bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth and spread of human malignant T lymphoblasts in immunosuppressed nude mice: a model for meningeal leukemia
Authors: Cavallo F , Forni M , Riccardi C , Soleti A , Di Pierro F , Forni G
Published: October 2019

Previous work has shown that nude (nu/nu) mice additionally immunosuppressed by splenectomy, sublethal irradiation, and treatment with antiasialo GM1 antiserum (SIA-nu/nu mice) have no detectable natural killer activity and allow the growth of human malignant lymphoblasts. We show here that all SIA-nu/nu mice engrafted intravenously with 5 x 10(6) malignant lymphoblasts originally derived from a child with a T-cell acute lymphoblastic leukemia (PF382) and from a boy with a T-cell lymphoma (ST-4) develop lethal meningeal leukemia and die within 35 days. Histologic examination of moribund SIA- nu/nu mice showed that vertebral and skull bone marrow was always replaced by proliferating human T lymphoblasts. From the spinal canal, lymphoblasts spread to the meninges, causing hind leg paralysis. Leaving the skull, they permeated the meninges and then invaded the nervous parenchyma. This efficient and reproducible experimental model may be suitable for experimental studies on the pathogenesis of meningeal leukemia.
Follicular dendritic cells inhibit human B-lymphocyte proliferation
Authors: Freedman AS , Munro JM , Rhynhart K , Schow P , Daley J , Lee N , Svahn J , Eliseo L , Nadler LM
Published: October 2019

In germinal centers, B lymphocytes are intimately associated with follicular dendritic cells (FDCs). It has been hypothesized that FDCs are involved in the regulation of B-cell growth and differentiation through cell-cell interactions. In this study, highly enriched preparations of FDCs were isolated by cell sorting using the FDC restricted monoclonal antibody DRC-1. When irradiated FDCs were cultured with mitogen stimulated B cells, B cell 3H-TdR uptake was inhibited by up to 80%. This inhibitory effect was not seen when paraformaldehyde fixed FDCs were added to B-cell cultures, suggesting that the FDCs needed to be metabolically active. Moreover, supernatants from cultured FDCs were similarly able to inhibit B-cell proliferation. These results demonstrate that FDCs may downregulate the clonal expansion of B cells that occurs within lymphoid follicles as part of the normal physiologic immune response. Potentially, the loss of the inhibitory role of FDCs in vivo may be of importance in certain infectious and neoplastic processes in which germinal centers are affected.
Human B-cell interleukin-10: B-cell lines derived from patients with acquired immunodeficiency syndrome and Burkitt's lymphoma constitutively secrete large quantities of interleukin-10 [see comments]
Authors: Benjamin D , Knobloch TJ , Dayton MA
Published: October 2019

A recent addition to the lymphokine network is human IL-10 (hIL-10). This novel lymphokine has striking homology to BCRF1 protein, the product of a previously uncharacterized open-reading frame in the Epstein-Barr virus (EBV) genome. To date, IL-10 expression has been described in several T clones induced with anti-CD3 and phorbol myristate acetate (PMA), in monocytes stimulated with lipopolysaccharide (LPS), and in murine B-cell lymphomas. We sought to determine whether human B cells express hIL-10 and, if so, its relationship to EBV and to other B-cell lymphokines. We studied 21 EBV- positive B-cell lines derived from patients with acquired immunodeficiency syndrome (AIDS) and Burkitt's lymphoma (n = 6), American Burkitt's (n = 3), African Burkitt's (n = 5), and normal lymphoblastoid cell lines (n = 7), in comparison with seven EBV- negative cell lines. All cell lines were activated with the tumor promoters PMA and teleocidin and were studied by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoadsorbent assay (ELISA). We demonstrated that EBV- positive cell lines derived from patients with American Burkitt's lymphoma, and especially those from patients with AIDS, constitutively express large quantities of hIL-10 by Northern blot analysis and ELISA (range, 3,101 to 25,915 pg/mL), and that both teleocidin and PMA induce hIL-10 in these cell lines. In contrast, six of seven EBV-negative cell lines did not express hIL-10 even by RT-PCR, and hIL-10 was not triggered by PMA or teleocidin. To assure that the 350 bp amplified by PCR was hIL-10 and not BCRF1, we used PCR primers, which do not amplify a fragment from plasmid templates containing BCRF1. Cloning and sequencing of the 350 bp product also demonstrated that B-cell IL-10 is identical to hIL-10 from the T-cell clone B21. Correlation of hIL-10 with other B-cell lymphokines secreted by these B-cell lines demonstrated that hIL-10 secretor cell lines also constitutively secrete or can be induced to secrete IL-6, although to a much lesser amount. Since both lymphokines influence B-cell growth and differentiation, we suggest that hIL-10 may contribute to the polyclonal B-cell activation and hyperglobulinemia seen in AIDS patients. Finally, several reports support the hypothesis that EBV is an important cofactor in the development of human immunodeficiency virus type 1 (HIV-1)-related B-cell lymphomas. Detection of large quantities of hIL-10 in B-cell lines derived from AIDS patients, the close association between EBV and hIL-10 shown in this report, and the ability of BCRF1 to capture hIL-10 activities, make hIL-10/BCRF1 an attractive candidate as a factor causing B-cell growth and immortalization in patients with AIDS and B-cell lymphomas.
Accumulation of high levels of methotrexate polyglutamates in lymphoblasts from children with hyperdiploid (greater than 50 chromosomes) B-lineage acute lymphoblastic leukemia: a Pediatric Oncology Group study
Authors: Whitehead VM , Vuchich MJ , Lauer SJ , Mahoney D , Carroll AJ , Shuster JJ , Esseltine DW , Payment C , Look AT , Akabutu J
Published: October 2019

