bluuebunny/crossref_metadata_2025 huggingface dataset:
Pentoxifylline inhibits integrin-mediated adherence of interleukin-2- activated human peripheral blood lymphocytes to human umbilical vein endothelial cells, matrix components, and cultured tumor cells
Authors: Kovach NL , Lindgren CG , Fefer A , Thompson JA , Yednock T , Harlan JM
Published: October 2019

Peripheral blood lymphocytes (PBLs) cultured in the presence of recombinant human interleukin-2 (rhIL-2) develop a natural killer (NK) cell phenotype (CD16+, CD56+, CD3-) and are referred to as lymphokine- activated killer cells (LAK). In developing the LAK phenotype, enhanced adherence to matrix components and endothelial cells have been described. In this report we investigated the functional behavior of adhesion receptors in rhIL-2-activated PBLs by in vitro adhesion assay and by flow cytometry. Compared to PBLs, IL-2-activated PBLs had increased integrin-mediated adherence to: (1) fibronectin (FN), (2) human umbilical vein endothelial (HUVE) cells, and (3) cultured melanoma and pancreatic tumor cell lines. This increase in adherence was mediated by increased surface expression of members of the beta 1 and beta 2 integrin subfamilies, as determined by flow cytometric analysis. No induction of an activation-dependent beta 1 (CD29) epitope was detected. We also investigated the effects of the methylxanthine derivative pentoxifylline (PTX) on PBLs and rhIL-2-activated PBL adhesion. PBLs co-cultivated in the presence of rhIL-2 (1,000 U/mL) and PTX exhibited reduced adherence to FN, HUVE and cultured tumor cell lines. This inhibition by PTX was concentration- and time-dependent. The increased expression of integrins induced by rhIL-2 was only in part inhibited by PTX, suggesting that PTX induced a subpopulation of integrins that are expressed but functionally inactive.
Interleukin-13 inhibits the proliferation of normal and leukemic human B-cell precursors
Authors: Renard N , Duvert V , Banchereau J , Saeland S
Published: October 2019

Interleukin-13 (IL-13) is a T-cell-derived cytokine that displays homology with IL-4 and shares some of its biologic functions. We investigated the effects of IL-13 on normal human B-cell precursors (BCP) and their malignant counterparts in B-lineage acute lymphoblastic leukemia (BCP-ALL). IL-13 inhibited growth of CD19+ slg- normal BCP cultured in the presence or absence of bone marrow accessory stromal cells and IL-7. In addition, IL-13 inhibited proliferation of blasts isolated from leukemic patients and cells from established BCP-ALL lines. Differences were observed in a number of cases with respect to growth inhibition in response to IL-13 and IL-4. These results suggest heterogeneity in the expression of IL-13 and IL-4 receptors in B-cell ontogeny. Growth-inhibition by IL-13 could be reverted by anti-IL-4 receptor antibody, indicating that the IL-13 and IL-4 binding chains can be closely associated on BCP. We further showed that the inhibitory effect of IL-13 results from decreased cell-cycle activity. Finally, whereas IL-13 induced CD23 expression on BCP-ALL cells, it did not promote differentiation into slg+ B lymphocytes.
Hereditary overexpression of adenosine deaminase in erythrocytes: studies in erythroid cell lines and transgenic mice
Authors: Chen EH , Mitchell BS
Published: October 2019

Overexpression of adenosine deaminase (ADA) in red blood cells is characterized by a marked, tissue-specific increase in levels of structurally normal ADA mRNA and enzymatic activity in the erythrocytes of affected individuals, leading to adenosine triphosphate (ATP) depletion and hemolytic anemia. This autosomal dominant trait is linked to the ADA gene. To investigate the molecular mechanism responsible for this disorder, we examined relative reporter gene activity using constructs containing 10.6 kb of 5′ flanking sequence and 12.3 kb of the first intron of the ADA gene from the normal and mutant alleles. No differences in chloramphenicol acetyltransferase (CAT) activity were found in transient transfection experiments using erythroleukemia cell lines. Transgenic mice containing the ADA constructs expressed CAT in the appropriate tissue-specific fashion, with 10(2)- to 10(4)-fold higher activity in the thymus. However, CAT activities in erythrocytes and bone marrow of mice containing high transgene copy numbers did not differ between the normal and mutant alleles. These results indicate that the mutation responsible for ADA overexpression is unlikely to reside in the 5′ and promoter regions or in the regulatory regions of the first intron. It is possible that the erythroid-specific overexpression of ADA results from a mutation at some distance from the gene or requires an interaction of a proximal mutation with more distal DNA elements.
Prenatal diagnosis of pyruvate kinase deficiency
Authors: Baronciani L , Beutler E
Published: October 2019

Prenatal testing for pyruvate kinase deficiency is often requested by parents who already have an affected child. However, before the development of molecular biologic techniques there were no suitable diagnostic methods. We present here two cases in which the diagnosis was established, one using amniotic fluid cells, the other cord blood. Two different approaches were used. The first, using a direct method of PCR amplification and restriction endonuclease analysis, detected mutations in fetus genomic DNA. The second method, using two polymorphic sites linked to the PKRL gene, enabled us to establish which chromosome had been inherited from each parent.
Busulfan pharmacokinetics using a single daily high-dose regimen in children with acute leukemia
Authors: Shaw PJ , Scharping CE , Brian RJ , Earl JW
Published: October 2019

