C
Published: October 2019

The possibility of inducing transplantation tolerance by somatic gene transfer is under investigation in our miniature swine model. As a crucial step in this project, we have used a retroviral vector engineered to express both a drug-resistance gene (Neo) and a swine class II DRB cDNA to transduce porcine bone marrow (BM) cells. Analysis of cultured swine fibroblasts exposed to high-titer viral supernatants demonstrated that drug resistance had been conferred and that transferred vector sequences were transcribed appropriately. Similar transduction studies with swine BM demonstrated the transfer of drug resistance to as high as 14% of colony-forming unit-granulocyte- macrophage (CFU-GM). Using polymerase chain reaction (PCR) of cDNA, vector-derived allogeneic DRB transcripts were detected in colonies derived from primitive CFU-Mix and high proliferative potential-colony- forming cell (HPP-CFC), as well as in drug-resistant GM colonies grown from transduced bone marrow (BM) maintained in long-term BM cultures (LTBMCs) for up to 5 weeks. These results indicate that a significant proportion of both colony-forming progenitors and LTBMC-initiating cells were transduced with the DRB-recombinant retroviral vector and that both vector-derived genes were expressed in the differentiated progeny of these cells.
Cloning of the complete Ly-6E.1 gene and identification of DNase I hypersensitive sites corresponding to expression in hematopoietic cells
Authors: Sinclair AM , Dzierzak EA
Published: October 2019

The Sca-1 antibody recognizes antigens encoded by members of the Ly-6 multigene family. These antigens are expressed on fetal and adult hematopoietic stem cells, progenitor cells, mature activated T cells, and some nonhematopoietic cells and are most likely encoded by the Ly- 6E.1 and Ly-6A.2 genes. Characterization and isolation of regulatory elements of Ly-6E.1 and A.2 genes that govern tissue-specific and high levels of expression in the cells of the hematopoietic system (particularly stem cells) are of considerable interest. To characterize the control elements of this gene, we have cloned a 30-kb fragment encoding a fully functional Ly-6E.1 gene and 13 kb of 5′ and 13 kb of 3′ flanking sequence. Transfection studies in murine erythroleukemia (MEL) cells show that a 14-kb BamHI fragment from this clone is sufficient to confer Ly-6E.1 gene expression at levels equivalent to those of the endogenous gene. By mapping regions of chromatin sensitive to DNase I digestion, we have located hypersensitive sites in the 5′ and 3′ regions of the gene in FDCP-1 cells, MEL cells, and various T- cell lines. The appearance of two 5′ hypersensitive sites in hematopoietic cells correlates with Ly-6E.1 expression after gamma- interferon induction. We show that the presence of hypersensitive sites in the 5′ and 3′ regions corresponds to Sca-1 expression, and we also discuss the localization of putative regulatory control elements.
Common clonal origin of chronic lymphocytic leukemia and high-grade lymphoma of Richter's syndrome
Authors: Cherepakhin V , Baird SM , Meisenholder GW , Kipps TJ
Published: October 2019

Patients with B-cell chronic lymphocytic leukemia (CLL) infrequently may develop high-grade B-cell lymphoma, or Richter's syndrome lymphoma (RS lymphoma). Such lymphomas differ from the original leukemia in both histology and clinical behavior. Studies seeking to define the clonal relationship between the cells of the two malignancies in any one patient have yielded conflicting reports. We examined the clonal relationship between the early and late neoplastic cells of a patient who underwent Richter's transformation. In contrast to the original leukemia cells, the secondary high-grade lymphoma was CD5-. However, both the leukemia cells and the evolved RS lymphoma expressed surface IgM lambda reactive with Lc1, a murine monoclonal antibody specific for a supratypic cross-reactive idiotype encoded by a subset of human Ig variable region genes of the VH4 subgroup. Nucleic acid sequence analyses of the heavy and light chain variable region genes expressed by both leukemia and lymphoma cells show that the CD5- B-cell lymphoma constitutes a clonal expansion of mutant cells derived from the original CD5+ B-cell leukemia. Moreover, certain sets of somatic mutations distinguish the Ig variable region genes used by RS lymphoma from those expressed by the CLL B cells. This is the first study to establish the clonal relationship between CLL and RS lymphoma through primary structural analyses of the expressed Ig genes.
The novel subset of CD14+/CD16+ blood monocytes is expanded in sepsis patients
Authors: Fingerle G , Pforte A , Passlick B , Blumenstein M , Strobel M , Ziegler- Heitbrock HW
Published: October 2019

