bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis and demonstration of FGF receptor expression in megakaryocytes and megakaryocytic-like cells
Authors: Bikfalvi A , Han ZC , Fuhrmann G
Published: October 2019

We have investigated the interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis. Acidic FGF (aFGF) stimulated the proliferation of murine megakaryocytes and human erythroleukemia (HEL) cells in a concentration-dependent manner. The concentrations of aFGF required to elicit half-maximum and maximum effects were similar for HEL and megakaryocytic colony formation. The effect of aFGF was comparable to that of basic FGF (bFGF) in both cell types. The effect of both FGFs was found to be synergistic with interleukin-3 (IL-3), and was abrogated by a monoclonal anti-IL-6 antibody. A specific cell surface receptor complex of approximately 120 Kd was detected for FGF by crosslinking experiments on HEL cells and total bone marrow (BM) cells. Single-cell autoradiography of megakaryocytes in BM smears and BM cultures showed binding sites for 125I-aFGF. Northern blot analysis of messenger RNA (mRNA) from total BM and HEL cells showed a 4.4-kb mRNA specific for FGF receptors type 1 (flg) and type 2 (bek). This was confirmed by polymerase chain reaction, which also showed the presence of FGF receptor mRNA in megakaryocytic-like cells, normal megakaryocytes, and platelets. Together, these results indicate that FGF is involved in megakaryocytopoiesis and suggest that this interaction may be mediated via FGF receptor type 1 and type 2 located on the megakaryocytic lineage or on accessory cells responsible for the release of megakaryocytic growth-promoting activities.
Erythropoietin-induced tyrosine phosphorylations in a high erythropoietin receptor-expressing lymphoid cell line
Authors: Damen J , Mui AL , Hughes P , Humphries K , Krystal G
Published: October 2019

Retroviral gene transfer of the murine erythropoietin receptor (EpR) cDNA into the pro-B-cell line, Ba/F3, was used to generate cells expressing high EpR levels. One of the resulting clones, Ba/F3 clone C5, contained 5 integrated copies of the gene and expressed, at the cell surface, a single affinity class of EpRs at 10 to 15 times the level present on spleen cells from phenylhydrazine-treated mice. Cross- linking studies with clone C5, using 125I-Ep, yielded the same two 105- and 88-Kd major species as that seen with typical erythroid cells. This was distinct from that obtained with EpR-transfected COS cells or L cells, which gave species of 88 and 65 Kd. This suggests that the biologically active EpR complex generated in this Ba/F3 cell line may closely resemble that present in native Ep-responsive erythroid progenitor cells. Tyrosine phosphorylation experiments showed that several proteins in clone C5 cells were rapidly phosphorylated on tyrosine residues in response to Ep, one being the EpR itself. The proportion of cell surface EpRs tyrosine phosphorylated in response to Ep was substantial, reaching a maximum of approximately 10% within 30 minutes of incubation at 37 degrees C. A comparison of Ep- and murine interleukin-3 (mIL-3)-induced tyrosine phosphorylation patterns in clone C5 cells showed that both growth factors stimulated the tyrosine phosphorylation of proteins with molecular weights of 135, 93, 70, and 55 Kd. This could suggest that the Ep and mIL-3 receptors are capable of using the same tyrosine kinase in these cells.
Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1- negative subset
Authors: Spangrude GJ , Brooks DM
Published: October 2019

Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the majority of hematopoietic stem cell activity, as defined by long-term competitive repopulation of irradiated animals and by a secondary transplant assay for spleen colony-forming units (CFU- S), could be localized in Ly-6b haplotype mice to a fraction of bone marrow cells that expresses the Ly-6A/E (Sca-1) molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains expressing the Thy-1.1 allele indicated that the vast majority of activity was included in the Thy-1low population. In contrast, hematopoietic stem cell activity found in the bone marrow of Thy-1.2 genotype mouse strains was recovered in both the Thy-1neg and the Thy- 1low populations. However, similar to Thy-1.1 strains, most activity was localized to the Ly-6A/E+ population of cells. The difference in Thy-1 phenotype of hematopoietic stem cell activity apparent between Thy-1.1- and Thy-1.2-expressing mouse strains was not caused by differences in the staining intensity of monoclonal antibodies (MoAbs) specific for the Thy-1 alleles. Furthermore, an antiframework MoAb that stains both alleles of Thy-1 separated hematopoietic stem cell activity from mice expressing the two alleles in the same manner as did allele- specific MoAb. The results of this study show that Thy-1 expression is not an invariant characteristic of mouse hematopoietic stem cells, and that mice expressing the Thy-1.1 allele are unique in that hematopoietic stem cell activity is found exclusively in the Thy-1low population.
Increased thromboxane biosynthesis in patients with polycythemia vera: evidence for aspirin-suppressible platelet activation in vivo [see comments]
Authors: Landolfi R , Ciabattoni G , Patrignani P , Castellana MA , Pogliani E , Bizzi B , Patrono C
Published: October 2019