Hyperdiploidy (greater than 50 chromosomes, or a DNA index greater than 1.16) confers a favorable prognosis in B-lineage acute lymphoblastic leukemia of childhood. Children with B-lineage acute lymphoblastic leukemia whose lymphoblasts at diagnosis accumulate high levels of methotrexate (MTX) and MTX polyglutamates (MTXPGs) in vitro experience a better event-free survival than those whose lymphoblasts do not (Blood 76:44, 1990). Lymphoblasts from 13 children with hyperdiploidy (greater than 50 chromosomes) accumulated high levels of MTX-PGs (1,095 and 571 to 2,346 pmol/10(9) cells [median and 25% to 75% intraquartile range]). These levels were higher than those in B-lineage lymphoblasts from 19 children with other aneuploidy (326 and 159 to 775 pmol/10(9) cells) and 15 children with diploidy (393 and 204 to 571 pmol/10(9) cells) (P = .0015). Chromosomal trisomies in hyperdiploid cases were highly nonrandom. Chromosome 9 was not one of the chromosomes involved in trisomies, even though this chromosome contains the gene for folate polyglutamate synthetase, which is the enzyme required for MTXPG synthesis. The correlation between MTXPG level and percentage of S- phase cells was weak, suggesting that increased levels of MTXPGs could not be attributed to elevated proportions of cells in active DNA synthesis. The ability of hyperdiploid lymphoblasts to accumulate high levels of MTXPGs may increase their sensitivity to MTX cytotoxicity, accounting in part for the improved outlook for hyperdiploid patients treated with regimens that emphasize MTX as a primary component of continuation therapy.
Effect of herbimycin A, an antagonist of tyrosine kinase, on bcr/abl oncoprotein-associated cell proliferations: abrogative effect on the transformation of murine hematopoietic cells by transfection of a retroviral vector expressing oncoprotein P210bcr/abl and preferential inhibition on Ph1-positive leukemia cell growth
Authors: Okabe M , Uehara Y , Miyagishima T , Itaya T , Tanaka M , Kuni-Eda Y , Kurosawa M , Miyazaki T
Published: October 2019

Herbimycin A, a benzoquinoid ansamycin antibiotic, was demonstrated to decrease intracellular phosphorylation by protein tyrosine kinase (PTK). In Philadelphia chromosome (Ph1)-positive leukemias such as chronic myelogenous leukemia (CML) and Ph1-positive acute lymphoblastic leukemia (ALL), both of which express bcr-abl fused gene products (P210bcr-abl or P190bcr-abl protein kinase) with augmented tyrosine kinase activities, herbimycin A markedly inhibited the in vitro growth of the Ph1-positive ALL cells and the leukemic cells derived from CML blast crisis. However, the same dose of herbimycin A did not inhibit in vitro growth of a broad spectrum of Ph1-negative human leukemia cells, and several other protein kinase antagonists also displayed no preferential inhibition. Furthermore, we demonstrated that herbimycin A has an antagonizing effect on the growth of transformed cells by a transfection of retroviral amphotrophic vector expressing P210bcr/abl into a murine interleukin (IL)-3-dependent myeloid FDC-P2 cell line. This inhibition was abrogated by the addition of sulfhydryl compounds, similar to the reaction previously described for Rous sarcoma virus transformation. The inhibitory effect of herbimycin A on the growth of Ph1-positive cells was associated with decreased bcr/abl tyrosine kinase activity, but no decrease of bcr-abl mRNA and protein, suggesting that the inactivation of bcr-abl tyrosine kinase activity by herbimycin A may be induced by its binding to the bcr-abl protein portion that is rich with sulfhydryl groups. The present study indicates that herbimycin A is a beneficial agent for the investigation of the role of the bcr-abl gene in Ph1-positive leukemias and further suggests that the development of agents inhibiting the bcr-abl gene product may offer a new therapeutic potential for Ph1-positive leukemias.
The type B receptor for tumor necrosis factor-alpha mediates DNA fragmentation in HL-60 and U937 cells and differentiation in HL-60 cells
Authors: Greenblatt MS , Elias L
Published: October 2019

Tumor necrosis factor-alpha (TNF) binds to two specific cell surface receptors, types A and B, which are both present on HL-60 and U937 cells, and induces monocytoid differentiation in HL-60 cells and early DNA fragmentation in HL-60 and U937 cells. To further define the receptors' roles, we studied how monoclonal antibodies (MoAbs) against each receptor affected TNF-induced cellular responses. HTR-9, an MoAb against the type B (low affinity, 55 Kd) receptor, reproduced all of these effects in a dose-dependent manner. UTR-1, an MoAb against the type A (high affinity, 75 Kd) receptor, had no effect in saturating doses, but supersaturating doses enhanced DNA fragmentation threefold. TNF and interferon gamma (IFN-gamma) synergistically induced morphologic differentiation and monocytic antigen expression, while the antitype B receptor MoAb was synergistic for morphologic response, but not antigen expression. Our results indicate that (1) the type B receptor mediates some responses to TNF in HL-60 and U937 cells, (2) the type A receptor does not stimulate these responses, (3) the TNF molecule is not necessary for some of these actions, and (4) TNF- induced morphologic changes and surface antigen expression in HL-60 cells may be regulated by separate postreceptor pathways.
Treatment of chronic myeloid leukemia with allogeneic bone marrow transplantation after preparation with BuCy2
Authors: Biggs JC , Szer J , Crilley P , Atkinson K , Downs K , Dodds A , Concannon AJ , Avalos B , Tutschka P , Kapoor N
Published: October 2019