Staining with CD14 and CD16 monoclonal antibodies will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the CD14 antigen (CD14++), and a minor population with weak expression of CD14 and expression of the CD16 antigen (CD14+/CD16+ cells). As shown herein, the latter cells account for 45 +/- 22 cells/microL and 9% +/- 5% of the monocytes in healthy control donors (n = 35). In septicemia patients, the CD14+/CD16+ cells can become a major population, with more than 50% of all monocytes in 3 of 18 patients and with more than 500 cells in 4 of 18 cases. There was no correlation of CD14+/CD16+ cells to any clinical parameter except for CD14+/CD16+ percentage and body temperature (P = .013). The CD14++ regular monocytes showed a substantial decrease in CD14 antigen density in 9 of 11 patients. Three-color immunofluorescence shows that the CD14+/CD16+ monocytes in septicemia patients when compared with the CD14++ monocytes exhibit a higher level of class II antigen and a lower level of CD11b and CD33 antigens, consistent with a more mature nature of the CD14+/CD16+ cells. Levels of interleukin-6 (IL-6) were increased in septicemia patients; 3 of 5 patients with high numbers of CD14+/CD16+ cells (> 200/microL) had high levels of IL-6 (> 250/U/mL). These data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as IL-6.
G6PD Nara: a new class 1 glucose-6-phosphate dehydrogenase variant with an eight amino acid deletion
Authors: Hirono A , Fujii H , Shima M , Miwa S
Published: October 2019

In the course of molecular studies on Japanese glucose-6-phosphate dehydrogenase (G6PD) variants using single-strand conformation polymorphisms (SSCP) analysis, we found an unusual class 1 G6PD variant that had nucleotide deletion in exon 9. The patient showed chronic nonspherocytic hemolytic anemia associated with frequent episodes of severe hemolytic attack. The hemolysate exhibited no measurable activity. Although the partially purified enzyme had detectable activity, we could not perform kinetic studies because of its extreme instability. Nucleotide sequencing showed a unique 24 bp deletion at nucleotide 953–976 that predicts an eight amino acid deletion of TKGYLDDP at residue 319–326. While this is one of the most drastic structural alterations found in G6PD variants, the region with the amino acid deletion was distant from both the G6P and NADP+ binding sites and was located in a domain with low sequence homology among species. The comparatively low functional importance of the deleted region may have saved the patient from lethal tissue dysfunction.
Direct and reversible inhibitory effect of the tetrapeptide acetyl-N- Ser-Asp-Lys-Pro (Seraspenide) on the growth of human CD34+ subpopulations in response to growth factors
Authors: Bonnet D , Lemoine FM , Pontvert-Delucq S , Baillou C , Najman A , Guigon M
Published: October 2019

The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP, Seraspenide; Ipsen- Biotech, Paris, France), an inhibitor of murine spleen colony-forming units reduces the number and the percentage in DNA synthesis of progenitors from human unfractionated bone marrow. To determine whether AcSDKP may directly affect the growth potential of purified progenitors even at the most primitive level, CD34+HLA-DRhigh and CD34++HLA-DRlow cells were highly purified by cell sorting. Then, CD34+ subsets were stimulated in liquid culture with combinations of growth factors (GFs) and AcSDKP was added for 20 hours or 6 days and cells plated in methylcellulose. After a 20-hour incubation, we show that AcSDKP (at 10(-10) mol/L) significantly inhibits the colony formation of both CD34+ subsets. Moreover, when added daily for 6 days, AcSDKP: (1) reduces the proliferation of both CD34+ cell fractions stimulated by 3 or 7 GFs, and (2) decreases the number of progenitors generated from the CD34+HLA-DRhigh and CD34++HLA-DRlow cell fractions. Furthermore, we show for the first time, using both high proliferative potential cell and long-term culture initiating cell assays, that AcSDKP inhibits the most primitive cells contained in the CD34++HLA-DRlow subpopulation. Finally, by using limiting dilution assays we demonstrated that AcSDKP acts directly at a single cell level and that its inhibitory effect is reversible and dose dependent.
Comparison of the human and mouse erythropoietin genes shows extensive homology in the flanking regions
Authors: Galson DL , Tan CC , Ratcliffe PJ , Bunn HF
Published: October 2019

Considerable insights into important cis regulatory elements in a gene can be gleaned from the identification of sequence homologies among different species. To extend and optimize the sequence comparison between human and mouse erythropoietin (Epo) genes, we have obtained new human sequence from 5,547 to 385 bp upstream of the cap site and extended the 3′ flank by 489 bp. In addition, we have obtained new sequence information on the mouse Epo gene extending from within the 3′ untranslated region (UTR) to 1,001 bp downstream of the polyadenylation site. Analysis of these additional sequences shows considerable homology between human and mouse Epo genes as far as 4 kb (human) or 3 kb (mouse) upstream of the cap sites, as well as far more homology at the 3′ end than was previously realized. In addition, both species were found to have a high frequency of short interspersed (SINE) repetitive sequences that interrupt homologies in both the 5′ flank and within the transcription unit.
Cross-linking of the beta-glucan receptor on human monocytes results in interleukin-1 receptor antagonist but not interleukin-1 production
Authors: Poutsiaka DD , Mengozzi M , Vannier E , Sinha B , Dinarello CA
Published: October 2019