Increased thromboxane (TX) production and modified aspirin sensitivity has been detected in vitro in platelets isolated from patients with polycythemia vera. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo and its suppression by oral aspirin, we have investigated the urinary excretion of major enzymatic metabolites of TXB2 in 17 patients with polycythemia vera and 23 gender- and age-matched controls. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. In addition, urinary immunoreactive leukotriene (LT) E4 was measured to explore the 5-lipoxygenase pathway of arachidonate metabolism. Polycythemic patients had significantly (P < .001) higher excretion rates of both 11-dehydro-TXB2 (1,033 +/- 1,050 v 117 +/- 45 pmol/mmol creatinine; mean +/- SD) and 2,3-dinor-TXB2 (725 +/- 676 v 82 +/- 43 pmol/mmol creatinine) than controls. In contrast, urinary LTE4 was not significantly different. Enhanced metabolite excretion did not correlate with the platelet count or with the hematocrit value, and was not related to the current treatment or to a clinical history of thrombotic complications. Platelet TX receptor studies did not show any significant changes in the binding characteristics of two different ligands. A platelet-selective regimen of aspirin therapy (50 mg/d for 7 to 14 days) was associated with greater than 80% suppression in metabolite excretion in nine patients. These results are consistent with abnormal stimuli operating in polycythemia vera to induce a selective enhancement in the platelet biosynthesis of TXA2 without changes in receptor binding. This in vivo abnormality in platelet biochemistry can be largely suppressed by low doses of aspirin.
Generation of vasoactive peptide bradykinin from human umbilical vein endothelium-bound high molecular weight kininogen by plasma kallikrein
Authors: Nishikawa K , Shibayama Y , Kuna P , Calcaterra E , Kaplan AP , Reddigari SR
Published: October 2019

High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. It is an inhibitor of sulfhydryl proteases and has procoagulant properties. It is also a source of the vasoactive peptide bradykinin (BK). It has been previously shown that HK binds to human umbilical vein endothelial cells (HUVEC) in culture. We have further characterized that interaction herein. Immunohistochemical experiments have indicated that when freshly obtained umbilical vein segments were treated with HK, washed, and probed with anti-HK antibodies, HK was localized on the endothelium. We next determined whether HUVEC-bound HK can be cleaved by plasma kallikrein to release BK. Cultured HUVEC were incubated with unlabeled HK for varying times, washed, and the kinetics of BK release by plasma kallikrein were assayed by radioimmunoassay. Results indicated that kallikrein released BK from HUVEC in proportion to the initial amount of bound HK. No release of BK occurred in the absence of kallikrein. Also, there was no BK release upon kallikrein treatment of the HUVEC not treated with exogenous HK. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of HUVEC-bound 125I-HK indicated that addition of kallikrein resulted in cleavage of HK, thus corroborating the BK release experiments. Comparison of cleavage patterns has also indicated that cell-bound HK is slightly less susceptible to digestion by kallikrein than free HK. Therefore, our data suggest that human HK can bind to vascular endothelium in situ and that plasma kallikrein can recognize endothelial-bound HK as a substrate and liberate the vasoactive peptide BK.
Antiplatelet drugs and generation of thrombin in clotting blood
Authors: Szczeklik A , Krzanowski M , Gora P , Radwan J
Published: October 2019

Platelets participate in formation of thrombin through secretion of coagulation factors and by providing a catalytic surface on which prothrombinase complex is assembled. We studied the effects of four antiplatelet drugs on thrombin formation in healthy volunteers. Thrombin generation was monitored both in vitro--in recalcified plasma-- and ex vivo--in blood emerging from a standardized skin microvasculature injury, which also served to determine bleeding time. A mathematical model has been developed to describe the latter reaction. It is based on estimation of the rate of increase in fibrinopeptide A (FPA), a specific marker of thrombin activity, in blood emerging from skin incisions. Two hours after the ingestion of 500 mg of aspirin, thrombin formation became significantly impaired both in vitro and ex vivo. In contrast, 2 hours after the oral administration of placebo, indomethacin 50 mg, or OKY-046 (a thromboxane synthase inhibitor) 400 mg, thrombinogenesis remained unaltered. Ticlopidine, studied either 3 hours after 500 mg oral administration, or after 5 days of intake at a daily dose of 500 mg, had no effect on thrombin generation. Thus, aspirin, contrary to other antiplatelet drugs, depresses thrombin formation in clotting blood, a phenomenon that might be of clinical relevance. It is suggested that aspirin exerts this effect by acetylating prothrombin and/or macromolecules of platelet membrane.
Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products
Authors: Colotta F , Re F , Polentarutti N , Sozzani S , Mantovani A
Published: October 2019