One hundred fifteen patients with chronic myelocytic leukemia (CML) were administered busulphan 4 mg/kg for 4 days and cyclophosphamide 60 mg/kg on each of 2 days (BuCy2) followed by allogeneic bone marrow transplantation from histocompatible sibling donors. For 62 patients in chronic phase, 26 in accelerated phase, and 27 in blast transformation, the actuarial survival at 3 years was 58%, 41%, and 25%, respectively. Actuarial probability of relapse was 3%, 12%, and 27%, respectively. Only two patients in chronic phase showed a transient cytogenetic relapse and one of these died from subsequent transplant-related complications, whereas the other remains cytogenetically normal 697 days posttransplant. Patients who were transplanted within 1 year of diagnosis in chronic phase had a survival of 70% compared with 40% when transplanted beyond 1 year from diagnosis. This significant difference in survival was due to transplant-related complications and was correlated with previous exposure to high doses of busulphan. This study indicates that BuCy2 is a useful conditioning regimen for marrow transplantation in patients with CML and results in similar survival statistics and transplant-related mortality as would be expected with conditioning regimens containing total body irradiation. It is possible that relapse after BuCy2 may be lower than expected with regimens containing total body irradiation, but larger analyses are required.
Unexpected heterogeneity in E2A/PBX1 fusion messenger RNA detected by the polymerase chain reaction in pediatric patients with acute lymphoblastic leukemia
Authors: Izraeli S , Kovar H , Gadner H , Lion T
Published: October 2019

The t(1;19)(q23;p13) is the most common recurring chromosomal translocation in childhood acute lymphoblastic leukemia (ALL) and has been associated with adverse prognosis. It involves the rearrangement of two genes, PBX1 and E2A, resulting in the production of transforming chimeric DNA-binding proteins. In all previous reports in which the presence of a chimeric transcript was described, the fusion point between the coding sequences of E2A and PBX1 was found to be constant at the RNA level. We have used RNA-based polymerase chain reaction (PCR) for the detection of E2A/PBX1 messenger RNAs (mRNAs) in children with ALL at the time of diagnosis. Of 21 patients exhibiting this rearrangement, 3 (14%) expressed a variant E2A/PBX1 transcript in addition to the expected one. The relative amounts of the two chimeric mRNAs varied between the patients, but remained constant in the same patient during different stages of the disease. Sequence analysis showed an identical insertion of 27 bp at the E2A/PBX1 junction of the variant RNA species, the translation of which would result in the replacement of Val478 by 10 amino acids. The inserted sequence has not been detected in any other human transcript besides the variant E2A/PBX1 RNA species and probably represents a splicing variant of the chimeric RNA. We conclude that a subset of pediatric patients with ALL that carry the E2A/PBX1 rearrangement express two types of the chimeric mRNA. The biologic significance of this additional E2A/PBX1 transcript is discussed.
Granulocyte colony-stimulating factor to prevent dose-limiting neutropenia in non-Hodgkin's lymphoma: a randomized controlled trial
Authors: Pettengell R , Gurney H , Radford JA , Deakin DP , James R , Wilkinson PM , Kane K , Bentley J , Crowther D
Published: October 2019

The effect of granulocyte colony-stimulating factor (G-CSF) on neutropenia, infection, and cytotoxic chemotherapy administration was studied in a randomized trial in patients receiving intensive weekly chemotherapy for non-Hodgkinxybs lymphoma (NHL). Eighty patients (aged 16 to 71 years) with high-grade NHL (Kiel) of any stage were randomized to receive VAPEC-B chemotherapy alone (39 patients) or with G-CSF administered as a daily subcutaneous dose of 230 micrograms/m2 (41 patients). Prophylactic ketoconazole and cotrimoxazole were administered to all patients throughout treatment. The protocol specified identical dose modification and antibiotic treatment criteria bor both groups. Neutropenia (absolute neutrophil count [ANC] less than 1.0 x 10(9)/L) occurred in 15 of 41 (37%) of the G-CSF-treated patients and in 33 of 39 (85%) of the controls, giving a relative risk for control patients of 2.31 (95% confidence interval [CI], [1.51, 3.54]; P = .00001). Fever (greater than or equal to 37.5 degrees C) with neutropenia (ANC less than 1.0 x 10(9)/L) occurred in 9 of 41 (22%) of the G-CSF group and in 17 of 39 (44%) of the controls (relative risk for control, 2.26; 95% CI [1.01, 5.06]; P = .04). There were fewer treatment delays, with shorter duration (P = .01) in patients receiving G-CSF. Chemotherapy doses were reduced in 4 of 41 (10%) of the G-CSF patients and 13 of 39 (33%) of the controls (P = .01). The dose intensity of cytotoxic chemotherapy was significantly increased in patients receiving G-CSF (median of 95% in G-CSF group compared with 83% in control patients). Three vascular deaths occurred in the G-CSF group. Delays in the control group most commonly resulted from neutropenia (19 patients, compared with 2 patients in the G-CSF-treated group, P = .000007). Severe mucositis was the major dose-limiting toxicity in G-CSF-treated patients, but did not occur more frequently than in controls (15 patients in each group). Overall, patients randomized to receive G-CSF achieved a greater dose intensity than control patients, but this did not result in significant differences in drug toxicity (other than neutropenia), intravenous antibiotic usage, or hospitalization between the two groups.
Growth hormone exerts hematopoietic growth-promoting effects in vivo and partially counteracts the myelosuppressive effects of azidothymidine
Authors: Murphy WJ , Tsarfaty G , Longo DL
Published: October 2019