The beta-glucan receptor, found on monocytes and neutrophils, binds glucose polymers derived from fungi. Ligands for the receptor have various immunomodulatory effects, including increased microbicidal killing activity. We have investigated the effect of beta-glucans on the production of interleukin-1 (IL-1) and its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1Ra). Particulate beta- glucan induced IL-1Ra production from human peripheral blood mononuclear cells (PBMC) but did not stimulate IL-1 beta synthesis or gene expression in these same cells. Monomeric (soluble) beta-glucan did not induce IL-1Ra production. However, when preincubated with PBMC, monomeric beta-glucan significantly (P <.01) reduced particulate beta- glucan induction of IL-1Ra by 40%, suggesting that crosslinking of beta- glucan receptors is required for induction of IL-1Ra. In support of this, monomeric beta-glucan immobilized on plastic surfaces stimulated IL-1Ra production. Vitamin D3, which increases the functional capacity of beta-glucan receptors, increased IL-1Ra production induced by particulate beta-glucan, whereas dexamethasone suppressed IL-1Ra synthesis. Because of their differential effects on cytokine production, beta-glucans may be used to therapeutic advantage in the diseases in which IL-1 is implicated.
Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease
Authors: Kenney RT , Malech HL , Epstein ND , Roberts RL , Leto TL
Published: October 2019

The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.
Randomized comparison of busulfan and hydroxyurea in chronic myelogenous leukemia: prolongation of survival by hydroxyurea. The German CML Study Group
Authors: Hehlmann R , Heimpel H , Hasford J , Kolb HJ , Pralle H , Hossfeld DK , Queisser W , Loffler H , Heinze B , Georgii A
Published: October 2019

In a randomized multicenter study the influence of hydroxyurea versus busulfan on the duration of the chronic phase and on survival of chronic myelogenous leukemia (CML) was determined. In addition cross resistance and adverse reactions of the drugs were analyzed. From July 1983 to January 1991, 441 CML patients were randomized to receive hydroxyurea or busulfan. Of these, 90.7% were Philadelphia positive; 25.7% were low, 38.2% intermediate, and 36.2% high risk patients according to Sokal's score. The median survival of the busulfan treated Philadelphia-positive patients is 45.4 months and of the hydroxyurea group 58.2 months (P = .008). The survival advantage for the hydroxyurea treated patients is recognized in all risk groups. Sixty four patients reached therapy resistance before blast crisis and were crossed over to the alternative drug. The 23 patients with primary hydroxyurea had a median survival of 5.6 years, the 41 patients with primary busulfan therapy a median survival of 2.7 years (P = .02). Adverse reactions were less frequent with hydroxyurea with no severe adverse effects (lung fibrosis, long lasting bone marrow aplasia). The analysis of white blood cell counts in the course of treatment showed lower counts in the hydroxyurea patients. We conclude that hydroxyurea is superior to busulfan in therapy of CML in chronic phase and should be used as first line therapy. Busulfan may have a role as secondary therapy after hydroxyurea resistance or intolerance.
ALL-1 gene at chromosome 11q23 is consistently altered in acute leukemia of early infancy
Authors: Cimino G , Lo Coco F , Biondi A , Elia L , Luciano A , Croce CM , Masera G , Mandelli F , Canaani E
Published: October 2019

Early infancy (< 1 year of age), massive tumor cell burden, and extremely poor prognosis are characteristic features of a particular subset of childhood acute leukemias (AL). In these cases, chromosome aberrations at the 11q23 band are the most frequently reported cytogenetic abnormalities. We have recently cloned a genetic locus named ALL-1, in which DNA breakpoints are clustered in leukemic patients with 11q23 aberrations. Analysis of the ALL-1 genomic configuration in DNA from 15 infants with AL showed specific ALL-1 rearrangements in 12 cases (80%), including 5 with normal karyotypes. These findings indicate that a consistent genetic defect underlies this particular leukemic subset.
Priming of polymorphonuclear neutrophils by tumor necrosis factor alpha in whole blood: identification of two polymorphonuclear neutrophil subpopulations in response to formyl-peptides
Authors: Elbim C , Chollet-Martin S , Bailly S , Hakim J , Gougerot-Pocidalo MA
Published: October 2019