The pharmacokinetics of busulfan, given as a single daily dose (either 4 mg/kg or 150 mg/m2), was determined in 22 children undergoing bone marrow transplantation for acute leukemia. The single daily dose regimen showed similar pharmacokinetics to previously reported regimens of 4 x 1 mg/kg, except for fourfold higher mean peak plasma levels and negligible trough levels. Daily systemic exposure for single dose regimens based on weight (4 mg/kg) or surface area (150 mg/m2), respectively were very similar to regimens of (4 x 1 mg/kg) or (4 x 37.5 mg/m2). Dose (milligrams per kilogram), peak plasma level, and area under the curve (AUC) were all higher in 12 children treated with 150 mg/m2 busulfan than in 9 children treated with 4 mg/kg. AUC was age dependent for the 4 mg/kg dose but not for the 150 mg/m2 dose. The use of a 150 mg/m2 dose allows escalation of the dose above 4 mg/kg, eliminating the tendency for younger children to receive lower systemic exposure. Little toxicity was observed in this study. Clearance and distribution volume correlated negatively with age, and AUC correlated positively with dose (milligram per kilogram). Administration of busulfan as crushed rather than whole tablets reduced the delay time for appearance of busulfan in plasma but had no effect on absorption or other pharmacokinetic parameters.
Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells
Authors: Zeigler FC , Bennett BD , Jordan CT , Spencer SD , Baumhueter S , Carroll KJ , Hooley J , Bauer K , Matthews W
Published: October 2019

The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor. Cell cycle analysis shows that stem cells that do not express the receptor have a greater percentage of the population in G0 when compared with the flk-2/flt-3- positive population. Development of agonist antibodies to the receptor shows a proliferative role for the receptor in stem cell populations. Stimulation with an agonist antibody gives rise to an expansion of both myeloid and lymphoid cells and this effect is enhanced by the addition of kit ligand. These studies serve to further illustrate the importance of the flk-2/flt-3 receptor in the regulation of the hematopoietic stem cell.
Persistence of the same viral strain in early and late relapses of Epstein-Barr virus-associated Hodgkin's disease
Authors: Brousset P , Schlaifer D , Meggetto F , Bachmann E , Rothenberger S , Pris J , Delsol G , Knecht H
Published: October 2019

Twelve cases of relapsing Hodgkin's disease were investigated for the presence of Epstein-Barr virus (EBV). Of these, 7 cases contained EBV gene products (LMP1, EBER RNA) in the diagnostic Reed-Sternberg cells and variants at first presentation and at relapse(s), whereas 5 cases were negative at both first diagnosis and relapse. Among the 7 EBV- positive cases, material for DNA extraction was available in 2 cases at both diagnosis and relapse(s). Ig and T-cell receptor gene rearrangements displayed a germline configuration in the 2 cases. However, Southern blot analysis of the terminal repeats (TR) of EBV genome showed that, in 1 of the 2 cases, the fragment was of the same size at diagnosis and in the subsequent two relapses (1 early and 1 late). The second case contained monoclonal EBV genome at diagnosis, but the Southern analysis of the TR was negative at relapse. The latent membrane protein (LMP1) sequence analysis confirmed the persistence of a distinctive viral strain in each of the 2 cases with individual abnormalities within the carboxy terminal region (5 point mutations and a 30-bp deletion for the first case and 6 point mutations for the second case). The persistence of a given strain in early and late relapses is evidence towards the view that in Hodgkin's disease such relapses are related to a single residual tumor cell clone.
Tumor necrosis factor alpha (TNF alpha) downregulates c-kit proto- oncogene product expression in normal and acute myeloid leukemia CD34+ cells via p55 TNF alpha receptors
Authors: Khoury E , Andre C , Pontvert-Delucq S , Drenou B , Baillou C , Guigon M , Najman A , Lemoine FM
Published: October 2019

Tumor necrosis factor alpha (TNF alpha), as a modulator of hematopoiesis, interacts with many growth factor receptors, such as interleukin-3, granulocyte-macrophage colony-stimulating factor (CSF), and granulocyte-CSF receptors. Here, we studied the interactions between TNF alpha and the stem cell factor (SCF) receptor, c-kit, in normal CD34+ hematopoietic progenitors and their leukemic counterpart, ie, acute myeloid leukemic (AML) CD34+ cells coexpressing c-kit antigen. The results showed that (1) incubation of normal bone marrow mononuclear cells with 200 U/mL rhTNF alpha for 20 hours induced a diminution of 31.2% +/- 5.2% of CD34+ cells coexpressing c-kit; (2) the same decrease was observed using purified CD34+ cells and, furthermore, their proliferative response to SCF was inhibited by 31.5% +/- 7.3% after exposure to TNF alpha; (3) similar experiments performed on CD34+ c-kit+ AML cells from 11 patients gave comparable results. Further analysis at the mRNA level indicated that TNF alpha decreased c-kit mRNA transcripts. Moreover, using monoclonal antibodies against the two types of TNF alpha receptors, p75 and p55, we showed that the downregulation of c-kit proto-oncogene product by TNF alpha, on normal and leukemic CD34+ cells, was exclusively mediated by the TNF alpha p55 receptor. Therefore, we conclude that TNF alpha acts as a downregulator of the SCF receptor expression.
Therapeutic efficacy of recombinant interleukin-6 (IL-6) alone and combined with recombinant human IL-3 in a nonhuman primate model of high-dose, sublethal radiation-induced marrow aplasia
Authors: MacVittie TJ , Farese AM , Patchen ML , Myers LA
Published: October 2019