Recombinant human growth hormone (rhGH) was administered to mice to determine its effect on hematopoiesis. BALB/c mice and mice with severe combined immune deficiency (SCID), which lack T cells and B cells, were administered intraperitoneal injections of rhGH for 7 days. Upon analysis, both strains of mice exhibited an increase in splenic and bone marrow hematopoietic progenitor cell content and cellularity, indicating that rhGH can act as a hematopoietic growth factor. C57BL/6 mice were then placed on azidothymidine (AZT). AZT is a reverse transcriptase inhibitor currently used as a treatment for acquired immune deficiency syndrome (AIDS), but which also produces significant myelotoxic effects. Treatment of mice with rhGH partially counteracted the myelosuppressive properties of AZT. Bone marrow cellularity, hematocrit values, white blood cell counts, and splenic hematopoietic progenitor cell content were all significantly increased if rhGH (20 micrograms injected intraperitoneally every other day) was concurrently administered with AZT. Administration of ovine GH (ovGH), which, unlike rhGH, has no effect on murine prolactin receptors, also prevented the erythroid-suppressive effects of AZT in mice, but had no significant effect on granulocyte counts. Thus, the effects of GH are mediated at least in part through GH receptors in vivo. Additionally, when mice were initially myelosuppressed by several weeks of AZT treatment, the subsequent administration of ovGH resulted in an increase in splenic hematopoietic progenitor cells. No significant pathologic effects were observed in mice receiving either repeated rhGH or ovGH injections. Thus, GH exerts significant direct hematopoietic growth-promoting effects in vivo and may be of potential clinical use to promote hematopoiesis in the face of myelotoxic therapy.
Heparin enhances active site-dependent binding of tissue-type plasminogen activator to endothelial cells
Authors: Rosenfeld L , Kuo A , Hirsh J , Klugherz B , Gardell SJ , Cines DB , Barnathan ES
Published: October 2019

Human umbilical vein endothelial cells (HUVEC) in culture express two classes of binding sites for tissue-type plasminogen activator (t-PA). The high-affinity binding site has been identified as PA inhibitor type 1 (PAI-1), which binds to the catalytic portion of the molecule, while the second site binds t-PA through an active-site independent domain. Because recombinant t-PA (rt-PA) is often administered concomitantly with heparin, we investigated the effects of heparin on rt-PA binding to HUVEC. Preincubation of HUVEC with heparin at 4 degrees C increased the binding of radiolabeled rt-PA in a time- and dose-dependent manner. One-half maximal increase in binding was observed within 10 minutes of heparin addition. When HUVEC were preincubated with optimal concentrations (5 U/mL) of heparin for 4 hours at 4 degrees C, a 2.5- +/- 0.2-fold increase in specific binding was observed (mean +/- SEM, n = 12, P less than .01). Other highly sulfated glycosaminoglycans and fucoidan (a sulfated polymer of fucose) stimulated rt-PA binding as well, whereas glycosaminoglycans with lower sulfate content than heparin did not. Several results suggested that heparin increased the binding of rt-PA to “cell-associated” PAI-1. First, only active-site- dependent binding was enhanced by heparin, whereas binding of active- site blocked rt-PA was not affected. Second, extracts from HUVEC preincubated with heparin contained increased amounts of rt-PA-PAI-1 complexes as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Third, antibodies to PAI-1 blocked the increased binding entirely. HUVEC preincubated with heparin also bound increased amounts of enzymatically active radiolabeled urokinase-type PAs. However, HUVEC preincubated with heparin did not express increased amounts of immunoreactive PAI-1. Therefore, heparin, at therapeutic concentrations, may enhance or stabilize the association of PAs with endothelial cell-associated PAI-1.
Unusual diffuse liver fibrosis accompanying transient myeloproliferative disorder in Down's syndrome: a report of four autopsy cases and proposal of a hypothesis
Authors: Miyauchi J , Ito Y , Kawano T , Tsunematsu Y , Shimizu K
Published: October 2019