We have used a cytofluorometric method to study the effect of tumor necrosis factor alpha (TNF) priming on the oxidative burst, FMLP- receptor expression and actin polymerization of whole blood polymorphonuclear neutrophils (PMN). This technique permits the study of single cells and, thus, allowed us to examine the responsiveness of PMN to TNF in whole blood. We found that TNF in whole blood strongly primed a subpopulation of PMN to produce H2O2 in response to FMLP stimulation, whereas TNF and FMLP alone did not have a significant effect. Furthermore, adding TNF to whole blood increased the capacity of a subpopulation of PMN to bind N-formyl peptides at 4 degrees C, a phenomenon that could account, at least in part, for the strong H2O2 production in response to FMLP after TNF priming. Dual-color cytometric analysis showed that TNF primed actin polymerization on the same subpopulation in response to FMLP. Because the PMN subpopulation, which strongly bound N-formyl peptides at 4 degrees C, was no longer detectable after 1 minute of incubation at 37 degrees C, our data suggest that TNF treatment of PMN in whole blood primes a subpopulation that actively cycles FMLP receptors. These results suggest that PMN in the circulation may respond weakly to bacterial peptides and that TNF may play a critical role in the induction of the oxidative burst in vivo.
Human plasma alpha 2-macroglobulin and von Willebrand factor possess covalently linked ABO(H) blood group antigens in subjects with corresponding ABO phenotype
Authors: Matsui T , Fujimura Y , Nishida S , Titani K
Published: October 2019

We recently identified ABO(H) blood group structures in Asn-linked sugar chains of human von Willebrand factor (vWF) purified from factor VIII concentrates (J Biol Chem 267:8723, 1992). We surveyed plasma glycoproteins carrying ABO(H) blood group antigens by Western blotting analysis and sandwich enzyme-linked immunosorbent assay using blood group-specific monoclonal antibodies (MoAbs) and a lectin. Two major plasma proteins showing apparent molecular weight of about 180 Kd and 270 Kd by sodium dodecyl sulfate polyacrylamide gel electrophoresis reacted with blood group-specific MoAbs and Ulex europaeus lectin I in accordance with donor blood group. Direct sequence analysis of the protein bands showed their identity with the N-terminal sequences of alpha 2-macroglobulin (alpha 2M) and vWF, respectively. The two bands also reacted with anti-alpha 2M and anti-vWF antibodies. The alpha 2M and vWF prepared from plasma by immunoprecipitation showed the appropriate blood group antigenicity. After incubation with endoglycosidase F, both alpha 2M and vWF lost almost all reactivity with anti-blood group reagents. About 90% of plasma vWF, but only approximately 10% of alpha 2M, was immunoprecipitated with anti-blood group antibody. These results indicate that at least two plasma glycoproteins, vWF and alpha 2M, possess Asn-linked ABO(H) blood group antigens in normal individuals with corresponding ABO phenotype. Therefore, ABO(H) blood group antigens in plasma glycoproteins should be considered during preparation of plasma materials for therapeutic use.
Characterization of cells emerging at the time of graft failure after bone marrow transplantation from an unrelated marrow donor
Authors: Donohue J , Homge M , Kernan NA
Published: October 2019

To help elucidate the mechanism responsible for graft failure (GF) following a T-cell depleted bone marrow transplant (BMT) from an unrelated donor, five patients (2 chronic myelogenous leukemia, 1 acute undifferentiated leukemia, 2 myelodysplastic syndrome) who experienced this complication were studied. All patients were HLA class I identical with their donors as determined by serology and one-dimensional isoelectric focusing (IEF); two were serologically matched with their donors for HLA class II antigens, whereas three donor-recipient pairs were serologically mismatched for one HLA-DR antigen. All patients received total body irradiation (fractionated, 1,500 rads), VP-16 (750 mg/m2), and cyclophosphamide (120 mg/kg) pre-BMT and antithymocyte globulin (15 mg/kg every other day) and methylprednisolone (2 mg/kg) post-BMT. Three patients experienced primary nonengraftment and two experienced secondary GF. Peripheral blood mononuclear cells obtained from the patients at the time of GF were studied to examine their functional and phenotypic characteristics. Emerging cells were of host origin and were found to be specifically cytotoxic to donor target cells and suppressive to the in vitro growth of donor BM, especially in the cases of primary nonengraftment. Peripheral blood mononuclear cells from these patients were expanded to form T-cell lines (TcLs). The cytotoxic activities of TcLs were tested in the presence of blocking MoAbs directed against various HLA determinants in an attempt to determine if HLA antigens expressed on donor cells were the target for cytotoxicity. The observed cytotoxic activity was blocked by antibodies to HLA-B, -C (1 patient), HLA-DR (1 patient), and HLA-DQ (1 patient). In two cases, antidonor cytotoxicity could not be blocked by MoAb directed against HLA-A, -B, -C, or -DR. Phenotypic characterization of four successfully maintained TcLs showed 100% CD3+ cells with 100% CD4+ (3 patients) or 50% CD4+/50% CD8+ (1 patient). In two of the three patients with 100% CD4+ cells, antidonor cytotoxicity was blocked by an anti-HLA class II MoAb. In contrast to our previous findings in cases of GF following T-cell-depleted HLA nonidentical family member BMT in which host T cells were CD8+ and cytotoxicity was directed against HLA class I antigens, our present study indicates host T cells emerging at the time of GF following BMT from an HLA class I IEF-identical unrelated donor can be of the CD4+ subset and seem to be capable of recognizing antigenic disparities in the HLA class II region.
Busulfan disposition below the age of three: alteration in children with lysosomal storage disease
Authors: Vassal G , Fischer A , Challine D , Boland I , Ledheist F , Lemerle S , Vilmer E , Rahimy C , Souillet G , Gluckman E
Published: October 2019