Using a nonhuman-primate model of radiation-induced bone marrow aplasia, we examined whether the single, concomitant, or sequential administration of recombinant human interleukin-3 (IL-3) and IL-6 would promote bone marrow regeneration measured by an increase in circulating platelets (PLT) and neutrophils (PMN). Rhesus monkeys were irradiated at 450 cGy and were randomly assigned to one of five treatment protocols, receiving IL-6; IL-3; combined IL-6 and IL-3; sequential IL- 3 and IL-6; or human serum albumin (HSA) as a control. Cytokines or HSA were administered at total dosages of 15 micrograms/kg/day. Complete blood counts and white blood cell differentials were monitored for 60 days postirradiation. Both IL-3 and IL-6 significantly enhanced the regeneration of PLTs and decreased the duration of thrombocytopenia (P = .005) without affecting PMN recovery. The radiation-induced anemia that was observed in the HSA-treated controls was less severe and resolved more quickly in the IL-6 treated animals. Sequential IL-3/IL-6 significantly increased the production of PLTs when compared with the HSA-treated controls (P = .003) and monkeys receiving concomitant IL- 3/IL-6 (P = .041) but did not alter PMN levels significantly (P = .80). Coadministration of IL-6 and IL-3 did not enhance PLT but improved PMN recovery over IL-6 alone. In this primate model of marrow aplasia, IL-6 significantly enhanced the regeneration of PLTs but had no significant effect on PMN production, and did not exacerbate radiation-induced anemia. Furthermore, the use of sequentially administered IL-3 and IL-6 may improve PLT recovery as compared with concurrent IL-3/IL-6 administration, although this protocol is not significantly different in effect than either cytokine alone.
Characterization of nuclear factors that bind to a critical positive regulatory element of the human granulocyte-macrophage colony- stimulating factor promoter
Authors: Fraser JK , Tran S , Nimer SD , Gasson JC
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates the proliferation, maturation, and functional activity of myeloid cells in peripheral blood and bone marrow. Expression of GM-CSF is tightly regulated and is limited to cells stimulated directly (T cells, macrophages) or indirectly (fibroblasts, endothelial cells) by immune challenge. Several studies of the transcriptional control of GM-CSF expression have elucidated a region of the GM-CSF promoter that mediates positive regulatory activity in a number of cell types. This region contains a direct repeat of the sequence CATTA/T that extends from nucleotides -37 to -48 upstream of the start of mRNA synthesis. Although specific DNA:protein interactions have been shown within this region, neither the nature nor the number of nuclear factors responsible for these interactions have been characterized. In this study, we use DNase I footprinting analysis to demonstrate that point mutations, which inactivate the GM-CSF promoter, disrupt DNA:protein interactions within this region. By combined electrophoretic mobility shift and ultraviolet cross-linking analysis, we have detected several protein species that bind specifically to the positive regulatory sequence.
Pharmacokinetic and antithrombotic properties of two pentasaccharides with high affinity to antithrombin III in the rabbit: comparison with CY216
Authors: Carrie D , Caranobe C , Saivin S , Houin G , Petitou M , Lormeau JC , Van Boeckel C , Meuleman D , Boneu B
Published: October 2019

This study compares the pharmacokinetic and the antithrombotic properties of two pentasaccharides with high affinity to antithrombin III with those of a conventional low molecular weight heparin, CY216, in the rabbit. On a weight basis, SR 90107A/ORG 31540 (natural pentasaccharide [NPS]) and SR 80027A/ORG 31550 (sulfated pentasaccharide [SPS]) were, respectively, 4.7 and 26 times more potent antifactor Xa inhibitory agents than CY216. They were devoid of antithrombin activity, whereas the antifactor Xa/antithrombin ratio of CY216 was 3.8. After bolus intravenous administration, the clearance (mL/kg/h) of CY216 decreased from 91 +/- 27 for the dose of 12.5 U/kg to 49 +/- 14 for the dose of 50 U/kg and then remained constant up to the highest dose tested (500 U/kg). The clearance of NPS was unrelated to the dose and comparable to that of CY216 over 50 U/kg, whereas that of SPS was 10 times lower. Consistent results were observed after continuous intravenous infusions for 9 hours and subcutaneous administration. The duration of the antithrombotic effect was compared after a single subcutaneous injection of 250 U/kg of either compound in the stasis-Wessler model using human serum as thrombogenic stimulus. Two hours after the injection, the three compounds provided a thrombus prevention of greater than 95% and mean plasma activities of 0.8, 0.9, and 1.9 U/mL for CY216, NPS, and SPS, respectively. Twelve hours after injection, the antithrombotic effects of CY216 and NPS had totally vanished, whereas that of SPS was 68%. At that time, the plasma anti-Xa activities were less than 0.06 U/mL for CY216 and NPS, but 1.1 U/mL for SPS. For the latter compound, significant antithrombotic effects and detectable anti-Xa activities were still recorded 48 hours after the injection. The antithrombotic potency of the three compounds was also compared as their ability to inhibit the growth of a standardized venous thrombosis during 4 hours. The lowest total doses providing the maximum inhibitory effect were 3,125, 1,428, and 62 micrograms/kg for CY216, NPS, and SPS, respectively. These doses generated mean steady state antifactor Xa activities of 1.06, 1.5, and 1.2 anti-Xa U/mL, respectively. These observations indicate that the amplification mechanisms triggered by thrombin bound to fibrin and leading to the generation of new thrombin are essential to ensure venous thrombosis growth and that these mechanisms may be efficiently inhibited by pure antifactor Xa targeting agents.
Expression of CD28 and CD40 in human myeloma cells: a comparative study with normal plasma cells
Authors: Pellat-Deceunynck C , Bataille R , Robillard N , Harousseau JL , Rapp MJ , Juge- Morineau N , Wijdenes J , Amiot M
Published: October 2019