Mature circulating polymorphonuclear cells (PMN) have the shortest half- life among leukocytes and undergo rapid programmed cell death in vitro. In this study, we have examined the possibility that inflammatory signals (cytokines and bacterial products) can regulate PMN survival. PMN in culture were found to rapidly die, with percentages of survival at 24, 48, 72, and 96 hours of 97.3% +/- 1.9%, 36.8% +/- 5.3%, 14.5% +/- 3.1%, and 4.2% +/- 2.9%, respectively (mean +/- SE of 20 different donors). PMN incubated with interleukin-1 beta (IL-1 beta), tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and interferon-gamma (IFN-gamma), but not with prototypic chemoattractants (fMLP, recombinant C5a, and IL-8), showed a marked increase in survival, with values ranging at 72 hours of incubation from 89.5% +/- 5.8% for IL-1 beta to 47.6% +/- 6.4% for IFN- gamma. The calculated half-life was 35 hours for untreated and 115 hours for IL-1-treated PMN. PMN activated with lipopolysaccharide (LPS) or inactivated streptococci also showed a longer survival compared with untreated cells (94.4% +/- 3.2% and 95.5% +/- 2.4%, respectively, at 72 hours). PMN surviving in response to LPS or IL-1 beta retained the capacity to produce superoxide anion when treated with phorbol esters or fMLP. All inducers of PMN survival protect these cells from programmed cell death because they reduced cells with morphologic features of apoptosis and the fragmentation of DNA in multiples of 180 bp. Thus, certain cytokines and bacterial products can prolong PMN survival by interfering with the physiologic process of apoptosis. Prolongation of survival may be important for the regulation of host resistance and inflammation, and may represent a crucial permissive step for certain cytokines and microbial products that activate gene expression and function in PMN.
A switch toward demethylation is associated with the expression of myeloperoxidase in acute myeloblastic and promyelocytic leukemias
Authors: Lubbert M , Oster W , Ludwig WD , Ganser A , Mertelsmann R , Herrmann F
Published: October 2019

Expression of numerous genes encoding myeloid-specific proteins is tightly regulated during normal myeloid differentiation. This heterogeneous group of genes thus offers a tool for dissection of different maturational steps of myelopoiesis. We have previously shown that the human myeloperoxidase (MPO) gene is modified at numerous CpG residues in normal myeloid cells and in myeloid cell lines in a development-specific and expression-associated manner. In the present work, we have applied this type of methylation analysis to primary leukemia myeloid cell samples. We found that expression of MPO messenger RNA (mRNA) is grossly limited to leukemias of late myeloblastic and promyelocytic stages; expression is thus associated with progressive demethylation in the 5′ region of the MPO gene. This modification was not global. Low-level induction of MPO mRNA expression in very immature leukemic cells using phorbol ester was not accompanied by progressive demethylation. This type of methylation analysis of a myeloid-specific gene may yield a molecular indicator for different types of myeloid leukemias.
Correlation of messenger RNA levels with protein defects in specific granule deficiency
Authors: Johnston JJ , Boxer LA , Berliner N
Published: October 2019

Neutrophil specific granule deficiency (SGD) is a rare congenital disorder of unknown cause associated with an impaired inflammatory response and an absence of neutrophil secondary granules. Reduced levels of several neutrophil proteins have led to the suggestion that the defect may lie at the level of transcription, a hypothesis that is supported by abnormally low levels of lactoferrin message in the bone marrow of two SGD patients. We have examined the level of seven granule protein RNAs in one SGD patient and have compared them with reported protein levels. We have found the RNA levels for all of these genes to be reduced in proportion to the decreased levels of their respective proteins. These data further support the hypothesis that the reduced protein levels reflect a defect in transcriptional control.
Molecular basis of inherited human antithrombin deficiency
Authors: Blajchman MA , Austin RC , Fernandez-Rachubinski F , Sheffield WP
Published: October 2019

Figures 1 and 4 summarize the various AT mutations that have been described. The molecular elucidation, over the past decade, of the various AT deficiency types has provided important new insights into functional-structural relationships of AT. This knowledge, together with data provided by monoclonal antibodies and x-ray crystallographic studies of related molecules, has provided important new insights as to how the AT molecule functions in vivo. Finally, such knowledge might, in the foreseeable future, lead to the production of AT molecules that are specifically genetically engineered to be of use in a variety of clinical situations.
Breakpoints at 11q23 in infant leukemias with the t(11;19)(q23;p13) are clustered
Authors: Morgan GJ , Cotter F , Katz FE , Ridge SA , Domer P , Korsmeyer S , Wiedemann LM
Published: October 2019

We have analyzed a series of nine infant leukemias that carry a t(11;19)(q23;p13). They had the morphologic features of acute lymphoblastic leukemia (ALL) and expressed markers typical of B-cell progenitor ALL or pre-B ALL; one coexpressed myeloid markers in addition to lymphoid markers (biphenotypic). Two probes (P/S4 and 98.40) subcloned from a yeast artificial chromosome (YAC) known to span the breakpoint in the t(4;11) were used to investigate DNA isolated from the leukemic cells of these patients. A total of approximately 15 kb of genomic DNA in the vicinity of the probes was examined by conventional Southern blot analysis using a series of restriction enzymes. In eight of the nine cases, the breakpoint could be mapped to an approximately 10-kb BamHI fragment disclosed by hybridization to the P/S4 probe.
All-transretinoic acid followed by intensive chemotherapy gives a high complete remission rate and may prolong remissions in newly diagnosed acute promyelocytic leukemia: a pilot study on 26 cases
Authors: Fenaux P , Castaigne S , Dombret H , Archimbaud E , Duarte M , Morel P , Lamy T , Tilly H , Guerci A , Maloisel F
Published: October 2019

