bluuebunny/crossref_metadata_2025 huggingface dataset:
Splice site mutation in the human protein C gene associated with venous thrombosis: demonstration of exon skipping by ectopic transcript analysis
Authors: Lind B , van Solinge WW , Schwartz M , Thorsen S
Published: October 2019

Heterozygosity for a G-->C mutation converting the highly conserved Gln184 (CAG) to His (CAC) was identified at the last nucleotide of exon 7 of the protein C gene in two family members with deep vein thrombosis. As the nucleotide is a part of the 5 splice site of intron G, it was examined how the mutation affected splicing of protein C pre- mRNA. Relevant protein C cDNA fragments were amplified with polymerase chain reaction after reverse transcription of ectopic mRNA from peripheral blood lymphocytes. Southern blot analysis and nucleotide sequencing of these fragments showed a fragment (A) corresponding to correctly spliced mRNA originating from the normal allele and a fragment (B) corresponding to a truncated mRNA lacking exon 7, originating from the mutant allele. A third fragment (C) lacking exons 7 and 8 was identified in both affected and unaffected family members, as well as in normal controls. Analysis of human liver protein C mRNA indicated that the ectopic lymphocyte mRNA was qualitatively representative for the tissue-specific mRNA. In conclusion, evidence is provided showing that the mutation abolishes formation of correctly spliced mRNA. This agrees with the observation that the mutation results in a type 1 protein C deficiency.
Allelic loss of IRF1 in myelodysplasia and acute myeloid leukemia: retention of IRF1 on the 5q- chromosome in some patients with the 5q- syndrome
Authors: Boultwood J , Fidler C , Lewis S , MacCarthy A , Sheridan H , Kelly S , Oscier D , Buckle VJ , Wainscoat JS
Published: October 2019

Acquired interstitial deletions of the long arm of chromosome 5 occur frequently in the myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Recently IRF1, a putative tumor suppressor gene localized to the long arm of chromosome 5, has been shown to be deleted from the 5q- chromosome in a group of patients with MDS and AML. It has been suggested that the loss of IRF1 may be critical to the development of the 5q- syndrome. We have investigated the allelic loss of IRF1 in a group of 12 patients with MDS and a 5q deletion and 2 patients with AML and a 5q deletion. Gene dosage experiments demonstrated that 12 of 14 patients had loss of one allele of the IRF1 gene but no evidence of homozygous loss and that 2 patients with 5q- syndrome retained both copies of the gene. The retention of IRF1 on the 5q- chromosome in these two cases has been confirmed by fluorescent in situ hybridization localization using an IRF1 cosmid. Pulsed field gel electrophoresis was used to determine whether there was any evidence for structural rearrangement in the region encompassing the IRF1 gene in these two patients. No aberrant bands were detected with a range of rare cutter enzyme digests. We conclude that IRF1 maps outside the commonly deleted segment of the 5q- chromosome and that loss of IRF1 is not solely responsible for the development of the 5q- syndrome.
Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines
Authors: Mayani H , Dragowska W , Lansdorp PM
Published: October 2019

We have previously identified, based on the expression of the CD45RA and CD71 antigens, three major subpopulations of CD34+ cells derived from human umbilical cord blood: CD34+ CD45RAloCD71lo cells (up to 42% multipotent progenitors), CD34+ CD45RA+ CD71lo cells (90% myeloid progenitors), and CD34+ CD45RAloCD71+ cells (70% erythroid progenitors). In the present study, we have investigated the long-term proliferation and differentiation of these subpopulations in response to hematopoietic cytokines. Cells from each subpopulation were cultured for 38 days in serum- and stroma-free liquid cultures supplemented with cytokine combinations that favor either erythropoiesis or myelopoiesis. In keeping with their high content of primitive progenitors, CD34+ CD45RAloCD71lo cells showed the highest CD34+ cell expansion (up to 532- fold) throughout the culture period, followed by CD34+ CD45RA+ CD71lo (130-fold) and CD34+ CD45RAloCD71+ (28-fold) cells. Interestingly, the cytokine combination favoring myelopoiesis was always more efficient in inducing CD34+ cell expansion than the one favoring erythropoiesis. In all but one of the cultures, a predominance of myelopoiesis was observed after 2 weeks, even in those supplemented with the cytokine mixture that favors erythropoiesis. Only when CD34+ CD45RAloCD71+ cells were cultured in the presence of erythroid cytokine mixture, erythropoiesis was evident at all time points. However, such cultures could be sustained for only 29 days. The results of this study demonstrate that the cord blood-derived CD34+ cell compartment consists of functionally distinct cell subpopulations that possess different proliferative capacities in vitro. Our results also show that the cytokine combinations used here were able to modulate proliferation and, to a much lesser extent, differentiation of such subpopulations, probably by favoring the expansion of committed progenitors rather than by acting on uncommitted cells.
Acute thrombocytopenic purpura in relation to the use of drugs
Authors: Kaufman DW , Kelly JP , Johannes CB , Sandler A , Harmon D , Stolley PD , Shapiro S
Published: October 2019