CD28 and CD40 are important activation pathways for T and B lymphocytes, respectively. The aim of this study was to determine the phenotype of plasma cells (PCs) and the expression of these two molecules, CD28 and CD40. Therefore, we have compared their expression on normal PCs from bone marrows and tonsils with that of freshly explanted malignant PCs from 31 patients with multiple myeloma (MM) and those from 12 human myeloma cell lines. For this purpose, we first described a new approach to identify plasma cells in bone marrow using two-color immunofluorescence analysis with anti-CD38 and B-B4 antibodies. B-B4 specifically recognizes all PC; all B-B4 cells are located within the CD38 bright fraction and vice versa. CD19 and CD56 expression, which was previously shown to discriminate normal from malignant PCs, was also evaluated. In the current report, we show that normal PCs express CD19, CD40, and CD56 (weakly as a subset) and lack CD28. Regardless of whether they express CD19, CD56 is clearly upregulated during the medullary chronic and accelerated phases of MM, but is absent in patients with extramedullary involvement. Although the level of CD40 expression is variable, only patients in accelerated phases expressed high CD40 levels. Finally, whereas CD28 was negative in chronic phase (as in normal PCs), it was expressed in 63% of the patients in accelerated phases and 100% of cell lines. Our data strongly suggest that both disease activity and medullary homing (or not) are correlated with the expression of CD19, CD40, CD28, and CD56 on human myeloma cells.
Regulation of intercellular adhesion molecule-1 gene expression involves multiple mRNA stabilization mechanisms: effects of interferon- gamma and phorbol myristate acetate
Authors: Ohh M , Smith CA , Carpenito C , Takei F
Published: October 2019

Although the intercellular adhesion molecule-1 (ICAM-1) is constitutively expressed at a low level on a subpopulation of hematopoietic cells, on vascular endothelium, on fibroblasts, and on certain epithelial cells, it is dramatically increased at sites of inflammation. Interferon-gamma (IFN-gamma) and phorbol myristate acetate (PMA) are known to increase the expression of ICAM-1 on many cell types. Because both human and murine ICAM-1 mRNAs contain putative destabilizing AUUUA sequences in their 3′ untranslated regions (UTRs), we examined the role of mRNA stability in the regulation of ICAM-1 gene expression. The treatment of the murine monocytic cell line P388D1, which constitutively expresses ICAM-1 mRNA at a low level, with IFN- gamma or PMA rapidly enhanced the level of ICAM-1 mRNA and dramatically prolonged its half-life. To determine whether the putative destabilizing sequences are responsible for this effect of IFN-gamma and PMA, fibroblast L cells were transfected with either the full- length ICAM-1 cDNA or a truncated form (ICAM-1 delta 3) lacking the putative destabilizing AUUUA sequences. Although ICAM-1 delta 3 mRNA was more stable than the full-length ICAM-1 mRNA, IFN-gamma treatment induced the accumulation of both mRNA species and prolongation of their half-lives. The transplantation of the ICAM-1 delta 3′ UTR into a stable ICAM-2 mRNA rendered it unstable, and it was unresponsive to IFN- gamma. Therefore, the treatment with IFN-gamma stabilizes the otherwise labile ICAM-1 mRNA, but the IFN-gamma-responsive sequence may at least in part reside within the protein coding region. PMA also upregulated ICAM-1 gene expression by mRNA stabilization. However, unlike IFN- gamma, PMA treatment only increased the level of the full-length, but not of the truncated, ICAM-1 mRNA. This shows that the PMA-responsive element is located within the 3′UTR. Furthermore, the effect of PMA on ICAM-1 delta 3 mRNA was recovered by ligating multiple AUUUA sequences derived from a heterologous gene fragment. The stability of this chimeric mRNA and the full-length ICAM-1 mRNA was markedly increased by PMA treatment, indicating that the AUUUA multimers in the 3′UTR are important in the PMA-induced upregulation of ICAM-1 mRNA.
PRAD-1/cyclin D1 gene overexpression in chronic lymphoproliferative disorders: a highly specific marker of mantle cell lymphoma
Authors: Bosch F , Jares P , Campo E , Lopez-Guillermo A , Piris MA , Villamor N , Tassies D , Jaffe ES , Montserrat E , Rozman C
Published: October 2019