We entered 26 patients with newly diagnosed acute promyelocytic leukemia (APL) in a pilot study of all-transretinoic acid (ATRA) followed by intensive chemotherapy. Median age was 46 (range 25 to 63). No patient presented with leukocytes > 10 x 10(9)/L or had the microgranular APL variant. Cytogenetic analysis (25 patients) found a t(15;17) in 24 cases. Patients were scheduled to receive ATRA (45 mg/m2/d) until complete remission, followed by an intensive daunorubicin (DNR) + Ara C course (“4 + 7” course), then three “2 + 5” DNR + Ara C courses and maintenance chemotheapy. However, the “4 + 7” course was administered in emergency if hyperleukocytosis rapidly developed to prevent leukostasis. Twenty-five patients (96%) achieved CR, 14 with ATRA alone and 11 after the addition of the “4 + 7” course on day 2 to 30 of treatment, because leukocytes rapidly increased (9 cases), because of resistance to ATRA (1 case), and development of organomegaly (1 case). The remaining patient died on day 6, from CNS bleeding. Apart from hyperleukocytosis, side effects were usually moderate. In the 11 patients who could be studied in vitro, a very good correlation was found between in vivo and vitro differentiation and proliferation of APL blasts with ATRA. Three patients were allografted after the “4 + 7” course. Four patients did not receive this course but received the subsequent “2 + 5” courses and maintenance. The remaining patients followed the scheduled protocol. Three patients relapsed after 8, 11, and 15 months (including one allografted patient). Two patients died in CR, after 6 and 17 months. The other 20 patients remained in CR after 18+ to 34+ months (median 21). Actuarial disease free interval (DFI) and event free survival (EFS) were 87% and 77%, respectively, after 18 months. These results were compared to those obtained in our previous APL 84 trial with chemotherapy alone in newly diagnosed APL (after excluding patients included in this trial who presented with hyperleukocytosis). In APL 84 trial, the CR rate was 76%, the actuarial DFI and EFS were 59% and 48% after 18 months, respectively. Differences with the pilot study of ATRA followed by chemotherapy were significant for DFI (P = .02), EFS (P = .006), but not for CR rate (P = .08). Although this is a historical comparison, these results suggest that ATRA followed by chemotherapy may prove superior to chemotherapy alone in newly diagnosed APL, by slightly increasing the CR rate, but perhaps more importantly by reducing the relapse rate.(ABSTRACT TRUNCATED AT 400 WORDS)
A controlled trial of recombinant human granulocyte-macrophage colony- stimulating factor after total body irradiation, high-dose chemotherapy, and autologous bone marrow transplantation for acute lymphoblastic leukemia or malignant lymphoma
Authors: Link H , Boogaerts MA , Carella AM , Ferrant A , Gadner H , Gorin NC , Harabacz I , Harousseau JL , Herve P , Holldack J
Published: October 2019

Infections during granulocytopenia are major complications of autologous bone marrow transplantation (ABMT). Since recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) has proved to accelerate bone marrow recovery after cytostatic chemotherapy, we studied its effects on hematopoietic regeneration and on infectious complications after total body irradiation (TBI) and high-dose chemotherapy followed by ABMT. Eighty-one patients with acute lymphoblastic leukemia (ALL) in complete remission (CR) or with non- Hodgkin's lymphoma (NHL) in CR or partial remission were randomized in a double-blind, placebo-controlled trial. They received either rhuGM- CSF 250 micrograms/m2 (Escherichia coli-derived) daily by continuous infusion after ABMT, or placebo. Treatment was continued until the neutrophil counts reached greater than 500/microL for 1 week. The maximum treatment duration was 30 days. Thirty-nine patients in the rhuGM-CSF group and 40 patients in the placebo group were evaluable. The median time needed to reach a neutrophil count of 500/microL was 15 days with rhuGM-CSF and 28 days with placebo (P = .0001). Bacterial infections occurred in 14 (35.9%) of the patients with rhuGM-CSF and in 25 (62.5%) of the patients given the placebo (P = .024). Nine of the 14 bacterial infections in the rhuGM-CSF group and 20 of the 25 infections in the placebo group were diagnosed within the first 10 days after ABMT. Capillary leakage and a reversible fluid retention were seen in five of the rhuGM-CSF-treated patients. Patients treated with rhuGM-CSF had lower serum protein and albumin levels than patients in the placebo group. There was no statistically relevant difference in overall survival between the two groups (P = .47). Relapse occurred in 14 (34%) patients with rhuGM-CSF and in 18 (45%) patients with placebo. We conclude that continuous infusion of rhuGM-CSF after ABMT accelerates the regeneration of granulocytes and reduces the number of bacterial infections.
Changes in T-helper cell function in human immunodeficiency virus- infected children during didanosine therapy as a measure of antiretroviral activity
Authors: Clerici M , Roilides E , Butler KM , DePalma L , Venzon D , Shearer GM , Pizzo PA
Published: October 2019