Busulfan disposition is age-dependent with a higher clearance and a larger volume of distribution in children than in adults. The optimal dosage of busulfan needed to achieve bone marrow (BM) displacement in young children with malignant or nonmalignant disease remains to be defined. Using a gas chromatography-mass spectrometry assay, we evaluated plasma pharmacokinetics of busulfan in 33 children (median age, 9 months; range, 2 months to 2.75 years) with immune deficiencies, lysosomal storage diseases, acute leukemias, and malignant lymphohistiocytosis after an oral dose ranging from 0.9 to 2.6 mg/kg. The busulfan clearance (assuming a bioavailability of 1) ranged from 2.1 to 13.4 mL/min/kg with a mean of 6.8 mL/min/kg, which is higher than that reported in older children (4.5 mL/min/kg) and adults (2.9 mL/min/kg). Six children with lysosomal storage disease (5 with Hurler's disease, 1 with San Filippo's disease) had a prolonged elimination half-life (4.9 v 2.4 hours), a larger volume of distribution (3.4 v 1.2 L/kg) and a faster clearance (8.7 v 6.3 mL/min/kg) than the other 27 children. This suggests that a higher dose of busulfan will be required to achieve BM displacement in children with lysosomal storage disease. Over the dose range of 0.9 to 2.6 mg/kg, busulfan pharmacokinetics were linear. However, only 46% of the interpatient variation in systemic exposure could be ascribed to the dose. Given the wide interpatient variability in busulfan disposition, dose adjustment and drug monitoring will be needed to achieve the optimal dosage of busulfan in young children. The plasma busulfan levels required to achieve BM displacement need to be defined, especially in lysosomal storage diseases.
Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells
Authors: Beck I , Weinmann R , Caro J
Published: October 2019

Erythropoietin (Epo) production in response to hypoxia or cobalt is primarily mediated by activation of transcription of the Epo gene. Recently an hypoxia responsive enhancer was identified in the 3′ flanking region of the mouse and human Epo genes. Using functional analysis in Hep 3B cells we define here the minimal enhancer element as a 29-bp segment starting at the Apa1 site in the 3′ flanking region of the human Epo gene. Mutagenesis studies of the minimal element identified three different areas that are necessary for full enhancer activity. Electrophoretic mobility shift assays show the presence of hypoxia- and/or cobalt-inducible nuclear DNA-binding proteins that bind to one of the active sites of the enhancer. Induction of hypoxia- binding activity was abolished by Anisomycin, a potent protein synthesis inhibitor, suggesting that de novo protein synthesis is necessary for the activation process. Further characterization of DNA- binding proteins by use of UV light crosslinking identified a protein of molecular weight of approximately 120-Kd that was present only in hypoxic extracts. This protein was found to be present in hypoxic nuclear extracts from both Epo-producing and non-Epo-producing cells, suggesting that it may be involved in a more generalized mechanism of cellular response to hypoxia.
Treatment of the anemia of myelodysplastic syndromes using recombinant human granulocyte colony-stimulating factor in combination with erythropoietin [see comments]
Authors: Negrin RS , Stein R , Vardiman J , Doherty K , Cornwell J , Krantz S , Greenberg PL
Published: October 2019

We treated myelodysplastic syndrome patients (MDS) with both recombinant human granulocyte colony-stimulating factor (G-CSF) and recombinant human erythropoietin (EPO) to determine whether such combination therapy resulted in improvement of their anemias. Twenty- four of 28 patients begun on study completed the protocol and were evaluable for erythroid responses. Therapy was initiated with G-CSF at 1 micrograms/kg administered by daily subcutaneous injection and adjusted to either normalize or double the neutrophil count. EPO was then administered by daily subcutaneous injection at a dose of 100 U/kg and dose-escalated to 150 and 300 U/kg every 4 weeks while continuing the G-CSF. Changes in absolute reticulocyte count, hematocrit level, and need for RBC transfusions were compared with pretreatment values as well as other blood cell counts. Ten of 24 patients (42%) had erythroid responses, whereas all patients had neutrophil responses. Six previously transfused patients no longer required RBC transfusions during the treatment period. Erythroid responses were found to be independent of patient age, French-American-British subtype, duration of disease, prior RBC transfusion requirements, or cytogenetic abnormalities at presentation. Pretreatment serum EPO levels were lower in erythroid-responding as compared with nonresponding patients (median 157 v 600 U/L; P = .05). The combined treatment modality was generally well tolerated. We conclude that a substantial percentage of MDS patients had both erythroid and myeloid responses when treated with the combination of G-CSF and EPO.
Ontogeny of hematopoietic stem cell development: reciprocal expression of CD33 and a novel molecule by maturing myeloid and erythroid progenitors
Authors: Brashem-Stein C , Flowers DA , Smith FO , Staats SJ , Andrews RG , Bernstein ID
Published: October 2019