The t(11;14)(q13;q32) translocation and its molecular counterpart bcl-1 rearrangement are frequently associated with mantle cell lymphomas (MCLs) and only occasionally with other variants of B-cell lymphoid malignancies. This translocation seems to activate the expression of PRAD-1/cyclin D1 gene located downstream from the major breakpoint cluster region of this rearrangement. However, the possible overexpression of this gene in other lymphoproliferative disorders independently of bcl-1 rearrangement is unknown. We have examined the overexpression of PRAD-1 gene in a large series of 142 lymphoproliferative disorders including 20 MCLs by Northern blot analysis. Cytogenetic and/or bcl-1 rearrangement analysis with 2 probes (MTC, p94PS) were performed in 28 cases. Strong PRAD-1 overexpression was observed in 19 of the 20 MCLs including 3 gastrointestinal forms and 4 blastic variants. t(11;14) and/or bcl-1 rearrangement was detected in 6 of the 12 MCLs examined. No correlation was found between the different levels of mRNA expression and the pathologic characteristics of the lymphoma. Among chronic lymphoproliferative disorders other than MCL, only 1 atypical chronic lymphocytic leukemia (CLL) with a t(11;14) translocation and bcl-1 rearrangement and the 2 hairy cell leukemias (HCLs) analyzed showed upregulation of PRAD-1 gene. The expression in the 2 HCLs was lower than in MCL, and no bcl-1 rearrangement was observed. These findings indicate that PRAD-1 overexpression is a highly sensitive and specific molecular marker of MCL but it may also be upregulated in some B-CLLs and in HCL.
Modulation of rat peripheral polymorphonuclear leukocyte response by nitric oxide and arginine
Authors: Seth P , Kumari R , Dikshit M , Srimal RC
Published: October 2019

The effect of nitric oxide (NO) on the luminol-dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was analyzed by using sodium nitroprusside (SNP), a NO donor, and L-arginine (L-arg), a NO precursor. A significant reduction in the LCL intensity was observed in presence of SNP (100 mumol/L) or L-arg (5 or 10 mmol/L) in arachidonic acid (AA) phorbol ester (PMA) and formyl- methionyl-leucyl-phenylalanine stimulated PMNLs. However, opsonized zymosan-induced LCL was not attenuated significantly. Reduction in hydroxyl radical and superoxide generation was also observed in SNP- or L-arg-pretreated cells. D-Arg (10 mmol/L) pretreatment did not inhibit PMNLs' LCL response. Furthermore, methylene blue (5 mumol/L) and L-NG- mono methyl-L-arginine (100 or 300 mumol/L) significantly attenuated the LCL response, as induced by various agonists. Cyclic GMP did not alter the reactive oxygen species generation from rat PMNLs. In addition, AA-induced release of myeloperoxidase, a marker of azurophilic granules, was found to be enhanced in L-arg- (10 mmol/L) pretreated PMNLs. The results suggest that NO inhibits free radical generation from rat PMNLs.
Apoptosis and macrophage-mediated deletion of precursor B cells in the bone marrow of E mu-myc transgenic mice
Authors: Jacobsen KA , Prasad VS , Sidman CL , Osmond DG
Published: October 2019

Transgenic mice expressing the c-myc proto-oncogene under the control of the Ig heavy chain enhancer (E mu-myc) all eventually develop clonal pre-B- or B-cell tumors. The preneoplastic period is characterized by increased polyclonal proliferation of pro-B and pre-B cells in the bone marrow (BM) associated with a reduced number of B cells, suggesting a high degree of B-cell loss. To examine the mechanisms of this cell loss, we have identified B220+ B-lineage cells within the BM of pretumorous E mu-myc transgenic mice by in vivo radiolabeling and electron microscope radioautography. Large mitotic B220(+)-labeled cells form prominent clusters in the extravascular compartment of the BM. Some B220+ small lymphocytes, as well as large lymphoid cells, enter BM sinusoids. However, in addition, large numbers of B220+ cells exhibit nuclear chromatin condensation, fragmentation, and other morphologic features characteristic of apoptotic cell death. Propidium iodide staining and flow cytometry of BM cells from pretumorous E mu- myc transgenic mice, as well as agarose gel electrophoresis of DNA, confirm extensive apoptosis. Many B220+ apoptotic cells are closely associated with the extensive processes of prominent macrophages that contain numerous B220+ apoptotic bodies and complex lysosomal systems. These results suggest that the constitutive expression of c-myc oncogene in BM B-lineage cells, which increases the proliferation of precursor B cells, also leads to increased apoptotic cell death and rapid elimination by resident macrophages. Further mutations may be needed to block these protective mechanisms and permit surviving c-myc- dysregulated cells to leave the BM and to initiate tumorigenesis.
Hepatosplenic candidiasis--a contraindication to marrow transplantation?
Authors: Bjerke JW , Meyers JD , Bowden RA
Published: October 2019