Didanosine has shown activity against the human immunodeficiency virus (HIV) in both children and adults. We prospectively assessed T-helper cell (Th) function as determined by in vitro interleukin-2 (IL-2) production in response to a panel of T-cell stimuli in 22 HIV-infected children before and during didanosine therapy and we correlated the incidence of opportunistic and recurrent bacterial infections with changes in p24 antigen and CD4 counts. Didanosine (270, 360, or 540 mg/m2/d) was administered orally for periods ranging from 8 to 40 weeks (mean, 24 weeks). Five of six asymptomatic patients (Centers for Disease Control P-1) compared with 6 of 16 symptomatic (P-2) patients exhibited improved Th function (greater than threefold increase in IL-2 production to at least 2 of the 4 stimuli) during therapy. Of 12 patients without infections during therapy, 9 (75%) showed improvement in Th function, compared with only 2 of 10 patients with infections (P = .03). Notably, the incidence of infections was not correlated with improvements in CD4 count or decreases in p24 antigen. Improvement in Th function during didanosine therapy is correlated with decreased incidence of infections. Assessment of Th function may provide an additional measurement of immunologic response to antiretroviral therapy.
A single cycle of 2-chlorodeoxyadenosine results in complete remission in the majority of patients with hairy cell leukemia
Authors: Tallman MS , Hakimian D , Variakojis D , Koslow D , Sisney GA , Rademaker AW , Rose E , Kaul K
Published: October 2019

Twenty-six patients with hairy cell leukemia (HCL) were treated with 2- chlorodeoxyadenosine (2-CdA), a purine analogue resistant to adenosine deaminase, at 0.1 mg/kg/d for 7 days by continuous intravenous infusion. Fifteen patients were previously untreated, while 11 patients had received prior treatment with splenectomy alone (three patients), interferon alpha alone (four), splenectomy, then interferon alpha (two), or splenectomy, interferon alpha, then 2-deoxycoformycin (2-DCF) (two). Sixteen (80%) of 20 patients evaluable at 3 months achieved complete remission (CR), and four (20%) achieved partial remission (PR) following a single cycle of therapy. All four patients in PR had complete recovery of their peripheral blood counts (except one patient whose platelet count remained 84,000/microL), but had residual HCL in the bone marrow (three patients) or residual splenomegaly (one). Patients with bulky adenopathy, massive splenomegaly, and severe pancytopenia responded as well as those with only modest marrow involvement. The three patients with residual marrow disease received a second cycle of 2-CdA, and two have attained CR. Therefore, 18 of 20 (90%) achieved CR with either one or two cycles of therapy. No patient achieving CR has relapsed at a median follow-up of 12 (+/- 2.1) months. Toxicities included myelosuppression and culture-negative fever. A community-acquired pneumonia was the only infectious complication. Since a single cycle of 2-CdA induces sustained CR in the vast majority of patients with minimal toxicity, this agent is emerging as the treatment of choice for all patients with HCL.
Acute myeloid leukemia (AML) in Down's syndrome is highly responsive to chemotherapy: experience on Pediatric Oncology Group AML Study 8498 [see comments]
Authors: Ravindranath Y , Abella E , Krischer JP , Wiley J , Inoue S , Harris M , Chauvenet A , Alvarado CS , Dubowy R , Ritchey AK
Published: October 2019

The treatment of acute myeloid leukemia (AML) in children with Down's syndrome (DS) has engendered considerable controversy. Because of the concerns for toxicity and increased rate of infections, treatment approaches varied considerably in the past with mixed results. However, experience on the recently completed Pediatric Oncology Group (POG) 8498 AML study suggests that DS children with AML constitute a distinct subgroup that responds well to therapy. Twelve of 285 children on POG 8498 (protocol for newly diagnosed AML) had DS. Children with DS and AML were predominantly male (9 of 12) and were quite younger at diagnosis (< 24 months in 10). The white blood cell count was less than 50 x 10(3)/microL in all 12 and French-American-British types M6 and M7 were frequent (5 of 12). An abnormal cytogenetic marker, in addition to constitutional trisomy 21, was present in 9 of 12 and involved chromosome 8 in 4 of 9. All cases studied (n = 5) were positive for myeloid cell surface markers (CD33, CD13, or CD11b) and, interestingly, were also positive for the CD7 antigen. Chemotherapy included daunorubicin, cytarabine (Ara-C), and 6-thioguanine for remission induction and featured high-dose Ara-C (3 g/m2 per dose) with or without L-asparaginase early in remission. Compared with children without DS, children with DS had a superior event-free survival (EFS at 4 years 100% v 28% +/- 6.2%; P = .003). The EFS remained superior even when compared with non-DS children less than 2 years of age with a white blood cell count less than 10 x 100,000/microL (100% v 48% +/- 17.3%; P = .01).
Stem cell factor directly stimulates the development of enriched granulocyte-macrophage colony-forming cells and promotes the effects of other colony-stimulating factors
Authors: Heyworth CM , Whetton AD , Nicholls S , Zsebo K , Dexter TM
Published: October 2019

The effects of the c-kit ligand (stem cell factor [SCF]) on the development of a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) were assessed. In soft agar assays, both in serum-containing and in serum-deprived cultures, SCF promoted the formation of colonies that contained predominantly granulocytic cells with some blast cells also present. The size of these colonies was far smaller than observed in the presence of interleukin-3 (IL-3). In serum- deprived conditions, no colonies were formed in the presence of macrophage colony-stimulating factor (M-CSF), but when M-CSF was combined with SCF, a marked change was noted in that large colonies were produced containing predominantly macrophages. When GM-CFC were cultured in the presence of IL-3 and SCF, colonies were formed that contained blast cells, granulocytes, and macrophages. A synergistic interaction was also seen using a combination of G-CSF plus SCF in either serum-containing or serum-deprived cultures. The addition of SCF to colony-forming assays markedly reduced the concentration of IL-3 or G-CSF required for optimal levels of colony formation. Furthermore, SCF was capable of promoting the survival of GM-CFC for several days, after which large colonies containing mature cells were formed upon the addition of a secondary growth factor such as G-CSF or IL-3. Thus, SCF can directly act on highly enriched committed progenitor cells in serum- deprived conditions to promote survival, proliferation, and development.
Importance of platelets in experimental venous thrombosis in the rat
Authors: Herbert JM , Bernat A , Maffrand JP
Published: October 2019