We have identified a molecule expressed by human marrow granulocyte/monocyte colony-forming cells (CFU-GM), erythroid colony- forming cells (CFU-E), and erythroid burst-forming units (BFU-E), but not their precursors detectable in long-term bone marrow culture. This antigen, detected by flow microfluorimetry using monoclonal antibody 7B9, is coexpressed with CD33 on many CD34+ CFCs, although only the 7B9 antigen was detected on a portion of BFU-E and CFU-E, whereas only CD33 was found on a portion of CFU-GM. Antibody 7B9 appears to be useful for isolating subsets of progenitors based on their common or selective expression of 7B9 antigen and CD33.
Identification of three candidate mutations causing type IIA von Willebrand disease using a rapid, nonradioactive, allele-specific hybridization method
Authors: Inbal A , Englender T , Kornbrot N , Randi AM , Castaman G , Mannucci PM , Sadler JE
Published: October 2019

Type IIA von Willebrand disease (vWD), the most common type II vWD variant, is characterized by decreased binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (Gplb) and by a decrease in large and intermediate vWF multimers. Mutations reported to cause vWD type IIA are clustered within the A2 domain of vWF, which is encoded by exon 28. Genomic DNA from affected members of 12 unrelated families with type IIA vWD were screened for these mutations by a rapid, nonradioactive, allele-specific oligonucleotide (ASO) hybridization method. Oligonucleotides containing each of eight mutations were cross-linked onto a nylon membrane by UV irradiation. A fragment of vWF exon 28 was amplified from peripheral blood leukocyte DNA using biotinylated primers and hybridized to the immobilized oligonucleotides. Positive signals were detected with an avidin-alkaline phosphatase conjugate and chemiluminescent substrate. Thus, in a single hybridization reaction, a patient sample could be analyzed for a large number of mutations simultaneously. Polymerase chain reaction (PCR) products from four patients did not contain any of the tested mutations and therefore were sequenced. Three additional candidate missense mutations, two of them novel, were identified: Arg(834)-->Gln in one patient, Gly(846)-->Arg in one patient, and Val(902)-->Glu in three ostensibly unrelated patients. By ASO hybridization, the mutations were confirmed in the affected patients and excluded in unaffected relatives and 50 normal controls. In one family, the Val(902)-->Glu mutation was shown to be a de novo mutation. This rapid screening method is applicable to other subtypes of vWD for which mutations have been identified.
Autonomous growth of blast cells is associated with reduced survival in acute myeloblastic leukemia
Authors: Hunter AE , Rogers SY , Roberts IA , Barrett AJ , Russell N
Published: October 2019

We have previously classified the in vitro growth characteristics of clonogenic blasts from patients with acute myeloblastic leukemia (AML) according to their capacity to proliferate autonomously in a blast cell colony assay. Here we have analyzed whether the presence of in vitro autonomous growth characteristics has any clinical relevance in AML. We have studied 50 patients (age 2 to 64 years), all of whom were treated with combination chemotherapy, excluding patients with a history of antecedent myelodysplasia. Leukemic cells from 34 of 50 patients (68%) exhibited either partial or totally autonomous growth in a blast cell colony assay. Cells from the remaining patients exhibited nonautonomous growth and were either totally dependent on exogenous growth factor (n = 8) or failed to proliferate at all in the culture system used (n = 8). All 50 patients were treated by intensive chemotherapy and 69% achieved complete remission (CR). Patients whose blasts exhibited autonomous growth in vitro had a significantly lower CR rate (57%) compared with the 16 patients with nonautonomous growth (94%, P = .02). White blood cell count was the only other significant factor (P = .03), but in multivariate analysis growth characteristics remains the most important predictor of CR. Actuarial relapse risk is 80% and 42% at 5 years for autonomous and nonautonomous groups respectively (P = .1). Overall disease-free survival is 21.8% and is higher in the nonautonomous growth group at 54.2% compared with 11.3% at 5 years (P = .0015) in the autonomous growth characteristics was found to be the single most important indicator of CR and disease-free survival. Our data suggests that the suppression of autocrine growth factor production may be of value in the treatment of AML.
Alterations in the deleted in colorectal carcinoma gene in human primary leukemia
Authors: Miyake K , Inokuchi K , Dan K , Nomura T
Published: October 2019

