bluuebunny/crossref_metadata_2025 huggingface dataset:
Perturbation of red blood cell membrane rigidity by extracellular ligands
Authors: Paulitschke M , Nash GB , Anstee DJ , Tanner MJ , Gratzer WB
Published: October 2019

It is known that binding of extracellular antibodies against the major sialoglycoprotein, glycophorin A, reduced the deformability of the red blood cell membrane. This has been taken to result from new or altered interactions between the glycophorin A and the membrane skeleton. We have shown by means of the micropipette aspiration technique that antibodies against the preponderant transmembrane protein, band 3, induce similar effects. A definite but much smaller reduction in elasticity of the membrane is engendered by univalent Fab fragments of the anti-band 3 antibodies. By examining cells genetically devoid of glycophorin A or containing a variant of this constituent, truncated at the inner membrane surface, we have shown that the anti-band 3 antibodies do not act through the band 3-associated glycophorin A. We examined the effect of anti-glycophorin A antibodies on homozygous Wr(a+b-) cells, in which an amino acid replacement in band 3 annihilates the Wright b (Wrb) epitope (comprising sequence elements of glycophorin A and band 3) and thus, by implication disrupts or perturbs the band 3-glycophorin A interaction; these cells show a much smaller response to an anti-glycophorin A antibody than do normal controls. We infer that in this case anti-glycophorin A antibodies exert their rigidifying effect through the associated band 3. Another anti- glycophorin A antibody, directed against an epitope remote from the membrane surface, however, increases the rigidity of both Wr(a+b-) and normal cells. This implies that not all antibodies act in the same manner in modifying the membrane mechanical properties. The effect exerted by anti-band 3 antibodies appears not to be transmitted through the band 3-ankyrin-spectrin pathway because the rigidifying effect of the intact antibody persists at alkaline pH, at which there is evidence that the ankyrin-band 3 link is largely dissociated. The large difference between the effects of saturating concentrations of the divalent and univalent anti-band 3 antibodies implies the existence of an overriding effect on rigidity, resulting from the bifunctionality of the intact antigen. Freeze-fracture electron microscopy shows that the anti-band 3 promotes the formation of small clusters of intra-membrane proteins. Extracellular ligands may in general act by promoting strong or transient interactions between integral membrane proteins, thereby impeding local distortion of the membrane skeletal network in response to shear.
The severe combined immunodeficient-human peripheral blood stem cell (SCID-huPBSC) mouse: a xenotransplant model for huPBSC-initiated hematopoiesis
Authors: Goan SR , Fichtner I , Just U , Karawajew L , Schultze W , Krause KP , von Harsdorf S , von Schilling C , Herrmann F
Published: October 2019

Mononuclear cells (MNCs) containing peripheral blood stem cells (PBSCs) were obtained from solid-tumor patients undergoing mobilizing chemotherapy followed by granulocyte colony-stimulating factor for PBSC transplantation-supported dose-intensified anticancer chemotherapy and were transplanted into unconditioned “nonleaky” young severe combined immunodeficient mice. Multilineage engraftment was shown by flow cytometry and immunocytochemistry using monoclonal antibodies to various human cell surface antigens as well as identification of human immunoglobulin in murine sera. Within a dose range of MNCs suitable for transplantation (10 to 36 x 10(6) cells/graft) the number of CD34+ cells injected (optimal at > 0.7 x 10(6)/graft) determined the yield of human cells produced in recipient animals. Engraftment of hu PBSC preparations resulted in prolonged generation of physiologic levels of human cytokines including interleukin-3 (IL-3), IL-6, and granulocyte- macrophage colony-stimulating factor, which were detectable in the murine blood over a period of at least 4 months. In vivo survival of immature human progenitor cells was preserved even 9 months after transplantation. Because human IL-3 is known to stimulate early hematopoiesis, a rat fibroblast cell line was stably transfected with a retroviral vector carrying the human IL-3 gene and cotransplanted subcutaneously as additional source of growth factor. Cotransplants of this cell line producing sustained in vivo levels of circulating human IL-3 for at least 12 weeks significantly accelerated the process of engraftment of huPBSC and spurred the spread of mature human cells to the murine spleen, liver, thymus, and peripheral blood. Cotransplants of allogeneic human bone marrow stromal cells derived from long-term cultures resulted in a comparable--though less prominent--support of engraftment.
Loss of heterozygosity in the chromosomal region 12p12-13 is very common in childhood acute lymphoblastic leukemia and permits the precise localization of a tumor-suppressor gene distinct from p27KIP1
Authors: Cave H , Gerard B , Martin E , Guidal C , Devaux I , Weissenbach J , Elion J , Vilmer E , Grandchamp B
Published: October 2019