Venous thrombosis was induced by ligature of the inferior vena cava in rats whose blood was made hypercoagulable by intravenous (IV) administration of tissue thromboplastin. From a dose-response showing that the administration of increasing doses of tissue thromboplastin resulted in a subsequent progressive increase of thrombus weight, two concentrations of tissue thromboplastin were chosen: a high dose (550 microL/kg, IV) where thrombus formation was optimal and a concentration (7 microL/kg, IV) where tissue thromboplastin-hypercoagulability was intermediate. In both experimental conditions, leukopenia provoked by a myelotoxic drug did not influence the development of venous thrombosis. However, after thrombocytopenia induced by an antiplatelet antiserum, a dramatic decrease in thrombus formation was observed in animals that had been pre-challenged with the lower dose of tissue thromboplastin, whereas decrease in platelet count did not affect venous thrombosis under high thrombogenic challenge. When administered orally 2 hours before thrombosis induction, the ticlopidine analogue clopidogrel showed dose-dependent inhibition of thrombus formation in animals that were pre-challenged with a low dose of tissue thromboplastin (ED50 = 7.9 +/- 1.5 mg/kg, orally) but remained ineffective against high tissue thromboplastin-induced venous thrombosis. We further determined the effect of heparin and hirudin, and showed that both of these drugs exhibited a more potent antithrombotic activity after injection of the lower dose of tissue thromboplastin than after injection of a high dose of tissue thromboplastin. Therefore, using our model of stasis and hypercoagulability, platelet activation played a major role in the development of venous thrombosis when the thrombogenitic stimulus was mild.
Retinoic acid inhibits interleukin-6-induced macrophage differentiation and apoptosis in a murine hematopoietic cell line, Y6
Authors: Oritani K , Kaisho T , Nakajima K , Hirano T
Published: October 2019

We established a radiation-induced murine hematopoietic cell line, Y6, that could be induced to differentiate into macrophages by interleukin- 6 (IL-6). IL-6 also induced growth inhibition and apoptosis in Y6 cells. Retinoic acid (RA) inhibited such effects of IL-6 on Y6 cells. The inhibitory effect of RA on the effects of IL-6 was not caused by the downregulation of the IL-6 receptor, because RA neither affected the expression of IL-6 receptor mRNA nor the expression of IL-6 receptor molecule on the cell surface. Furthermore, RA did not inhibit the IL-6-induced expression of junB mRNA, indicating that the expression of functionally active IL-6 receptor and the signal transduction pathway activating the junB gene are not inhibited by RA. IL-6-induced macrophage differentiation of Y6 cells was preceded by the downregulation of the c-myc gene, which was also prevented by RA. Because the inhibitory effect of RA on Y6 cells was reversible and seemed not to require de novo protein synthesis, the RA receptor by itself might be directly involved in the inhibition of the IL-6 signal transduction pathway. The results indicated that the IL-6 signal transduction pathways leading to the induction of macrophage differentiation and junB gene expression can be dissected by RA.
Characterization of adhesion molecules on human myeloma cell lines
Authors: Uchiyama H , Barut BA , Chauhan D , Cannistra SA , Anderson KC
Published: October 2019

In multiple myeloma, malignant plasma cells are localized in marrow and rarely circulate in peripheral blood. To investigate the role of adhesion proteins in this process, we determined the expression and function of adhesion molecules on cell lines derived from patients with myeloma. The U266, ARH-77, IM-9, and HS-Sultan cell lines strongly expressed beta 1 and alpha 4 integrins (89% to 98% positive), confirming that VLA-4 is the principal integrin on these cell lines. The U266 and IM-9 cell lines also expressed alpha 3 integrin on 15% to 20% cells. In contrast, all lines lacked cell surface alpha 2, alpha 5, and alpha 6 integrin expression (< 5% positive). These cell lines adhered to fibronectin (20% to 40% specific binding), without significant binding to either collagen or laminin. Adhesion of these cell lines to fibronectin was partially blocked with either anti-beta 1 integrin monoclonal antibody (MoAb) (75% inhibition), anti-alpha 4 integrin MoAb (75% inhibition), or RGD peptide (50% inhibition), but was unaffected by anti-alpha v beta 3 or anti-alpha IIb beta 3 MoAbs. Moreover, the combination of anti-beta 1 plus RGD peptide or anti-alpha 4 plus RGD peptide inhibited binding to fibronectin by 80% and 95%, respectively. Finally, pretreatment and coculture of the IM-9 cell line with interleukin-6 (IL-6) resulted in a 52% decrease in specific binding to fibronectin (30% +/- 6% to 15% +/- 6%; P = .001), associated with a decrease in the number of cells expressing VLA-4 and a decrease in intensity of VLA-4 expression. These data suggest that myeloma cells adhere to fibronectin through VLA-4 as well as through RGD-dependent mechanisms, and that this binding can be downregulated by IL-6. Future studies of binding of both myeloma cell lines and freshly isolated tumor cells to extracellular matrix proteins and to marrow stroma may enhance our understanding of localization and trafficking of cells within the bone marrow microenvironment.
Cell cycle kinetics in malignant lymphoma studied with in vivo iododeoxyuridine administration, nuclear Ki-67 staining, and flow cytometry
Authors: Brons PP , Raemaekers JM , Bogman MJ , van Erp PE , Boezeman JB , Pennings AH , Wessels HM , Haanen C
Published: October 2019