Transient myeloproliferative disorder (TMD), an acute leukemia-like disorder in neonates with Down's syndrome, is characterized by spontaneous regression of abnormal blast growth. Because proliferating blasts frequently express phenotypes of megakaryocytic lineage and, as a result, this disorder resembles acute megakaryoblastic leukemia (AMKL), it would be of interest to determine whether myelofibrosis, a common complication of AMKL, is also present in TMD. Pathologic observations of four autopsy cases of TMD showed that myelofibrosis was not present in any of them, whereas intralobular diffuse liver fibrosis was present in all of them. Laboratory data of four additional cases showed hepatic dysfunction in all of them, suggesting a close association between hepatic lesions and TMD. From these results, we propose a hypothesis that the abnormal blasts with megakaryocytic properties in TMD originate from the fetal liver and cause liver fibrosis, as AMKL cells are thought to cause myelofibrosis by producing collagen-stimulating cytokines in the bone marrow. This hypothesis also seems to explain some other unique aspects of TMD.
Human lymphotropic retroviruses associated with mycosis fungoides: evidence that human T-cell lymphotropic virus type II (HTLV-II) as well as HTLV-I may play a role in the disease
Authors: Zucker-Franklin D , Hooper WC , Evatt BL
Published: October 2019

The human T-cell lymphotropic virus type I (HTLV-I) is causally associated with adult T-cell leukemia, but its role in mycosis fungoides (MF) has remained enigmatic. The virus is suspect because a small percentage of patients with MF have antibodies to it, the cells of others harbor deleted HTLV-I proviral sequences, and particles resembling HTLV-I emerge in cultured blood lymphocytes obtained from most patients. An alternative possibility is that disparate lymphotropic retroviruses may infect or affect a population of epidermotropic lymphocytes, leading to the same outcome, ie, MF. In studies designed to identify the particles detected in lymphocyte cultures of nine patients with a diagnosis of skin involvement characteristic of MF, this concept has gained support. While the cells of four patients provided evidence of HTLV-I infection, molecular hybridization with HTLV-II-specific pol probes showed HTLV-II in the cells of another patient. The 103-bp fragment amplified by the HTLV-II- specific probe was sequenced and proved to have greater than 90% homology with the same fragment amplified from cells known to be infected with HTLV-II. A role for HTLV-II in MF has not been suggested heretofore. Therefore, HTLV-I, HTLV-II, and their incomplete forms may be found in cells of MF patients, suggesting new theories regarding the pathogenesis of this disease.
Changes in Na,K-ATPase gene expression during granulocytic differentiation of HL60 cells
Authors: Chambers SK , Gilmore-Hebert M , Kacinski BM , Benz EJ Jr
Published: October 2019

During granulocytic differentiation of HL60 cells, immediate reduction of ouabain-sensitive potassium flux is observed within the first 12 hours of addition of dimethyl sulfoxide (DMSO). We show that gene expression of the alpha 3 isoform of Na+,K(+)-ATPase, which encodes an ouabain-inhibitable Na+,K(+)-ATPase activity, significantly declines during the first 24 hours of granulocytic differentiation by DMSO of HL60 cells. The more common alpha 1 isoform decreases, but more gradually over 72 hours of DMSO induction. Loss of alpha 3 and alpha 1 messenger RNA (mRNA) are due to changes in mRNA decay; their transcription is not altered. alpha 3 mRNA half-life is 3 hours in HL60 cells; upon induction by 16 hours of DMSO, it decreases to approximately 2 hours. alpha 1 transcripts are less sensitive to DMSO induction, with their half-life being 3.5 hours in HL60 cells; upon induction, their half-life decreases to 3 hours. Experiments measuring protein stability confirm that alpha 3 protein is more labile than alpha 1. In uninduced HL60 cells, alpha 3 membrane protein comprises 30% of the total alpha isoforms, and is less stable than alpha 1, with a protein half-life of only 9 hours. Upon DMSO induction, steady-state alpha 3 protein decreases markedly within 10 hours, whereas alpha 1 protein remains stable. These results show that posttranscriptional changes during induction play a major role in the differential regulation of alpha 1 and alpha 3 isoforms of Na+,K(+)-ATPase; regulation of the latter may be important for early granulocytic differentiation, or for one of the differentiated functions of mature granulocytes.
Specific marrow ablation before marrow transplantation using an aminophosphonic acid conjugate 166Ho-EDTMP
Authors: Appelbaum FR , Brown PA , Sandmaier BM , Storb R , Fisher DR , Shulman HM , Graham TC , Schuening FG , Deeg HJ , Bianco JA
Published: October 2019

166Holmium ethylenediaminetetramethylene phosphonic acid (166Ho-EDTMP) is a short-lived beta-emitting radionuclide complexed to an aminophosphonate ligand that we have investigated in a canine model as a potential agent for specific marrow ablation before marrow transplantation. After intravenous injections, 166Ho-EDTMP distributed principally to bone and after 24 hours the concentrations of 166Ho- EDTMP in bone were more than 200-fold higher than in any other organ. Increasing dosages of 166Ho-EDTMP led to increasingly prolonged and severe myelosuppression, but myeloablation was not achieved. Histologic examination of recovering animals suggested that the spleen may have acted as a reservoir for circulatory hematopoietic precursors. Four splenectomized animals administered 20 to 30 mCi/kg 166Ho-EDTMP without marrow transplantation died with marrow aplasia, while four splenectomized animals administered similar dosages of 166Ho-EDTMP followed by autologous transplantation recovered. The dose-limiting toxicity of 166Ho-EDTMP appeared to be marrow stromal damage resulting in myelofibrosis, which was reversible. These results suggest that 166Ho-EDTMP can be used to specifically ablate marrow function before marrow transplantation.
Low-risk intensive therapy for multiple myeloma with combined autologous bone marrow and blood stem cell support
Authors: Jagannath S , Vesole DH , Glenn L , Crowley J , Barlogie B
Published: October 2019