The relation of acute thrombocytopenic purpura (TP) to the use of drugs was investigated in a case-control study conducted in eastern Massachusetts, Rhode Island, and the Philadelphia region; 62 cases over the age of 16 years with acute onset and with a rapid recovery were compared with 2,625 hospital controls. After control for confounding by multiple logistic regression, use of the following drugs in the week before the onset of symptoms was significantly associated: trimethoprim/sulfamethoxazole (relative risk [RR] estimate, 124), quinidine/quinine (101), dipyridamole (14), sulfonylureas (4.8), and salicylates (2.6). The overall annual incidence of acute TP was estimated to be 18 cases per million population. The excess risks for the associated drugs were estimated to be 38 cases per million users of trimethoprim/sulfamethoxazole per week, 26 per million for quinidine/quinine, 3.9 per million for dipyridamole, 1.2 per million for sulfonylureas, and 0.4 per million for salicylates. Associations with sulfonamides, quinidine/quinine, sulfonylureas, and salicylates have been previously reported, but the present study has provided the first quantitative measures of the risk. The association with dipyridamole was unexpected. In general, despite large RRs, the incidence rates attributable to the drugs at issue (excess risks) were low, suggesting that TP is not an important consideration in the use of the various drugs.
Stimulation of the human neutrophil respiratory burst by formyl peptides is primed by a protein kinase inhibitor, staurosporine
Authors: Combadiere C , el Benna J , Pedruzzi E , Hakim J , Perianin A
Published: October 2019

Stimulation of polymorphonuclear neutrophils (PMN) by phorbol esters or formyl peptides (fMLP) generates large quantities of superoxide anion, the so-called respiratory burst (RB), a phenomenon associated with intense phosphorylation of a 47-kD protein (p47 phox). Staurosporine, a potent protein kinase C (PKC) antagonist, inhibits both responses when PMN are stimulated by phorbol myristate acetate (PMA), suggesting a positive role of PKC. In this study, we reassessed these PMN responses in fMLP-stimulated cells and found that staurosporine had opposite effects depending on the duration of PMN treatment with staurosporine. Short PMN incubation (0.5 to 3 minutes) with 25 to 100 nmol/L staurosporine inhibited the fMLP-induced RB, whereas longer treatment (15 to 20 minutes) enhanced it by up to about 200% relative to controls. In contrast, the PMA-mediated RB was depressed by staurosporine in a time-dependent manner. A primed fMLP-induced RB was also observed after long (15 minutes) PMN treatment with 5 to 100 mumol/L H-7, whereas shorter treatment (5 minutes) resulted in a small decrease in RB. By contrast, the tyrosine kinase inhibitor genistein (2 to 80 mumol/L) depressed fMLP-induced RB whatever the duration of PMN treatment. Analysis of 32P-phosphorylated proteins in fMLP-stimulated cells showed that short PMN treatment (< 8 minutes) with staurosporine abolished the phosphorylation of the 47-kD protein, which was identified as p47 phox, whereas long treatment partially restored p47 phox phosphorylation up to approximately 50% of the control value. In PMA-stimulated PMN, phosphorylation was reduced in a time-dependent manner. Furthermore, the staurosporine-primed RB and the staurosporine- induced recovery of phosphorylation were inhibited by sphingosine but not by genistein. Thus, in addition to its known depressive effect, staurosporine markedly potentiated fMLP-stimulated RB as a function of the duration of PMN treatment. The restoration of p47 phox phosphorylation suggests that staurosporine may alter the interactions between different protein kinases, producing marked time-dependent changes in signalling pathways. These data emphasize the care that should be taken in interpreting data obtained using this kinase inhibitor that may, however, be helpful analyzing in signalling pathways.
Fractionated total body irradiation and high-dose etoposide as a preparatory regimen for bone marrow transplantation for 99 patients with acute leukemia in first complete remission
Authors: Snyder DS , Chao NJ , Amylon MD , Taguchi J , Long GD , Negrin RS , Nademanee AP , O'Donnell MR , Schmidt GM , Stein AS
Published: October 2019

Ninety-nine consecutive patients with acute leukemia in first complete remission under age 50 (median age 27 years; age range 1 to 47 years) with a histocompatible sibling donor were treated with fractionated total body irradiation (1,320 cGy) and high-dose etoposide (60 mg/kg) followed by allogeneic bone marrow transplantation. Sixty-one patients were diagnosed with acute myelogenous leukemia (AML), 34 patients with acute lymphoblastic leukemia (ALL), 3 patients with biphenotypic acute leukemia, and 1 patient with acute undifferentiated leukemia. Thirty of the 34 patients with ALL had at least one of the following high-risk factors: age greater than 30, white blood cell count at presentation > 25,000/microL, extramedullary disease, certain chromosomal translocations, or the need for greater than 4 weeks of induction chemotherapy to achieve first complete remission. Cumulative probabilities of disease-free survival and relapse at 3 years were 61% and 12%, respectively, for the 61 patients with AML and 64% and 12%, respectively, for the 34 patients with ALL. By stepwise Cox regression analysis, significant prognostic variables for patients with acute myelogenous leukemia were the presence of acute graft-versus-host disease and increasing age, whereas for patients with acute lymphoblastic leukemia, significant variables were age and the development of cytomegalovirus-associated interstitial pneumonia. Complications related to graft-versus-host disease and relapse of leukemia were the major causes of death.
Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression
Authors: Peng J , Friese P , Heilmann E , George JN , Burstein SA , Dale GL
Published: October 2019