Cell cycle kinetics of malignant lymphoma were investigated using in vivo labeling with iododeoxyuridine (IdUrd) and subsequent flow cytometry (FCM) of IdUrd/DNA and Ki-67/DNA. This approach provides an extensive cell kinetic profile from only one single tumor biopsy, including data upon the percentage of S-phase cells, the IdUrd labeling index (LI), Ki-67-derived growth fraction, duration of the S-phase, duration of the G1-phase, potential doubling time, cell production rate, and total cell cycle time. Tissue samples from 33 patients were studied: non-Hodgkin's lymphoma (NHL; n = 22), Hodgkin's disease (HD; n = 7), and reactive hyperplasia (n = 4). In NHL, the percentage of S- phase cells, LI, growth fraction, duration of the S-phase, and cell production rate were significantly correlated with the histologic malignancy grade according to the Working Formulation (P < or = .02). Data found in HD were not essentially different from those in low-grade NHL and reactive hyperplasia. Remarkably, the duration of the S-phase, the duration of the G1-phase, and the total cell cycle time appeared to be rather independent of histologic malignancy grade within the NHL category. A significant correlation was observed between the IdUrd LI and the percentage of S-phase cells, the growth fraction, the potential doubling time, and the cell production rate (P < .001), but not with the duration of the separate cell cycle phases (P > .05). Our data show (1) that it is feasible to obtain detailed information on the in vivo growth characteristics of malignant lymphoma; and (2) that the transition time through the different cell cycle phases widely varies, even within distinct histologic subgroups.
Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway
Authors: Hansen-Hagge TE , Yokota S , Reuter HJ , Schwarz K , Bartram CR
Published: October 2019

Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3′ RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.
O-linked oligosaccharides of glycophorins A and B in erythrocytes of two individuals with the Tn polyagglutinability syndrome
Authors: Blumenfeld OO , Lalezari P , Khorshidi M , Puglia K , Fukuda M
Published: October 2019

Tn polyagglutinability syndrome is an acquired condition where erythrocytes express Tn neo-antigen and become susceptible to hemagglutination by the naturally occurring anti-Tn present in normal sera. Early studies had indicated that O-linked N-acetyl galactosamine was the sole serologic Tn determinant, but more recently O-linked NeuNAc alpha 2, 6GalNAc also has been implicated as a Tn antigen (sialosyl-Tn). However, none of these studies were performed on purified glycoproteins. In this report we examine oligosaccharides of glycophorins A and B purified from Tn erythrocytes of two affected individuals to establish how N- and O-linked saccharides differ from normal. Analysis of carbohydrate composition and treatment with N- glycanase showed that the Asn-linked unit of glycophorin A was not affected. O-linked oligosaccharides were obtained by beta-elimination in the presence of tritiated sodium borohydride. The reduced radiolabeled products were fractionated by Bio-Gel P-2 chromatography, and their structures were investigated by comparison with standards, by monosaccharide quantification, and by neuraminidases of known specificities. The results show that Tn glycophorins from both donors contain intact and truncated forms of trisaccharide and tetrasaccharide NeuNAc alpha 2,3Gal beta 1,3GalNAc and NeuNAc alpha 2,3Gal beta 1,3- (NeuNAc alpha 2,6)GalNAc usually present in glycophorins A and B. The truncated forms include the protein O-linked monosaccharide, GalNAc and disaccharide, NeuNAc alpha 2,6GalNAc (major isomer). The presence of intact glycans in the total population of Tn erythrocytes was confirmed by their susceptibility to T activation after treatment with neuraminidase. The proportion of the four species was not identical in glycophorins of these two donors but, in both, the truncated units predominated and the amount of the disaccharide was approximately one half of that of the monosaccharide. The data are consistent with alterations in UDPGal:GalNAc beta 1,3galactosyl transferase that may have multiple molecular origins and with induction of a specific GalNAc protein alpha 2,6 sialosyl transferase in Tn hematopoietic precursor cells. The molecular basis for these alterations awaits further study.
Use of recombinant human granulocyte-macrophage colony-stimulating factor in patients with lymphoid malignancies transplanted with unpurged or adjusted-dose mafosfamide-purged autologous marrow
Authors: Carlo-Stella C , Mangoni L , Almici C , Cottafavi L , Meloni G , Mandelli F , Rizzoli V
Published: October 2019