To determine whether a prior history of hepatosplenic candidiasis resulted in increased Candida-associated morbidity and mortality after marrow transplant, 15 consecutive patients with biopsy-proven hepatosplenic candidiasis were observed prospectively. All patients received amphotericin B before transplant. Amphotericin B was continued at a dose of 0.5 mg/kg/day from conditioning through marrow engraftment, at which time it was discontinued if computerized tomography (CT) evidence of disease was stable or improved. Patients were observed for progression of candidiasis for the first 100 days after transplant. The amount and duration of antifungal therapy received before transplant varied widely. The majority of patients (73%) had persistently abnormal CT scans before transplant. After transplant, 3 of 15 died (20%) with evidence of fungal disease, although fungal species differed from those diagnosed pretransplant, compared with a historical mortality rate of 90% in posttransplant patients with documented hepatosplenic candida. Comparison CT scans obtained before and after transplant showed improvement in 9 of 15 (60%), complete resolution in 2 of 15 (13%), and none showed progression. We conclude that hepatosplenic candidiasis is not an absolute contraindication to marrow transplant when patients receive amphotericin B therapy before transplant and continue therapy until engraftment is established.
Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells
Authors: Henschler R , Brugger W , Luft T , Frey T , Mertelsmann R , Kanz L
Published: October 2019

CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.
Cytokine therapy with gene-transfected cells: single injection of irradiated granulocyte-macrophage colony-stimulating factor-transduced cells accelerates hematopoietic recovery after cytotoxic chemotherapy in mice
Authors: Rosenthal FM , Fruh R , Henschler R , Veelken H , Kulmburg P , Mackensen A , Gansbacher B , Mertelsmann R , Lindemann A
Published: October 2019

Development of cell-based delivery systems that can release therapeutic levels of hematopoietic growth factors into the systemic circulation would facilitate treatment of patients requiring cytokine therapy. In this study, we have investigated the potential of granulocyte- macrophage colony-stimulating factor (GM-CSF)-secreting, irradiated syngeneic murine cells to accelerate hematopoietic recovery after cytotoxic chemotherapy. As a model, CMS-5 fibrosarcoma cells, were transduced with a retroviral vector containing the murine GM-CSF cDNA. Transduced cells secreted 38 ng GM-CSF/10(6) cells in 24 hours. After irradiation, in vitro GM-CSF production initially increased up to fivefold and was measurable for about 2 weeks. One and 2 days after injection of irradiated, GM-CSF-secreting CMS-5 cells (N2/CMVGM- CSF/CMS5 # 6 cells) into mice, GM-CSF serum levels of 405 +/- 58 pg/mL and 183 +/- 36 pg/mL were measured, respectively. Serum levels were comparable with levels detected 3 hours after injection of 100 ng recombinant murine GM-CSF (rmGM-CSF) subcutaneously (90 pg/mL). Injection of N2/CMVGM-CSF/CMS5 # 6 cells in cyclophosphamide-treated mice was as effective in accelerating neutrophil recovery as twice daily subcutaneous injections of rmGM-CSF. These data suggest that irradiated hematopoietic growth factor-secreting cells might offer an alternative to parenteral injections of recombinant cytokines in the treatment of neutropenic patients.
Genistein reduces tumor necrosis factor alpha-induced plasminogen activator inhibitor-1 transcription but not urokinase expression in human endothelial cells
Authors: van Hinsbergh VW , Vermeer M , Koolwijk P , Grimbergen J , Kooistra T
Published: October 2019

The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF- alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55- kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u- PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also inhibited PAI-1 induction by thrombin and IL-4, it is likely that genistein does not act on a TNF alpha-receptor- coupled protein kinase but on the signal transduction pathway enhancing PAI-1 transcription. Our results suggest that the TNF alpha-induced signal transduction pathway of PAI-1 transcription involves a genistein- sensitive step that is not involved in the induction of u-PA by TNF alpha. Given the limited sensitivity to several other tyrosine protein kinase inhibitors, this genistein-sensitive step may be a potential target for pharmacologic intervention to reduce elevated plasma PAI-1 levels.
Serum levels of soluble intercellular adhesion molecule 1 are increased in chronic B-lymphocytic leukemia and correlate with clinical stage and prognostic markers [see comments]
Authors: Christiansen I , Gidlof C , Wallgren AC , Simonsson B , Totterman TH
Published: October 2019

The serum levels of soluble intercellular adhesion molecule 1 (ICAM-1) were significantly elevated (P < .001) in patients with chronic B- lymphocytic leukemia (B-CLL, n = 113) compared with healthy controls (n = 31). sICAM-1 levels in B-CLL were positively correlated to the tumor mass as reflected by the modified Rai and the Binet staging systems, lymphocyte counts, and isolated spleno/hepatomegaly. During disease progression or regression on cytoreductive therapy, the circulating sICAM-1 levels changed accordingly. sICAM-1 was also correlated to a kinetic parameter such as the lymphocyte doubling time. Furthermore, the serum sICAM-1 levels were inversely correlated to hemoglobin levels in patients with early clinical stage, and this may turn out to be of prognostic value. sICAM-1 was compared with other serum markers said to reflect disease activity in B-CLL, ie, soluble CD23, thymidine kinase, lactate dehydrogenase (LDH), and beta 2-microglobulin. sICAM-1 was equally well or better correlated to clinical stage and lymphocyte doubling time. In univariate regression analysis, all serum markers but LDH correlated with survival, and in multivariate analysis, sICAM-1 was the only marker approaching significance for additional prognostic information when included after clinical stage and lymphocyte doubling time. Based on the present observations, it appears that prospective studies repeatedly monitoring serum sICAM-1 in B-CLL are justified.
Interleukin-6 prevents dexamethasone-induced myeloma cell death
Authors: Hardin J , MacLeod S , Grigorieva I , Chang R , Barlogie B , Xiao H , Epstein J
Published: October 2019