To evaluate the role of the deleted in colorectal carcinoma (DCC) gene in leukemogenesis, we examined loss of heterozygosity (LOH) in the DCC gene in 64 primary human leukemias using Southern blot analysis and examined the expression of the DCC gene using reverse transcriptase- polymerase chain reaction (RT-PCR) analysis. Allelic loss in the DCC gene was observed in two patients (6%, 2 of 35 informative cases), and expression of the DCC gene was reduced or absent in 8 of 26 (31%) patients with acute myelogenous leukemia (AML), 3 of 9 (33%) patients with acute lymphocytic leukemia (ALL), and 7 of 29 (24%) patients with chronic myelogenous leukemia (CML). Moreover, in one ALL patient with absent DCC expression at diagnosis, its expression became normal after performing chemotherapy and achieving remission. These findings suggest that inactivation of the DCC gene contributes to some instances of leukemogenesis.
Stimulation of monocyte chemotaxis by human growth hormone and its deactivation by somatostatin
Authors: Wiedermann CJ , Reinisch N , Braunsteiner H
Published: October 2019

Monocyte infiltration occurs early in the course of inflammation and is a prerequisite for optimal repair of tissue damage. In this study, human recombinant growth hormone was shown to be a potent chemoattractant for human monocytes, inducing migration at picomolar concentrations of recombinant human growth hormone. Chemotaxis of monocytes was measured in vitro by a modified Boyden chamber assay using nitrocellulose micropore filters and measuring microscopically the migration depth of the leading front of monocytes. Somatostatin, which inhibits the release of growth hormone, and its long-acting analogue, octreotide, also stimulated chemotaxis of monocytes; however, the effective peptide concentration was in the micromolar range. When tested for chemotaxis in combination or in experiments using pretreatment with somatostatin and washing of treated cells, somatostatin significantly antagonized the chemotactic responses of monocytes to growth hormone. The inhibitory effect on growth hormone- stimulated chemotaxis was dose dependent and occurred at concentrations severalfold lower than the chemotactically active concentration of somatostatin. Combinations of growth hormone with interferon or substance P also deactivated the chemotactic responses. These observations suggest that human growth hormone may have a regulatory role in monocyte chemotaxis.
Enhanced expression of the complement regulatory protein, membrane inhibitor of reactive lysis (CD59), is regulated at the level of transcription
Authors: Holguin MH , Martin CB , Weis JH , Parker CJ
Published: October 2019

The membrane inhibitor of reactive lysis (MIRL) is an 18-Kd glycosyl phosphatidylinositol anchored membrane glycoprotein that inhibits the cytolytic activity of complement. MIRL is expressed by all hematopoietic elements and by a wide variety of nonhematopoietic tissues. A deficiency of MIRL is primarily responsible for the greater sensitivity of the erythrocytes of paroxysmal nocturnal hemoglobinuria to complement mediated lysis. Because of its critical role in protecting host cells from injury by complement, we hypothesized that mechanisms exist that allow MIRL expression to be regulated. To investigate this hypothesis, both MIRL RNA and MIRL protein expression were analyzed following exposure of K562 erythroleukemia cells to a variety of potential stimulants. Incubation with dexamethasone, calcium ionophore, lipopolysaccharide, interleukin 1, tumor necrosis factor, hemin, and cyclic AMP had no effect on MIRL expression. However, incubation with phorbol 12-myristate 13 acetate (PMA), induced a marked increase in MIRL RNA as determined by Northern blot analysis. This enhanced expression of MIRL RNA was associated with an increase in MIRL protein expression as determined by immunoprecipitation of metabolically labeled proteins, Western blot analysis, and immunobinding assay. Enhanced MIRL RNA expression was first detected after 8 hours and increased through 24 hours of observation. Inhibitors of either protein synthesis or transcription abrogated the PMA-induced enhancement of MIRL RNA expression. Together, these results are consistent with a model in which PMA induces synthesis of a trans acting protein that enhances transcription of the MIRL gene.
Hematopoietic cells from mice deficient in wild-type p53 are more resistant to induction of apoptosis by some agents
Authors: Lotem J , Sachs L
Published: October 2019

Wild-type p53 is a tumor-suppressor gene that can induce cell death by apoptosis when expressed in myeloid leukemic and some other types of tumor cells. However, the question remained as to what extent wild-type p53 is a mediator of apoptosis in normal cells. We have used mice deficient in wild-type p53 to determine whether induction of apoptosis in hematopoietic cells from these p53 deficient mice is defective. We show here that bone marrow myeloid progenitor cells from p53-deficient mice are more resistant to induction of apoptosis when there was only a low concentration of the viability factors granulocyte-macrophage colony-stimulating factor; interleukins-1 alpha, -3, and -6; or stem cell factor; or when apoptosis was induced in these cells by irradiation or heat shock. The loss of one allele of wild-type p53 was sufficient for increased resistance. The higher resistance to apoptosis in p53-deficient mice was also found in irradiated thymocytes, but not in thymocytes treated with dexamethasone or in mature peritoneal granulocytes. The degree of resistance in irradiated myeloid progenitors and thymocytes showed a dosage effect of the number of wild- type p53 genes. The results show that wild-type p53 is involved in the induction of apoptosis by some agents in normal hematopoietic cells. Loss of wild-type p53 can, therefore, contribute to tumor development by decreasing cell death at low concentrations of viability factors and after exposure to a DNA-damaging agent. The results also show that there are wild-type p53-dependent and -independent pathways of normal cell apoptosis.
Prothymosin alpha gene expression correlates with proliferation, not differentiation, of HL-60 cells
Authors: Smith MR , al-Katib A , Mohammad R , Silverman A , Szabo P , Khilnani S , Kohler W , Nath R , Mutchnick MG
Published: October 2019