The neutropenia-related morbidity and mortality occurring after autologous bone marrow transplantation (ABMT) is increased by marrow purging procedures. While phase I through III clinical trials showed the enhancing activity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophil recovery after ABMT with unpurged marrow, controversial results have been reported when purged marrow was used. Therefore, it was the aim of the present study to evaluate the efficacy of rhGM-CSF administration in a group of patients (n = 15) with lymphoid malignancies transplanted in complete remission with mafosfamide-purged (n = 10) or unpurged (n = 5) marrow. Mafosfamide concentrations used for marrow purging were evaluated on an individual basis by means of a recently described technique that destroys the granulocyte-macrophage (granulocyte-macrophage colony- forming units [CFU-GM]) compartment, but spares 50% of the more primitive stroma adherent colony-forming cells (CFU-Blast). rhGM-CSF (10 micrograms/kg/d) was started within 24 hours of ABMT and administered in a 4-hour infusion daily until the absolute neutrophil count (ANC) reached 500 x 10(6)/L and then for 7 more days. Patients receiving mafosfamide-purged or unpurged marrow failed to show any difference in terms of median number of days required to achieve an ANC > or = 500 x 10(6) (13 v 14.0, P > .4) cells/L. As compared with retrospective controls, granulocytic recovery was reduced by a median time of 11 (P < or = .0005) and 5 (P < or = .0005) days for patients grafted with purged and unpurged marrow, respectively. The number of CFU-GM (mean +/- SD) infused per kilogram of body weight was significantly lower in patients who received purged autografts as compared with those receiving unpurged autografts (0.85 +/- 0.79 x 10(4) v 15.7 +/- 9.2 x 10(4), P < or = .0005). The dose of CFU-GM progenitors infused per kilogram of body weight did not correlate (r = .031, P > .05) with the time required to reach an ANC > or = 500 x 10(6) cells/L. The number of CFU-Blast (mean +/- SD) infused per kilogram of body weight was not significantly different between patients who received purged or unpurged autografts (5.05 +/- 2.51 x 10(3)/kg v 6.18 +/- 2.66 x 10(3)/kg, P < or = .375). A statistically significant correlation (r = -.658, P < or = .05) was observed between the number of CFU-Blast infused and the number of days required to reach an ANC > or = 500 x 10(6) cells/L.(ABSTRACT TRUNCATED AT 400 WORDS)
Analysis of T-cell receptor variability in transplanted patients with acute graft-versus-host disease
Authors: Dietrich PY , Caignard A , Diu A , Genevee C , Pico JL , Henry-Amar M , Bosq J , Angevin E , Triebel F , Hercend T
Published: October 2019

T lymphocytes play a pivotal role in graft-versus-host disease (GVHD) and largely contribute to the graft-versus-leukemia (GVL) effect. Most mature T lymphocytes specifically recognize antigens through the alpha/beta T-cell receptor (TCR). Each alpha/beta TCR chain includes a constant region and a variable region, the latter being encoded by V-J alpha or V-D-J beta rearranged gene segments. To better characterize T cells involved in GVHD, V alpha and V beta gene segment usage was analyzed, after cDNA amplification, in peripheral blood mononuclear cells (PBMC) and skin samples from three patients with grade II cutaneous GVHD. At time of GVHD diagnosis (days 11, 22, and 25), when first signs of engraftment were detectable, virtually all V alpha and V beta subfamilies were represented in PBMC RNAs of the three recipients. These results suggest that diversified TCR gene segment expression is observed early after allogenic bone marrow transplantation (alloBMT). Lymphocytes infiltrating GVHD skin also expressed a large series of V alpha and V beta subfamily specificities. However, analysis of the V alpha and V beta amplified products showed substantial differences between PBMC and the skin lymphocyte RNAs. These observations indicate that a large variety of T lymphocytes are present at the disease site, while some of them may be specifically amplified or decreased in response to minor histocompatibility antigens (miHA). Further characterization of the latter T-cell subpopulations should lead to a better understanding of human in vivo responses directed at miHA.
Biologic response to anti-CD16 monoclonal antibody therapy in a human immunodeficiency virus-related immune thrombocytopenic purpura patient
Authors: Soubrane C , Tourani JM , Andrieu JM , Visonneau S , Beldjord K , Israel-Biet D , Mouawad R , Bussel J , Weil M , Khayat D
Published: October 2019

A patient with refractory human immunodeficiency virus (HIV)-related immune thrombocytopenic purpura (ITP) was treated with 3G8 (anti-CD16) monoclonal antibody on days 1, 3, and 8 (25, 25, and 50 mg were administered intravenously, respectively). Side effects were those expected after the administration of a xenogenic protein, but a severe bone pain occurred from the second injection. At the time of the initiation of the treatment the platelet count was 20,000/mm3 and the absolute CD4 number was 100/mm3. We obtained a long-term correction of thrombocytopenia and, to a lesser extent, there was a stabilization of CD4 lymphocytes for 18 months. We observed a significant stimulation of natural killer (NK) function and an elevation in the serum level of tumor necrosis factor alpha, interferon gamma, and granulocyte- macrophage colony-stimulating factor. This suggests that in HIV-related ITP the removal of platelets is mediated by low-affinity Fc gamma receptors (CD16). The stimulation of NK function and elevation in CD4+ lymphocytes may be related to the production of cytokines by activated human NK cells through the interaction of their CD16-bearing receptor with the 3G8 monoclonal antibody. This observation warrants confirmation and further clinical trials.