After the intravenous infusion of N-hydroxysuccinimido biotin into dogs, 80.6% +/- 9.7% (n = 5) of platelets were covalently labeled with biotin. The in vivo survival of the biotinylated platelets was monitored by flow cytometry and was normal as compared with previous reports for dog platelets. The ability of the biotinylated platelets to be activated was analyzed by measuring the expression of cell-surface P- selectin after incubation with graded concentrations of thrombin. When P-selectin expression was examined 3 hours after labeling, biotinylated platelets were indistinguishable from the nonlabeled population of platelets, indicating that biotinylation did not adversely affect the cells. On consecutive days after biotinylation, the thrombin dose- response curves for biotinylated and nonbiotinylated platelets were repeated, and as the biotinylated-platelets aged, they became less responsive to thrombin. On days 3, 4, and 5, the thrombin EC50 for the aged, biotinylated platelets as compared with the total population of platelets was 136%, 150%, and 178%, respectively. Increasing age clearly impairs the reactivity of platelets towards thrombin as quantitated by the expression of cell-surface P-selectin.
Interleukin-12 induces tyrosine phosphorylation and activation of 44-kD mitogen-activated protein kinase in human T cells
Authors: Pignata C , Sanghera JS , Cossette L , Pelech SL , Ritz J
Published: October 2019

Interleukin-12 (IL-12) is a novel cytokine that enhances numerous functional activities of human T cells and natural killer (NK) cells. The present studies were undertaken to characterize some of the early signaling events following IL-12 stimulation of mitogen-activated normal T cells. In these cells, IL-12 induces rapid tyrosine phosphorylation of proteins of 21, 44, and 54 kD. However, IL-12 does not induce tyrosine phosphorylation in normal resting T cells. In conjunction with increased tyrosine phosphorylation of several substrates, IL-12 stimulation resulted in increased in vitro kinase activity of immunoprecipitated tyrosine phosphorylated proteins. The 44- kD protein has been characterized as one isoform of the mitogen- activated protein (MAP) kinase family. Increased tyrosine phosphorylation of MAP kinase following IL-12 stimulation was also associated with enhanced enzymatic activity of this protein in vitro as measured by myelin basic protein phosphotransferase assay. These studies identify MAP kinase as one of the intracellular elements of the IL-12 signaling pathway in human T cells.
Murine myeloid cells transformed by myb require fibroblast-derived or autocrine growth factors in addition to granulocyte-macrophage colony- stimulating factor for proliferation
Authors: Macmillan EM , Gonda TJ
Published: October 2019

Murine myeloid cells can be transformed in vitro by infection with recombinant retroviruses carrying activated myb genes. While these myb- transformed hematopoietic cells (MTHCs) can proliferate continuously in culture, they exhibit several characteristics of progenitor cells of the granulocyte-macrophage (GM) lineage, including an absolute dependence on hematopoietic growth factors (HGFs) such as GM colony- stimulating factor (GM-CSF) for survival and growth. Whereas we have previously shown that MTHCs respond synergistically to certain combinations of HGFs, we report here that MTHCs apparently require two HGFs for proliferation, because GM-CSF alone appears insufficient to promote growth when MTHCs are cultured at very low densities. However, proliferation can be stimulated by either increasing the density at which MTHCs are cultured (implying the production of an autocrine growth factor) or by the presence of a feeder layer of irradiated fibroblasts. We find that the activity of such feeder layers is greatest when the MTHCs are allowed to contact them directly; and by using mutant fibroblast lines, that it depends on the production of CSF- 1, but not Steel factor (SLF). In contrast, the autocrine factor appears not to be either CSF-1 or SLF, and the possibility is raised that it may represent a novel HGF activity. Potential implications of these results for normal and leukemic hematopoiesis are discussed.
Molecular cloning of the breakpoint for 3q27 translocation in B-cell lymphomas and leukemias
Authors: Miki T , Kawamata N , Arai A , Ohashi K , Nakamura Y , Kato A , Hirosawa S , Aoki N
Published: October 2019

Reciprocal exchanges between chromosomal region 3q27 and three loci of the Ig genes have been reported in cases of B-cell type non-Hodgkin's lymphoma. We have cloned a region containing a breakpoint junction of 3q27 from a cell line established from a patient with Burkitt's lymphoma carrying t(3;22)(q27;q11). The region cloned was shown to contain an Ig lambda light chain gene fused to a gene on chromosome 3q27. This finding was subsequently confirmed by fluorescence in situ hybridization. Extra nucleotides were present at the joining site. The heptamer-like and nonamer-like sequences separated by an intervening 24 bp were present in the region corresponding to the breakpoint of 3q27, suggesting that a misrecombination in Ig gene rearrangement may be involved in the translocation. Southern blot analysis with a 3q27- specific probe showed rearrangements in three additional patients with B-cell malignancies with the t(3;14)(q27;q32). The breakpoints of all four cases clustered within a limited 3-kb region on chromosome 3q27. The region of 3q27 involved in the translocation was designated as the BCL5 locus. The transcripts from the BCL5 locus were detected in normal tissues and hematopoietic cell lines, and the increased expression of transcript of aberrant size was detected in the established cell line carrying t(3;22). These observations suggest that a gene located at 3q27 is involved in the translocation and that its deregulation plays a role in the malignant transformation of B cells.
Thrombocytopenia after bone marrow transplantation caused by a recipient origin Br(a) allo-antibody: presence of mixed chimerism 3 years after the graft without hematologic relapse
Authors: Bierling P , Pignon JM , Kuentz M , Mitjavila MT , Fromont P , Barbu V , Goossens M , Duedari N , Vainchenker W , Vernant JP
Published: October 2019