Accumulating evidence suggests that prothymosin alpha has an as yet undefined intracellular, perhaps intranuclear, function related to cell proliferation. Prothymosin alpha mRNA and/or peptide levels increase when cells are stimulated to proliferate. Because proliferation and differentiation events are often inversely correlated, we examined prothymosin alpha gene expression during proliferation and differentiation of HL-60 myeloid leukemia cells. Steady-state levels of prothymosin alpha mRNA, which are high in exponentially growing HL-60, decrease within hours after induction of HL-60 to differentiate along the neutrophil pathway with dimethylsulfoxide (DMSO) or along the macrophage lineage with either tetradecanoylphorbol acetate (TPA) or bryostatin 1. The decline in prothymosin alpha mRNA in response to these differentiation signals parallels that of c-myc mRNA under the same conditions. We then determined whether the downregulation of prothymosin alpha and c-myc mRNA were due to differentiation or cessation or proliferation. Recombinant human gamma-interferon induces monocytic differentiation of HL-60, but permits continued proliferation, and, under these conditions, expression of prothymosin alpha, as well as of c-myc, mRNA remains elevated. We conclude that prothymosin alpha and c-myc expression are coregulated in differentiating HL-60 and that their expression correlates with the proliferative state of HL-60 cells, rather than with the differentiated state.
Enhancement of the biologic effects of interleukin-3 in vivo by anti- interleukin-3 antibodies
Authors: Jones AT , Ziltener HJ
Published: October 2019

Interleukin-3 (IL-3) has been shown to be a promising agent in the stimulation of bone marrow regeneration following myeloablative therapy. The biologic half-life of this agent is very short (5 to 15 minutes), which limits the effectiveness of low-dose therapy. Here we show that the biologic effects of low-dose IL-3 in mice may be enhanced by concurrent use of polyclonal anti-IL-3 antibodies. The biologic effects of IL-3 in vivo were enhanced dramatically by the combination of the cytokine and polyclonal rabbit anti-IL-3 antibodies, which recognized a peptide comprising the first 29 amino acids of the IL-3 molecule. Enhancing effects were not apparent in vitro, where weak neutralizing properties were observed for these antibodies. The mechanism of this enhancement by the antibody appears to be via a ninefold reduction in the total-body clearance of the cytokine in vivo. The apparent volumes of distribution for IL-3 and for the IL-3/antibody complex were surprisingly similar and exceeded the expected intravascular volume. The prolonged biologic half-life of IL-3 was reproducibly associated with a threefold to fivefold increase in splenic mast-cell precursors over levels observed in mice treated with IL-3 alone; increases in the numbers of mature mucosal-type mast cells in the spleen, but not in the jejunum or lung; increases in IL-3- dependent colony-forming unit-cell in the spleen; and an apparent redistribution of mast cells away from the bone marrow. These experiments demonstrate that antibodies to a cytokine can enhance the biologic activity of that cytokine in vivo.
Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener's granulomatosis
Authors: Mayet WJ , Csernok E , Szymkowiak C , Gross WL , Meyer zum Buschenfelde KH
Published: October 2019

Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (PR-3), have proved to be a useful clinical tool confirming the diagnosis or monitoring disease activity of Wegener's granulomatosis (WG). Although several concepts concerning the pathophysiologic potentials of ANCA have been discussed, only sparse data about ANCA-endothelium interactions have been available. In this study, we have investigated the expression of PR-3 in cytokine- treated human endothelial cells using purified anti-PR-3 antibodies of patients with WG, murine and human monoclonal anti-PR-3 antibodies as probes. We were able to show that tumor necrosis factor-alpha, interleukin-1 alpha/beta, and interferon-gamma led to an increased PR-3 expression in the cytoplasm of endothelial cells by performing polymerase chain reaction analysis, Western blot, cyto-enzyme-linked immunosorbent assays, and confocal laser scanning microscopy. Moreover, PR-3 was also translocated into the cell membrane, becoming accessible to ANCA. Our data suggest a possible direct pathogenic effect of anti- PR-3 antibodies in WG and other vasculitides. Anti-PR-3 antibodies represent an important missing link in ANCA-endothelial interactions.