Abnormalities of the short arm of chromosome 12 are relatively common in hematologic malignancies and deletions of the region. 12p12–13 are found in approximately 5% of the patients with acute lymphoblastic leukemia (ALL). As a potent inhibitor of cyclin-dependent kinases, p27KIP1 prevents the progression of the cell cycle and the gene encoding p27KIP1 represents a potential tumor-suppressor gene. Its recent assignment to the chromosomal region (12p12.3) prompted us to study the p27KIP1 gene in a series of 61 children with ALL. Microsatellite polymorphic markers flanking the p27KIP1 gene were analyzed to detect losses of heterozygosity (LOH). Eleven patients displayed LOH for at least one of the markers. The deleted are encompassed the p27KIP1 gene locus in 10 cases, but inactivation of the remaining allele by deletion, translocation, or mutation was never observed. In addition, in 1 patient, the p27KIP1 gene was situated outside of the region of LOH. Thus, p27KIP1 does not seem to be the target gene of 12p12–13 alterations. However, this study indicates that 12p12–13 alterations at the molecular level, which are present in about 27% of the children with B-lineage ALL, are much more common than had previously been reported by usual chromosome analysis. Moreover, LOH mapping allowed us to better define the location of a putative tumor- suppressor gene implicated in these malignancies and should therefore help in identifying this gene.
Late infections after allogeneic bone marrow transplantations: comparison of incidence in related and unrelated donor transplant recipients
Authors: Ochs L , Shu XO , Miller J , Enright H , Wagner J , Filipovich A , Miller W , Weisdorf D
Published: October 2019

Infectious complications are a major cause of morbidity and mortality after allogeneic bone marrow transplantation (BMT). We have evaluated the incidence of late infections (beyond day +50) in recipients of related (RD) and unrelated donor (URD) allogeneic BMT, factors associated with increased risks of infection, and the impact of the late infections on survival. Between 1989 and 1991, 249 patients received an RD (n = 151) or URD (n = 98) allogeneic BMT at the University of Minnesota and all late infections were investigated. Three hundred sixty-seven late infectious events developed in 162 patients between 50 days and 2 years after BMT. The incidence of any late infection was greater in URD versus RD recipients (84.7% v 68.2%, respectively; P = .009). In multivariate analysis, advanced graft- versus-host disease (GVHD) was significantly associated with late infections. The effect of GVHD was apparent only in RD recipients (relative risk [RR], 2.29; P = .003), whereas URD recipients, with or without GVHD, had more late infections compared with RD recipients without GVHD. Multivariate analysis showed that late posttransplantation infections were the dominant independent factor associated with increased nonrelapse mortality (RR, 5.5; P = .0001), resulting in improved 3-year survival for RD versus URD recipients (49.9% +/- 8% v 34.4% +/- 10%; P = .004). In this study, we observed that late infections are more frequent in URD recipients, resulting in substantially higher nonrelapse mortality. This prolonged period of increased infectious risk in URD recipients suggests the need for aggressive surveillance and therapy of late infections and perhaps prolonged antibiotic prophylaxis for all URD BMT recipients.
Carrier frequency of the IVS4 + 4 A-->T mutation of the Fanconi anemia gene FAC in the Ashkenazi Jewish population
Authors: Verlander PC , Kaporis A , Liu Q , Zhang Q , Seligsohn U , Auerbach AD
Published: October 2019