We report a case of mild, clinically asymptomatic, immune thrombocytopenia after allogenic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML) caused by the presence of a recipient- origin Br(a) antibody that recognized the donor platelets. Although the antibody titer decreased, it remained detectable more than 3 years after BMT. Chimerism studies were performed combining cytogenetics, blood cell phenotype studies, and genomic amplification of hypervariable sequences. Cytogenetic studies and molecular analysis of peripheral blood cells, purified B- and T-lymphocyte subpopulations, and bone marrow colonies showed the hematopoiesis to be of donor origin, but absorption-elution experiments with peripheral RBCs showed a small amount of recipient RBCs. The CML chimeric transcript was also detected by means of polymerase chain reaction on samples collected until day +867 post-BMT. This case shows that recipient-origin platelet alloantibodies can cause thrombocytopenia after BMT and that the persistence of small numbers of recipient cells (even leukemic) is not necessarily associated with hematologic relapse.
kat: a high-efficiency retroviral transduction system for primary human T lymphocytes
Authors: Finer MH , Dull TJ , Qin L , Farson D , Roberts MR
Published: October 2019

We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to be free of detectable replication competent retrovirus by an extended provirus mobilization assay, demonstrating that this system is as safe as currently available stable packaging lines. The kat virus production system should be of general use for the rapid production of high titer viral supernatants, as well as for high-efficiency transduction hematopoietic cell types refractory to retroviral transduction.
Hemorrhagic tumor necrosis during a pilot trial of tumor necrosis factor-alpha and anti-GD3 ganglioside monoclonal antibody in patients with metastatic melanoma
Authors: Minasian LM , Szatrowski TP , Rosenblum M , Steffens T , Morrison ME , Chapman PB , Williams L , Nathan CF , Houghton AN
Published: October 2019

Hemorrhagic tumor necrosis is an inflammatory event that leads to selective destruction of malignant tissues, with both potentially toxic and beneficial consequences. A pilot clinical trial was undertaken combining tumor necrosis factor-alpha (TNF-alpha) with the monoclonal antibody R24 (MoAb R24) against GD3 ganglioside in patients with metastatic melanoma. Patients received MoAb R24 to recruit leukocytes to the tumor followed by low doses of recombinant TNF-alpha to activate leukocytes. Eight patients were treated and seven patients had mild toxicity. One patient with extensive metastatic melanoma developed tumor lysis syndrome within hours after treatment with almost complete necrosis of bulky tumors in multiple visceral sites. To our knowledge, this is the first documented case of hemorrhagic tumor necrosis in a patient with metastatic cancer in multiple visceral sites.
Protection of murine bone marrow by dexamethasone during cytotoxic chemotherapy
Authors: Kriegler AB , Bernardo D , Verschoor SM
Published: October 2019

Corticosteroids have the ability to suppress the production of growth factors and cytokines and are thus implicated in the negative regulation of hematopoiesis. We have shown that the corticosteroids, prednisolone and dexamethasone, were able to effectively protect progenitor cells in four strains of mice against cell-cycle-specific antimetabolic chemotherapy agents. The highest levels of protection against 5-fluorouracil (FU; 200 mg/kg) were achieved when two or three intraperitoneal injections of dexamethasone were administered between - 7 and +3 hours at a dose of 7.5 mg/kg/injection (optimal dose) or by continuous infusion between -4 and +20 hours. This protective effect is manifested as an increase in the number of high proliferative potential colony-forming cells that survive in the bone marrow 3 days after treatment with FU from between 0.5% and 11% to between 10% and 34% of normal. The bone marrow progenitors and blood cell numbers return to normal from 3 to 5 days and 1 to 2 days earlier, respectively. Less dexamethasone than prednisolone is required to give an equivalent protective effect, which is consistent with their anti-inflammatory potency. These findings are further evidence of the negative regulatory role played by corticosteroids, and indicate that the treatment schedules of corticosteroids during cancer therapy need to be reexamined to obtain the maximum benefit from their use.
Effects of stem cell factor (kit-ligand) and interleukin-3 on the growth and serine proteinase expression of rat bone-marrow-derived or serosal mast cells
Authors: Haig DM , Huntley JF , MacKellar A , Newlands GF , Inglis L , Sangha R , Cohen D , Hapel A , Galli SJ , Miller HR
Published: October 2019

The effects of rat stem-cell factor (SCF) and interleukin-3 (IL-3), alone or in combination, on the in vitro growth and serine proteinase expression of rat serosal/connective-tissue mast cells (CTMC) or bone marrow-derived mast cells (BMMC) were examined. Rat SCF stimulated the growth of both CTMC and BMMC. IL-3 stimulated BMMC growth to a lesser extent than did SCF, whereas CTMC numbers did not increase in IL-3. However, SCF and IL-3 had synergistic effects on the growth of both BMMC and CTMC. SCF favoured the maintenance of rat mast cell proteinase- I (RMCP-I) in CTMC, but did not induce detectable production of RMCP-I in BMMC. In contrast, when IL-3 or lymph node-conditioned medium (LNCM) was added to SCF, a subpopulation of CTMC expressed and stored the soluble proteinase RMCP-II. In BMMC, the RMCP-II content of cells maintained in SCF was significantly less than that of cells maintained in IL-3 or LNCM. RMCP-II also appeared in the supernatants of BMMC, especially when BMMC numbers were increasing rapidly in SCF with or without IL-3 or LNCM. Thus, SCF and IL-3 can regulate the growth of rat BMMC and CTMC, as well as influence their production and release of proteinases.
Expression of sialosyl-Tn in colony-forming unit-erythroid, erythroblasts, B cells, and a subset of CD4+ cells
Authors: Muroi K , Suda T , Nakamura M , Okada S , Nojiri H , Amemiya Y , Miura Y , Hakomori S
Published: October 2019