To improve the safety of autotransplantation for myeloma, peripheral blood stem cell (PBSC) collection was attempted in 75 previously treated patients after the administration of high-dose cyclophosphamide (HD-CTX; 6 g/m2) with or without granulocyte-macrophage colony- stimulating factor (GM-CSF). Sixty patients subsequently received melphalan 200 mg/m2 (57 patients) or melphalan 140 mg/m2 and total body irradiation (850 cGy) (3 patients) supported by both autologous bone marrow and PBSC; 38 patients received GM-CSF posttransplantation. Among 72 patients undergoing PBSC apheresis, “good” mobilization (greater than 50 colony-forming units granulocyte-macrophage [CFU-GM] per 10(5) mononuclear cells) was achieved when prior chemotherapy did not exceed 1 year and when GM-CSF was used post-HD-CTX; similarly, rapid platelet recovery to 50,000/microL within 2 weeks was associated with “good” PBSC mobilization. These same variables also predicted for rapid engraftment after autotransplantation, so that hematologic recovery (granulocytes greater than 500/microL and platelets greater than 50,000/microL) proceeded within 2 weeks among the 37 patients with “good” PBSC collection. As a result of rapid neutrophil recovery (greater than 500/microL) within a median of 2 weeks, infectious complications both post-HD-CTX and posttransplant were readily manageable, resulting in only one treatment-related death post-HD-CTX. The cumulative response rate (greater than or equal to 75% cytoreduction) for all 75 patients was 68%, with 12-month event-free and overall survival projections of about 85%. Using both bone marrow and PBSC together with GM-CSF, autotransplants are safe and appear effective in myeloma, especially when prior therapy had been limited to less than 1 year. More than 80% of transplanted patients achieved complete hematologic recovery within a median of 1 month posttransplant (granulocytes greater than 1,500/microL; platelets greater than 100,000/microL; hemoglobin greater than 10 g%), thus providing sufficient hematopoietic reserve for further chemotherapy in the event of posttransplant relapse.
Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel factor to stimulate proliferation of the human factor-dependent cell line, M07e
Authors: Miyazawa K , Hendrie PC , Kim YJ , Mantel C , Yang YC , Kwon BS , Broxmeyer HE
Published: October 2019

Human interleukin-9 (IL-9) was originally identified and cloned based on its stimulatory effect on proliferation of human myeloid cell line, M07e. IL-9 synergized with Steel factor, the ligand for the c-kit product, to stimulate M07e cell proliferation. To investigate potential mechanisms for this, IL-9 was assessed for effects on protein tyrosine kinase activities in M07e cells by immunoblotting with anti- phosphotyrosine monoclonal antibody; results were compared with those of Steel factor alone and in combination with IL-9, and those of 12–0- tetradecanoyl phorbol-13-acetate (TPA). Recombinant human IL-9 (10 ng/mL) rapidly and transiently induced or enhanced at least four tyrosine phosphorylated protein bands with molecular weights of 105, 97, 85, and 81 Kd. This tyrosine phosphorylation pattern was different from that generated by recombinant murine Steel factor or TPA stimulation and the combination of IL-9 and Steel factor did not change the IL-9-induced pattern. IL-9-induced tyrosine phosphorylated bands were completely blocked by treatment of IL-9 with anti-IL-9 antibody under conditions that also neutralized the synergistic effect of IL-9 with Steel factor on M07e cell proliferation. Genistein, a tyrosine kinase inhibitor, blocked phosphorylation of IL-9 and Steel factor- induced bands. Unlike Steel factor or TPA, IL-9 did not appear to stimulate phosphorylation of 42-Kd mitogen-activated protein (MAP) kinase or Raf-1, or enhance MAP kinase activity. MAP kinase and Raf-1 are serine/threonine kinases that are phosphorylated and activated by many growth factors and by agonists for protein kinase C. While the combination of IL-9 plus SLF did not appear to induce phosphorylation of new bands not already seen with either IL-9 or SLF alone, or enhance the phosphorylation of those bands seen with either cytokine alone, the results suggest that IL-9 activates specific and unique signal transduction pathways.
CD34+ marrow cells, devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelopoiesis in lethally irradiated allogeneic baboons
Authors: Andrews RG , Bryant EM , Bartelmez SH , Muirhead DY , Knitter GH , Bensinger W , Strong DM , Bernstein ID
Published: October 2019