The effects of dexamethasone on the growth of four human multiple myeloma cell lines were studied. In addition, the effects on the expression of interleukin-6 (IL-6) and IL-6 receptor (IL-6R) genes were investigated by the use of reverse-transcriptase polymerase chain reaction. Dexamethasone (Dex) concentrations of 10(-7) to 10(-6) mol/L inhibited IL-6 gene expression in three of four cell lines studied, whereas the higher concentration of the hormone inhibited also IL-6R gene expression. Dex effects were modulated through the glucocorticoid receptor (GR). Dex treatment resulted in killing of sensitive cells associated with DNA fragmentation, which could be reversed by concomitant treatment with IL-6. The reversal of Dex-mediated effects by IL-6 did not result from an inhibition of GR function as measured by receptor nuclear translocation or Dex-regulated reporter gene function. These results indicate that blockage of the IL-6 signaling pathway is essential for effective myeloma cell kill by Dex.
Expression of the multidrug resistance associated protein and P- glycoprotein in doxorubicin-selected human myeloid leukemia cells
Authors: Slapak CA , Mizunuma N , Kufe DW
Published: October 2019

Drug-resistant sublines of the human U-937 myeloid leukemia cell line were selected in doxorubicin concentrations of 10, 40, and 200 ng/mL (designated U-A10, U-A40, and U-A200, respectively). Northern blot analysis showed overexpression of the multidrug resistance-associated protein (MRP) gene, but not MDR1, in U-A10 cells as compared with parental U-937 cells. Prolonged passage of U-A10 cells in 10 ng/mL of doxorubicin had little effect on MRP RNA levels, but increased MDR1 expression. The U-A40 and U-A200 cells, derived by selection of U-A10 cells, showed high levels of both MRP and MDR1 expression. None of the drug-resistant cell lines showed MRP or MDR1 gene amplification as judged by Southern blot analysis. U-A10 cells exhibited minimal decreased net accumulation of anthracycline, whereas U-A40 and U-A200 cells showed more significantly decreased drug accumulation as compared with U-937 cells. Subcellular anthracycline accumulation in U-937 cells as determined by fluorescence microscopy showed daunorubicin fluorescence predominately in the nucleus. However, the drug-resistant cell lines showed minimal nuclear drug accumulation with marked redistribution of drug into a vesicular compartment. Treatment with sodium azide/2-deoxyglucose, 2,4-dinitrophenol, or monensin, but not verapamil, abolished the vesicular accumulation. These studies in doxorubicin-selected U-937 cells indicate that induction of MRP overexpression occurs before that for the MDR1 gene. In addition, the drug-resistant cells possess an energy-dependent redistribution of anthracyclines into a nonnuclear vesicular compartment.
Chemotherapy in 998 unselected childhood acute lymphoblastic leukemia patients. Results and conclusions of the multicenter trial ALL-BFM 86
Authors: Reiter A , Schrappe M , Ludwig WD , Hiddemann W , Sauter S , Henze G , Zimmermann M , Lampert F , Havers W , Niethammer D
Published: October 2019

In trial ALL-BFM 86, the largest multicenter trial of the Berlin- Frankfurt-Munster (BFM) study group for childhood acute lymphoblastic leukemia (ALL), treatment response was used as an overriding stratification factor for the first time. In the previous trial ALL-BFM 83, the in vivo response to initial prednisone treatment was evaluated prospectively. A blast cell count of > or = 1,000/microL peripheral blood after a 7-day exposure to prednisone and one intrathecal dose of methotrexate (MTX) identified 10% of the patients as having a significantly worse prognosis. In trial ALL-BFM 86 patients with > or = 1,000/microL blood blasts on day 8 were included in an experimental branch EG. Patients with < 1,000/microL blood blasts on day 8 were stratified by their leukemic cell burden into two branches, Standard Risk Group (SRG) and Risk Group (RG). SRG patients received an eight- drug induction followed by consolidation protocol M (6-mercaptopurine, high-dose [HD] MTX 4 x 5 g/m2) and maintenance. RG patients were treated with an additional eight-drug reinduction element. For EG patients protocol M was replaced by protocol E (prednisone, HD-MTX, HD- cytarabine, ifosfamide, mitoxantrone). All patients received intrathecal MTX therapy; only those of branches RG and EG received cranial irradiation. In branch RG, patients were randomized to receive or not to receive late intensification (prednisone, vindesine, teniposide, ifosfamide, HD-cytarabine) in the 13th month. During the trial reinduction therapy was introduced in branch SRG, because in the follow-up of trial ALL-BFM 83 the randomized low-risk patients receiving reinduction did significantly better. Nine hundred ninety- eight evaluable patients were enrolled, 28.6% in SRG, 61.1% in RG, 10.3% in EG. At a median follow-up of 5.0 (range 3.4 to 6.9) years, the estimated 6-year event-free survival was 72% +/- 2% for the study population, 58% +/- 5% in branch SRG for the first 110 patients without reinduction therapy, 87% +/- 3% for the next 175 patients with reinduction therapy, 75% +/- 2% in branch RG, and 48% +/- 5% in branch EG. Late intensification did not significantly affect treatment outcome of RG patients; however, only 23% of the eligible patients were randomized. Prednisone poor response remained a negative prognostic parameter despite intensified therapy. The results confirmed the benefit of intensive reinduction therapy even for low-risk patients. The strategy of induction, consolidation, and intensive reinduction may offer roughly 75% of unselected childhood ALL patients the chance for an event-free survival.
p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies
Authors: Wattel E , Preudhomme C , Hecquet B , Vanrumbeke M , Quesnel B , Dervite I , Morel P , Fenaux P
Published: October 2019