The epitopes Tn and sialosyl-Tn are expressed on erythrocytes of individuals with a very rare blood group, who often suffer from “Tn syndrome.” We surveyed expression of Tn and sialosyl-Tn in normal blood cells, malignant transformed cells, and progenitor stem cells from bone marrow (BM). An anti-Tn antibody, IE3, and an anti-sialosyl-Tn antibody, TKH2, were used in this study. TKH2 reacted with erythroblasts, B cells, and a subset of CD4+ cells; but not with erythrocytes. Erythroblastic cell lines (K562, HEL, and UT7/EPO) and B- cell lines (Daudi, Raji, and B-cell lines transformed by Epstein-Barr virus) showed reactivity to TKH2. Similar results from the reactivity of TKH2 with transformed cells from leukemia patients and lymphoma patients were obtained; TKH2 reacted with blasts from erythroleukemia (M6; for 4 of 4 cases) and with lymphocytes from B-cell chronic lymphocytic leukemia (3 of 3), B-cell lymphoma (5 of 5), and CD4+ adult T-cell leukemia (4 of 4), but did not react with blasts from acute myeloid leukemia (M0 to M5; 0 of 22) or acute lymphoid leukemia (B- lymphoid leukemia, 0 of 11; T-lymphoid leukemia, 0 of 2; undifferentiated leukemia, 0 of 1). IE3 did not react with all of the tested cells. CD2-CD19-TKH2+ normal BM cells (BMC) contained blasts and various maturation stages of erythroblasts. The TKH2+ cells produced a large number of colony-forming unit-erythroid (CFU-E) colonies, whereas they produced a small number of burst-forming unit-erythroid colonies and CFU-granulocyte-macrophage colonies. CD34+ normal BMC did not express Tn and sialosyl-Tn. These findings suggest that sialosyl-Tn expresses in CFU-E to erythroblasts.
Circulating erythroid progenitors in polycythemia vera are hypersensitive to insulin-like growth factor-1 in vitro: studies in an improved serum-free medium [see comments]
Authors: Correa PN , Eskinazi D , Axelrad AA
Published: October 2019

We have investigated the question of erythropoietin (Epo) hypersensitivity versus Epo independence as the basis for the endogenous erythroid bursts (EEBs) that develop in cultures without added Epo from hematopoietic cells of polycythemia vera (PV) patients. Using an improved serum-free (SF) medium containing interleukin (IL)-3, but no insulin-like growth factor-1 (IGF-1), and devoid of contaminants that influence erythropoiesis, we compared circulating normal and PV early erythroid progenitors (BFU-E) with respect to their responses in vitro to recombinant human (rHu) Epo. Cultures were seeded with Ficoll- Hypaque density-separated peripheral blood (PB) mononuclear cells (MNCs), and erythroid bursts, together with their component colonies of > or = 50 cells, were scored in situ at 13 to 16 days of culture. The Epo dose-response curve of BFU-E from PV patients was found to be statistically indistinguishable from that of normal subjects. This observation provides compelling evidence against the Epo- hypersensitivity hypothesis. In the complete SF medium minus Epo, the sensitivity of BFU-E to IGF-1 was much greater in PV than in normals, the dose-response curve being shifted to the left by at least 2 orders of magnitude. These data show that the erythroid progenitor cell response in PV is hypersensitive to IGF-1, and independent of Epo. The data also emphasize the importance of truly SF medium conditions for assessment of progenitor cell sensitivities to recombinant growth factors. Depletion of adherent cells totally prevented erythroid burst formation by normal circulating progenitors, but did not prevent the hypersensitive response to IGF-1 of such cells from PV patients. Hence, again unlike its normal counterpart, the progenitor cell response in PV appears to be independent of adherent cell control.
Characterization of genomic PIG-A gene: a gene for glycosylphosphatidylinositol-anchor biosynthesis and paroxysmal nocturnal hemoglobinuria
Authors: Iida Y , Takeda J , Miyata T , Inoue N , Nishimura J , Kitani T , Maeda K , Kinoshita T
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by the presence of abnormal subpopulations of blood cells that are deficient in surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins. Recent studies showed that the gene termed PIG-A, which participates in the first step of GPI-anchor biosynthesis, is mutated in the abnormal blood cells from patients with PNH. In this study the genomic PIG-A gene was cloned and characterized to obtain nucleotide sequence information for analyzing somatic mutations of PIG-A in patients with PNH. The PIG-A gene is at least 17 kb long and has six exons. The exon-intron boundaries and 583 bp of the 5′ flanking region were sequenced. The 5′ flanking region has no TATA-like sequence, but includes four CAAT boxes, two AP-2 sequences, and a CRE sequence, some of which are present in regions necessary for the promoter activity. We report pairs of oligonucleotide primers for polymerase chain reaction that should be useful to amplify and analyze various regions of the PIG-A gene in patients with PNH.
Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells
Authors: Iwama A , Okano K , Sudo T , Matsuda Y , Suda T
Published: October 2019