CD34+ cells devoid of detectable mature and immature T and B lymphocytes, expressing the CD2, CD10, and CD20 antigens, were isolated from marrows of three pairs of sex-mismatched, mixed lymphocyte culture (MLC) nonreactive, sibling baboons. Reciprocal transplants were performed between members of each pair, using the sex chromosomes, identified by standard cytogenetic techniques, as markers of the transplanted cells. Five animals from these three pairs were transplanted with 0.6 to 2.1 x 10(6)/kg of isolated cryopreserved and/or fresh isolated cells that were greater than 95% to 97% CD34+. Before transplantation, animals were treated with either single (920 or 1,020 cGy) or split (700 cGy x 2) dose total body irradiation. All animals engrafted with donor cells, as demonstrated by cytogenetic analysis of bone marrow metaphase cells 4 weeks after transplantation, with days to white blood cell count (WBC) greater than 500 being 19 +/- 2, to WBC greater than 1,000 23 +/- 2, to absolute neutrophil count greater than 500 24 +/- 3, and to platelets greater than 20,000 30 +/- 7. Three animals died of infectious-related complications at 34, 42, and 109 days after transplantation with evidence of host and donor cells (mixed chimerism) in marrow. Two animals remain alive and healthy more than 545 and 455 days after transplantation with stable mixed chimerism in marrow and blood. For these two animals, cytogenetic analysis of granulocyte/macrophage and erythroid colonies derived from marrow precursors between weeks 25 and 42 posttransplant showed evidence of mixed chimerism. Cytogenetic studies of CD2+ T cells and CD20+ B cells isolated from blood of these two animals between weeks 21 and 51 posttransplant showed the presence of mixed chimerism in both lymphocyte populations. Thus, isolated allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes can engraft and reconstitute stable long-term myelopoiesis and lymphopoiesis in lethally irradiated baboons. These results are consistent with the hypothesis that CD34+ marrow cells contain pluripotent hematopoietic stem cells capable of fully reconstituting lymphohematopoiesis in the transplanted host.
The role of macrophages in the regulation of erythroid colony growth in vitro
Authors: Wang CQ , Udupa KB , Lipschitz DA
Published: October 2019

Depletion of macrophages from murine marrow by the use of a monoclonal anti-macrophage antibody resulted in a significant increase in the number of erythroid burst forming units (BFU-E). This increase could be neutralized by the addition back to culture of macrophages or macrophage conditioned medium indicating that the suppression was mediated by soluble factors. To further characterize this effect, the addition to culture, either alone or in combination, of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the growth of BFU-E and the colony-forming unit granulocyte-macrophage (CFU-GM) was examined in macrophage-containing and macrophage-depleted cultures. The addition of IL-1 alpha to culture stimulated the release of both TNF alpha and GM-CSF and acted synergistically with both cytokines, resulting in a dose-dependent suppression of BFU-E and stimulation of CFU-GM growth. The increase in CFU-GM caused by the addition of IL-1 alpha was mediated by GM-CSF but not by TNF alpha as the increase was prevented by the addition of a monoclonal anti-GM-CSF antibody but not by anti-TNF alpha. When either TNF alpha or GM-CSF was neutralized by monoclonal antibodies the addition of IL-1 alpha resulted in a significant increase in BFU-E growth. The addition of GM-CSF to culture caused a dose-dependent suppression of BFU-E that was mediated by TNF alpha, as colony number was not reduced when GM-CSF and a monoclonal anti-TNF alpha antibody were simultaneously added to culture. TNF alpha- induced suppression of BFU-E only occurred in the presence of macrophages. In macrophage-depleted cultures, a dose-dependent suppression of BFU-E could be induced if subinhibitory concentrations of IL-1 alpha or GM-CSF were simultaneously added with increasing concentrations of TNF alpha. The effects of IL-1 alpha or GM-CSF and TNF alpha were markedly synergistic so that the doses required to induce suppression when added simultaneously was only 10% of that required when either were added to culture alone. Suppression of BFU-E by GM-CSF or the combined addition of GM-CSF and TNF alpha did not require IL-1 alpha because inhibition was not neutralized by the addition of anti-IL-1 alpha antibody.
CD4 is expressed on murine pluripotent hematopoietic stem cells
Authors: Wineman JP , Gilmore GL , Gritzmacher C , Torbett BE , Muller-Sieburg CE
Published: October 2019

We show here for the first time that pluripotent hematopoietic stem cells express the CD4 antigen. CD4+ cells isolated from mouse marrow repopulated all hematopoietic lineages in both the long-term repopulation assay and the competitive repopulation assay. This finding indicates that the CD4+ population contains primitive stem cells with extensive repopulation capacity. Interestingly, the CD4- population had significant life-sparing activity, even though this population was depleted of long-term repopulating stem cells when compared with CD4+ cells. The majority of the cells that respond to the stroma in Whitlock- Witte cultures with B-cell differentiation were recovered in the CD4- population. Thus, this bone marrow (BM)-derived B-cell precursor lacks CD4, which is in contrast to myeloid precursors and thymus-derived lymphoid precursors that reportedly express CD4. We show further that the CD4 molecule expressed on BM cells is similar in molecular weight and epitope makeup to the CD4 antigen found on thymocytes. Detection of CD4 on BM cells is dependent on using high concentrations of antibodies. Thus, it is not surprising that expression of CD4 on pluripotent stem cells has been missed previously. Taken together, our data suggest that the CD4 molecule may play an important role in lineage definition in early hematopoietic differentiation.
A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells
Authors: Scott LM , Civin CI , Rorth P , Friedman AD
Published: October 2019

Members of the CCAAT/enhancer binding protein (C/EBP) family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBP alpha are found only in mature, nondividing cells. Using Western blotting and immunohistochemical staining, we have determined the temporal order of expression for C/EBP alpha, C/EBP beta, and C/EBP delta in differentiating myelomonocytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBP alpha expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While we have detected C/EBP alpha, C/EBP beta, and C/EBP delta in multiple myeloid leukemia cell lines, and C/EBP alpha in normal myeloid cells and in de novo human myeloid leukemias, we have not detected these C/EBP isoforms in either erythroid or lymphoid cells. Finally, we show that C/EBP alpha, C/EBP beta, and C/EBP delta protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes.
Stem cell factor enhances proliferation, but not maturation, of murine megakaryocytic progenitors in serum-free culture
Authors: Tanaka R , Koike K , Imai T , Shiohara M , Kubo T , Amano Y , Komiyama A , Nakahata T
Published: October 2019