We analyzed the prognostic value of p53 mutations for response to chemotherapy and survival in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic lymphocytic leukemia (CLL). Mutations were detected by single-stranded conformation polymorphism (SSCP) analysis of exons 4 to 10 of the P53 gene, and confirmed by direct sequencing. A p53 mutation was found in 16 of 107 (15%) AML, 20 of 182 (11%) MDS, and 9 of 81 (11%) CLL tested. In AML, three of nine (33%) mutated cases and 66 of 81 (81%) nonmutated cases treated with intensive chemotherapy achieved complete remission (CR) (P = .005) and none of five mutated cases and three of six nonmutated cases treated by low-dose Ara C achieved CR or partial remission (PR) (P = .06). Median actuarial survival was 2.5 months in mutated cases, and 15 months in nonmutated cases (P < 10(-5)). In the MDS patients who received chemotherapy (intensive chemotherapy or low-dose Ara C), 1 of 13 (8%) mutated cases and 23 of 38 (60%) nonmutated cases achieved CR or PR (P = .004), and median actuarial survival was 2.5 and 13.5 months, respectively (P < 10(-5)). In all MDS cases (treated and untreated), the survival difference between mutated cases and nonmutated cases was also highly significant. In CLL, 1 of 8 (12.5%) mutated cases treated by chemotherapy (chlorambucil and/or CHOP and/or fludarabine) responded, as compared with 29 of 36 (80%) nonmutated cases (P = .02). In all CLL cases, survival from p53 analysis was significantly shorter in mutated cases (median 7 months) than in nonmutated cases (median not reached) (P < 10(-5)). In 35 of the 45 mutated cases of AML, MDS, and CLL, cytogenetic analysis or SSCP and sequence findings showed loss of the nonmutated P53 allele. Our findings show that p53 mutations are a strong prognostic indicator of response to chemotherapy and survival in AML, MDS, and CLL. The usual association of p53 mutations to loss of the nonmutated P53 allele, in those disorders, ie, to absence of normal p53 in tumor cells, suggests that p53 mutations could induce drug resistance, at least in part, by interfering with normal apoptotic pathways in tumor cells.
Hemoglobin stimulates mononuclear leukocytes to release interleukin-8 and tumor necrosis factor alpha
Authors: McFaul SJ , Bowman PD , Villa VM , Gutierrez-Ibanez MJ , Johnson M , Smith D
Published: October 2019

Incubation of human mononuclear leukocytes (MNL) with human stroma-free hemolysate (SFH), purified adult hemoglobin Ao (HbAo), and oxidized HbAo (METHb) caused MNL to release compounds into the supernate that mediated neutrophil (polymorphonuclear leukocytes, PMN) chemotaxis and PMN adherence to human umbilical vein endothelial cells (HUVEC). Chemotaxis and PMN adherence to HUVEC were reduced significantly when supernates were preincubated with neutralizing antibodies to interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha), respectively, suggesting that IL-8 and TNF-alpha played significant roles in mediating these activities. Greatest chemotactic activity was observed in supernates of MNL treated with HbAo; while greatest PMN/endothelial cell (EC) adherence activity was observed in supernates of MNL treated with METHb. Furthermore, PMN/EC adherence activity was a function of METHb content in each hemoglobin solution. PMN chemotaxis, PMN adherence to HUVEC, and cytokine release increased as a function of increasing incubation time. Chemotactic activity was detected in HbAo- treated and METHb-treated MNL supernates after incubation for 6 hours and was maximal by 10 hours. IL-8 was detected in both HbAo and METHb- MNL supernates by 4 hours. PMN/EC adherence activity was detected in HbAo-MNL supernates at 10 hours and in METHb-MNL supernates at 4 hours. TNF-alpha was detected in METHb and HbAo-MNL supernates at 4 and 12 hours, respectively. These results suggest that hemoglobin solutions stimulate MNL to release IL-8 and TNF-alpha in quantities sufficient to induce PMN chemotaxis and PMN adherence to HUVEC. This is a US government work. There are no restrictions on its use.