To identify the novel receptor tyrosine kinases (RTKs) critical to the proliferation of hematopoietic stem cells, we performed polymerase chain reaction-based cloning from highly purified murine hematopoietic stem cells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- or Sca- 1-positive (Lin-c-KIT+Sca-1+) cells were sorted by a fluorescence- activated cell sorter. Two sets of degenerate oligonucleotide primers were directed to the conserved sequences of the catalytic domain, and were used to amplify cDNAs that encode protein tyrosine kinases (PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified, as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs were identical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK, FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK). Six novel cDNA sequences (stk series) were identified. However, three of them turned out to be BPK, RYK, and TEK. The remaining three showed high homology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively. Characterization of full-length cDNA sequence of the v- SEA/cMET-related gene showed that this was a novel RTK gene and we named this gene STK (stem cell-derived tyrosine kinase). We identified two distinct forms of STK cDNA; the short one encoded a putative truncated protein that lacked most of the extracellular domain. STK was expressed at various stages of hematopoietic cells, including stem cells, but we could not detect any apparent expression in other adult tissues. The expression of the truncated form of mRNA was more predominant than that of the complete form. STK was assigned by fluorescent in situ hybridization to the R-positive F1 band of chromosome 9, the same region to which hepatic growth factor-like protein has been assigned. Characterization of these PTKs, including STK, will be helpful to elucidate the molecular mechanism of the growth regulation of hematopoietic stem cells.
Number and location of AUUUA motifs: role in regulating transiently expressed RNAs
Authors: Akashi M , Shaw G , Hachiya M , Elstner E , Suzuki G , Koeffler P
Published: October 2019

Many RNAs coding for either cytokines or oncogenes are unstable and have a short half-life (t1/2). The AUUUA motif is a highly conserved sequence and is repeated three or more times in the 3′ untranslated region (3′UTR) of RNAs encoding many of these short-lived cytokines and oncogenes. These sequences can confer instability. In this study, we investigated the role of number and location of AUUUA motifs in stabilization of RNA. We introduced 1xATTTA, 2xATTTA, ATTTTTTTA (second adenosine of 2xATTTA was substituted with a thymidine), 3xATTTA, 5xATTTA, 7xATTTA [AT-rich sequence from granulocyte-macrophage colony- stimulating factor [GM-CSF] gene (AT-62)], and GC-62 (GC sequences were substituted for ATTTA sequences in the 7xATTTA) into the 3′UTR of rabbit beta-globin (R beta G) gene. This construct also contained the neomycin-resistance gene. These expression vectors were transfected into human lung fibroblasts (W138), which constitutively expressed low levels of GM-CSF mRNA. Stable transfectants were selected by growth in G418. Northern blot analysis of actinomycin D-treated, stably transfected cells demonstrated that the number of AUUUA sequences correlated with rapidity of turnover of the chimeric R beta G mRNA. The rank order of stability was GC-62 = 1xATTTA = 2xATTTA (no RNA decay at 4 hours) > 3xATTTA = 5xATTTA (t1/2, 4 hours) > 7xATTTA (t1/2, 2 hours). Stability of mRNA of R beta G also was reduced (t1/2, 2 to 4 hours) when AT-62 was introduced into the second exon of R beta G gene. In these same cells, the t1/2 of GM-CSF RNA was approximately 10 to 15 minutes, suggesting that the AUUUA motifs cannot alone account for the rapid degradation of this cytokine mRNA. Phorbol diesters, including 12- 0-tetradecanoyl phorbol 13-acetate (TPA), stabilize a variety of transiently expressed RNAs, including GM-CSF RNA. We found that TPA markedly increased (> 30-fold) the accumulation of GM-CSF RNA. In contrast, TPA was unable to stimulate the levels of the chimeric R beta G when either 1x, 2x, 3x, or 5xATTTA motifs were fused to 3′UTR, or when either AT-62 or GC-62 control sequences were fused to the second exon. The chimeric beta-globin construct with either AT-62 or ATTTTTTTA in the 3′UTR had only an approximately twofold to threefold increase in accumulation.
Enhanced production of nitric oxide by bone marrow cells and increased sensitivity to macrophage colony-stimulating factor (CSF) and granulocyte-macrophage CSF after benzene treatment of mice
Authors: Punjabi CJ , Laskin JD , Hwang SM , MacEachern L , Laskin DL
Published: October 2019

Nitric oxide is a short-lived reactive mediator that inhibits bone marrow (BM) cell proliferation induced by granulocyte-macrophage colony- stimulating factor (GM-CSF). The present studies show that nitric oxide also inhibits macrophage colony-stimulating factor (M-CSF)-induced growth of mouse BM cells, an effect that was dependent on the presence of an inflammatory mediator and blocked by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (L-NMA). Treatment of mice with the hematotoxicant benzene (800 mg/kg, intraperitoneally, two times per day, for 2 days) resulted in a significant increase in nitric oxide production by BM cells stimulated with lipopolysaccharide (LPS) and interferon gamma alone or in combination with M-CSF or GM-CSF. Cells from benzene-treated mice also displayed increased sensitivity to the growth-promoting effects of M-CSF and GM-CSF. These results suggest that benzene treatment of mice primes BM cells to inducers of nitric oxide. Northern blot analysis showed that this was, at least in part, caused by increased expression of mRNA for inducible nitric oxide synthase (iNOS). Surprisingly, treatment of mice with L-NMA was found to cause a depression in BM cell proliferation and to potentiate benzene-induced decreases in BM cellularity and increases in nitric oxide production. L-NMA administration also augmented nitric oxide production by BM cells. These data indicate that L-NMA is hematotoxic and suggest that it may have actions distinct from inhibition of nitric oxide synthase in the BM.
Hematopoietic growth factor expression and ATRA sensitivity in acute promyelocytic blast cells
Authors: Dubois C , Schlageter MH , de Gentile A , Guidez F , Balitrand N , Toubert ME , Krawice I , Fenaux P , Castaigne S , Najean Y
Published: October 2019