The effects of recombinant rat stem cell factor (SCF/c-kit ligand) on murine megakaryocytopoiesis were studied using partially purified bone marrow cells derived from normal and 5-fluorouracil (5-FU)-treated mice in a serum-free culture system. SCF alone did not support the formation of megakaryocyte (M) and granulocyte-macrophage-megakaryocyte (GMM) colonies. However, the addition of SCF to cultures containing interleukin-3 (IL-3) resulted in a significant increase in the number of M and GMM colonies formed by bone marrow cells from normal mice, whereas IL-6 augmented only M colony growth. The stimulatory effect of SCF was approximately three to four times as high as that of IL-6 on the primitive progenitors capable of megakaryocytic-lineage expression derived from 5-FU-treated mice. In addition, SCF, but not IL-6, significantly increased the number of constituent cells in the individual M colonies supported by IL-3. On the other hand, SCF did not exert any effect on the size and DNA content of megakaryocytes in IL-3- dependent M and GMM colonies, whereas IL-6 enhanced the maturation of megakaryocytes. These results suggest that SCF stimulates the proliferative process in megakaryocytic progenitors and that the main activity of IL-6 is the promotion of megakaryocyte maturation.
Clonal involvement of granulocytes and monocytes, but not of T and B lymphocytes and natural killer cells in patients with myelodysplasia: analysis by X-linked restriction fragment length polymorphisms and polymerase chain reaction of the phosphoglycerate kinase gene
Authors: van Kamp H , Fibbe WE , Jansen RP , van der Keur M , de Graaff E , Willemze R , Landegent JE
Published: October 2019

To determine the clonal nature of hematopoiesis and to assess lineage involvement in patients with myelodysplastic syndromes (MDS), we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Eleven female MDS patients heterozygous for at least one of these probes were studied: 3 with refractory anemia (RA), 2 with RA with ringed sideroblasts (RARS), 2 with chronic myelomonocytic leukemia (CMML), and 4 with RA with excess of blasts in transformation (RAEB-t). All exhibited clonal hematopoiesis as determined by Southern analysis of DNA prepared from peripheral blood (PB) and/or bone marrow (BM) cells. In three of the six patients heterozygous for the PGK1 gene, purified cell suspensions of polymorphonuclear cells (PMN), monocytes, lymphocytes, and/or T cells prepared from PB were tested. In addition, five of these patients were analyzed by a polymerase chain reaction (PCR)-based procedure as described recently. This method was slightly adapted to facilitate the analysis of cell lysates of fluorescence- activated cell sorted (FACS) monocytes, T and B lymphocytes, and natural killer (NK) cells. The outcome of Southern and PCR analysis was concordant, showing that PMN and monocytes were clonally derived, whereas circulating T and B lymphocytes and NK cells exhibited random X- chromosome inactivation compatible with a polyclonal pattern. To address the question of whether T cells are derived from unaffected progenitor cells or that their origin had antedated the onset of MDS, naive and memory T cells were analyzed separately. Both subsets showed a polyclonal pattern. However, in one patient analysis of constitutive DNA suggested a skewed methylation, and the presence of clonal lymphocytes against a background of polyclonal lymphoid cells cannot be ruled out in this patient. PCR analysis of PB and BM cells showed a nonrandom, unilateral pattern of X-inactivation, compatible with a mixture of clonally (myeloid) and polyclonally (lymphoid) derived cells. In conclusion, in some patients, MDS represents a disorder with clonal hematopoiesis restricted to cells of myeloid origin, whereas a random X-inactivation pattern is found in lymphoid cells.
Deletions involving two distinct regions of 6q in B-cell non-Hodgkin lymphoma
Authors: Gaidano G , Hauptschein RS , Parsa NZ , Offit K , Rao PH , Lenoir G , Knowles DM , Chaganti RS , Dalla-Favera R
Published: October 2019

The recurrent loss of genetic material from a specific chromosomal region in a given tumor type suggests the presence of a tumor- suppressor gene, the loss or inactivation of which may be relevant for tumorigenesis. In this study, we provide molecular evidence for the recurrent association between deletions on the long arm of chromosome 6 and B-cell non-Hodgkin lymphoma (B-NHL). Normal and tumor DNAs from 71 cases of B-NHL were studied for loss of constitutional heterozygosity (LOH) at 19 loci on chromosome 6 using a panel of restriction fragment length polymorphism (RFLP) probes. LOH, indicating deletion of all or part of 6q, was detected in 16 of 71 cases (22.5%), ranging from low- grade to high-grade B-NHL. The isolated loss of 6p or the loss of other chromosomes (8, 17, 22) tested as controls for specificity was not observed in any case. Comparison of the extent of the deletions among different cases allowed the identification of two distinct regions of minimal deletion (RMD) at 6q25 to 6q27 (RMD-1) and at 6q21 to 6q23 (RMD- 2), respectively, suggesting the existence of two tumor-suppressor genes. These data support a role for 6q deletions in B-NHL pathogenesis and provide a basis for identifying the corresponding tumor-suppressor genes.
Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells
Authors: Vyas P , Sharpe JA , Watt P , Higgs DR , Wood WG
Published: October 2019

Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.