Acute promyelocytic leukemia (APL) is a homogeneous subgroup of acute myeloid leukemias (AMLs) characterized by the presence of the t(15,17) translocation and the resulting promyelocytic myeloid leukemia/retinoic acid receptor alpha (PML/RAR alpha) fusion proteins. To date APL is the only AML that is sufficiently sensitive to all-trans retinoic acid's (ATRA) differentiating effect. In vivo ATRA alone achieves complete remission in most APL patients. However, failure or partial responses are observed and the molecular basis of the absence of ATRA response in these patients has not been determined. To gain insights in the cell growth and differentiation of APL cells, expression of hematopoietic growth factors (HGF) shown to be produced by leukemic cells (interleukin-1 beta [IL-1 beta], IL-6, tumor necrosis factor alpha (TNF alpha), granulocyte colony-stimulating factor [G-CSF], granulocyte- macrophage colony-stimulating factor [GM-CSF], and IL-3) was studied in 16 APL samples. Twelve APL cases expressed IL-1 beta, IL-6, and TNF alpha, but not G-CSF, GM-CSF, and IL-3. These cases achieved complete remission with ATRA therapy. The four remaining patients (either TNF alpha negative or G-CSF, GM-CSF or IL-3 positive) did not achieve complete remission with ATRA. In all cases, in vivo response to ATRA therapy was correlated to the in vitro differentiation effect of all- trans retinoic acid 10(-6) mol/L. Thus, ATRA differentiation induction was strongly correlated to the HGF expression (P < .0001). These results suggest that the presence or absence of HGF's expression by APL cells may contribute to the therapeutic effect of ATRA in this disease.
C3a activates the respiratory burst in human polymorphonuclear neutrophilic leukocytes via pertussis toxin-sensitive G-proteins
Authors: Elsner J , Oppermann M , Czech W , Kapp A
Published: October 2019

In contrast to C5a, which represents a well-established potent activator of the respiratory burst in polymorphonuclear neutrophilic granulocytes (PMN), the functional role of C3a in the activation of PMN is, so far, poorly understood. Herein, the potential role of human C3a in the activation of the respiratory burst in human PMN was investigated. The release of reactive oxygen species (ROS) of PMN from healthy donors was measured by lucigenin-dependent chemiluminescence. C3a dose-dependently induced the production of ROS in human PMN in the range between 10 ng/mL and 1,000 ng/mL, whereas C3a-desArg was inactive. Flow cytometric measurement of H2O2 by dihydrorhodamine-123 labeling of anti-CD16-stained PMN showed that predominantly neutrophilic PMN are responsible for the C3a-induced activation of the respiratory burst. To exclude that C3a stimulation was caused by contamination with C5a, the specificity of C3a-induced activation of PMN was shown using monoclonal antibodies (MoAbs). Accordingly, the effect of C3a was completely abolished in the presence of Fab fragments of a blocking anti-C3a MoAb. In addition, blockade of the C5a receptor by the anti-C5a receptor (anti-C5aR) MoAb, S5/1, totally inhibited the C5a-induced production of ROS, whereas the C3a response in the presence of the anti-C5aR MoAb was unaffected. The specificity of the response was further confirmed by homologous desensitization after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. The C3a-induced ROS production by PMN was inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi proteins) in the signal transduction process initiated by C3a. In addition, stimulation of PMN by C3a resulted in a transient increase in the cytosolic free calcium concentration ([Ca2+]i) in a dose-dependent manner. In contrast to C3a- induced ROS production, C3a did not induce a chemotactic response in PMN, indicating functional qualitative differences as compared with C5a. In summary, these results show that C3a is a potent activator of the respiratory burst in human PMN. Therefore, these findings point to a novel role of C3a in the pathogenesis of inflammatory diseases associated with increased C3a levels and PMN activation.
Interaction of rare illegitimate recombination event and a poly A addition site mutation resulting in a severe form of alpha thalassemia
Authors: Fortina P , Parrella T , Sartore M , Gottardi E , Gabutti V , Delgrosso K , Mansfield E , Rappaport E , Schwartz E , Camaschella C
Published: October 2019

The clinical diversity of thalassemia depends on interaction of diverse genetic defects. We have characterized a severe form of alpha thalassemia caused by coinheritance of a rare alpha-globin gene deletion and a nondeletional defect in a southern Italian family. The proband, a 7-year-old girl, exhibited an abnormal hemoglobin electrophoresis pattern with hemoglobin H and hemoglobin Barts, indicating inheritance of H and hemoglobin Barts, indicating inheritance of a severe form of alpha thalassemia. Southern blot analysis of DNA showed normal as well as aberrant alpha-globin gene fragments indicating heterozygosity for a deletional form of alpha thalassemia in the proband and her mother. The coinheritance of a nondeletional form of alpha thalassemia (alpha alpha T) was suspected because of the severity of the proband's phenotype and the presence of normal alpha-globin gene fragments in the father. Selective polymerase chain reaction of the paternal alpha 1- and alpha 2-globin genes in the proband followed by DNA sequence analysis showed an AATAAA to AATGAA mutation in the polyadenylation signal sequence of the alpha 2-globin gene. Genomic DNA mapping and sequence analysis of a unique polymerase chain reaction product generated across the deletion breakpoint of the maternal allele showed a 5,201-bp deletion extending from 870 nucleotides 5′ of the alpha 2-globin gene to nucleotide +519 in the alpha 1-globin gene. This deletion is similar to that previously suggested by blotting studies in a Greek family (Pressley et al, Nucleic Acids Res 8:4889, 1980) and removes the entire alpha 2-globin gene and a portion of the 5′ end of the alpha 1-globin gene. Sequence characterization of the resultant aberrant truncated alpha 1-globin gene from the proband showed a 27 nucleotide duplication corresponding to the 3′ end of the alpha-globin gene IVS-2 region separated by the insertion of a tetranucleotide (GGTT), suggesting that this deletion is caused by an illegitimate recombination event.
Glycophorin He(Sta) of the human red blood cell membrane is encoded by a complex hybrid gene resulting from two recombinational events
Authors: Huang CH , Lomas C , Daniels G , Blumenfeld OO
Published: October 2019

A complex glycophorin (GP) variant of the human red blood cell membrane exhibiting both He and Sta antigens was characterized at the molecular level. Restriction mapping identified two novel Msp I fragments derived from the 5′ and 3′ portions of the GPHe(Sta) gene, respectively. Genomic DNA, including exons II-V and their splice junctions, was amplified by polymerase chain reaction, and the nucleotide sequences were determined. Comparison with the GPA and GPB sequences showed the presence in GPHe(Sta) of multiple recombinational breakpoints. In the 5′ region of the variant gene, a sequence covering a portion of exon II to intron 2 had been transferred from GPA to GPB, resulting in a B-A-B hybrid structure. Such a gene conversion-like event introduced a number of templated and untemplated nucleotide replacements and was the direct cause for the expression of the He antigen. In the 3′ region of the variant gene, an unequal crossover from GPB to GPA took place in the third intron at a recombination site apparently identical to that observed in the B-A hybrid GPSta type A gene. These results indicated that GPHe(Sta) occurs as a B-A-B-A hybrid gene, most likely originating from a two-step mechanism of homologous recombination. Transcript analysis showed the maturation from the GPHe(Sta) pre-mRNA of two shortened mRNAs of which the exon III-deleted species encodes both the He and Sta antigens.
Association of p72 tyrosine kinase with Stat factors and its activation by interleukin-3, interleukin-6, and granulocyte colony-stimulating factor
Authors: Matsuda T , Hirano T
Published: October 2019

Hematopoietic cytokines, including interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF), induce the proliferation, differentiation, and activation of hematopoietic lineage cells. These cytokines activate the Jak/Stat-mediated signal transduction pathway that is important in the biologic activities of these cytokines. In this study, we showed that hematopoietic cytokines, such as IL-3, IL-6, and G-CSF, all induced tyrosine-phosphorylation of Stat family proteins and Stat-associated 150-kD and 72-kD molecules in hematopoietic lineage cell lines. Furthermore, we showed that the 72-kD molecule had tyrosine kinase activity. The tyrosine kinase activity of the 72-kD molecule was enhanced by the stimulation through an IL-6 signal transducer, gp130, that was shared among the receptors for the IL-6-related cytokine subfamily, such as leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor. Because 72-kD tyrosine kinase was distinct from Syk, Tec, and Btk and coimmunoprecipitated with anti-Stat antiserum, we termed it Stat- associated 72-kD tyrosine kinase (p72sak). p72sak may directly activate Stat family proteins or other signal transducing molecules for IL-3, G- CSF, and the IL-6-related cytokine subfamily.
Total marrow failure induced by pegylated stem-cell factor administered before 5-fluorouracil
Authors: Molineux G , Migdalska A , Haley J , Evans GS , Dexter TM
Published: October 2019

To examine the potential role of stem-cell factor (SCF) in cancer chemotherapy, we have administered it to mice either before or after 5- fluorouracil (5-FU). When polyethylene glycolated (PEG-ylated) SCF was administered to mice before 5-FU, it had a significant sensitizing effect on primitive bone marrow cells. Examination of the hematopoietic status of these mice showed that the damage caused by 5-FU to both bone marrow and spleen hematopoiesis was exaggerated when it was preceded by SCF. SCF given before each of two 5-FU treatments at 7-day intervals resulted in the death of all treated mice. The time of death and hematopoietic status of these animals are compatible with the onset of hypoplastic marrow failure leading to pancytopenia and death. SCF given after 5-FU had little impact either on the initial degree of hematopoietic damage or subsequent recovery. Gut populations were similarly sensitized to 5-FU by prior treatment with SCF, and the damage caused to intestinal populations was greater than that resulting from 5-FU alone. This indicates that the different tissues may be similarly sensitized by SCF. The sensitizing effect of SCF was reversed by concurrent administration of transforming growth factor (TGF)-beta 3, and survival of the majority of the mice was ensured. Examination of hematopoiesis in mice treated concurrently with SCF and TGF-beta 3 showed that the degree of marrow and spleen damage had reverted to that caused by 5-FU alone. In further experiments, 100% survival and normal hematopoiesis could be attained by transplantation of 1 million syngeneic bone marrow cells 24 hours after 5-FU treatment following SCF sensitization. These data indicate that PEG-ylated SCF can sensitize normally resistant hematopoietic and gut stem cells to the effects of 5- FU. This sensitization resulted in effective eradication of hematopoiesis in SCF-pretreated/5-FU-treated animals and their subsequent death from marrow failure. These findings imply that SCF pretreatment may represent a novel method of increasing the effectiveness of conventional chemotherapy, making marrow ablation more effective without drug dose escalation and perhaps sensitizing some tumor cells to the effects of therapy.