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous autosomal recessive disorder defined by a cellular hypersensitivity to DNA cross-linking agents. One of the FA genes, FAC, has been cloned and the genomic structure of the coding region has been characterized. We have developed amplification refractory mutation system (ARMS) assays for five known mutations in FAC, and have applied these assays to determine the carrier frequency of the IVS4 + 4 A-->T (IVS4) mutation in an Ashkenazi Jewish population. We tested 3,104 Jewish individuals, primarily of Ashkenazi descent, for the two most common FAC mutations, IVS4 and 322delG. Thirty-five IVS4 carriers were identified, for a carrier frequency of 1 in 89 (1.1%; 95% confidence interval 0.79% to 1.56%); no 322delG carriers were found. To determine if the IVS4 mutation was confined to the Ashkenazi Jewish population, we tested 563 Iraqi Jews for IVS4, and no carriers were found. Because the IVS4 mutation has only been found on chromosomes of Ashkenazi Jewish origin and is the only FAC mutation found on these chromosomes, we suggest that a founder effect is responsible for the high frequency of this mutation. With a carrier frequency greater than 1% and simple testing available, the IVS4 mutation merits inclusion in the battery of tests routinely provided to the Jewish population.
Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes
Authors: Brunetti M , Martelli N , Colasante A , Piantelli M , Musiani P , Aiello FB
Published: October 2019

Glucocorticoid (GC)-induced apoptosis is a well-recognized physiologic regulator of murine T-cell number and function. We have analyzed its mechanisms in human mature T cells, which have been thought to be insensitive until recently. Peripheral blood T cells showed sensitivity to GC-induced apoptosis soon after the proliferative response to a mitogenic stimulation, and were also sensitive to spontaneous (ie, growth factor deprivation-dependent) apoptosis. CD8+ T cells were more sensitive to both forms than CD4+ T cells. Acquisition of sensitivity to GC-induced apoptosis was not associated with any change in number or affinity of GC receptors. Both spontaneous and GC-induced apoptosis were increased by the macromolecular synthesis inhibitors, cycloheximide (CHX) and puromycin. A positive correlation between the degree of protein synthesis inhibition and the extent of apoptosis was observed. Interleukin-2 (IL-2) IL-4, and IL-10 protected (IL-2 > IL-10 > IL-4) T cells from both forms of apoptosis in a dose-dependent manner. Our data suggest that spontaneous and GC-induced apoptosis regulate the human mature T-cell repertoire by acting early after the immune response and differentially affecting T-cell subsets.
Differential expression of integrins on human thymocyte subpopulations
Authors: Mojcik CF , Salomon DR , Chang AC , Shevach EM
Published: October 2019

Integrins represent a candidate group of cell surface receptors that may control the homing and population of the thymus by T-cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. We have used multiparameter flow cytometric methods to characterize the expression of several members of the integrin family (alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1, and alpha L beta 2) on thymocyte subpopulations and have correlated integrin expression with other well-defined thymocyte differentiation markers. alpha 4 beta 1 was expressed by all thymocytes, but expression was highest on CD4-CD8- double-negative (DN) cells, high on CD+CD8+ double-positive (DP) cells, and lowest on mature single-positive (SP) cells, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 were present on 13%, 63%, and 26% of thymocytes, respectively, with maximal levels of expression on DN and SP cells, and low levels of expression on DP cells. Simultaneous analysis of alpha 4 beta 1, alpha 5 beta 1, and CD3 expression suggested a pathway of T-cell differentiation in the thymus in which the majority of the DN cells were alpha 4 beta 1hi alpha 5 beta 1hi, the DP cells alpha 4 beta 1hi alpha 5 beta 1lo/-, and the most mature SP cells were alpha 4 beta 1int. The stage-specific expression of integrins strongly implies their functional involvement during T-cell maturation in the thymus.
The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents
Authors: Richardson DR , Tran EH , Ponka P
Published: October 2019

Numerous studies have suggested that iron (Fe) chelators such as desferrioxamine (DFO) may be useful antitumor agents (Blatt and Stitely, Cancer Res 47:1749, 1987; Becton and Bryles, Cancer Res 48:7189, 1988). Recent work with several analogues of the lipophilic Fe chelator, pyridoxal isonicotinoyl hydrazone (PIH), indicate that some of these ligands are considerably more efficient than DFO both in terms of their Fe chelation efficacy and at preventing 3H-thymidine incorporation by neuroblastoma (NB) cells (Richardson and Ponka, J Lab Clin Med 124:660, 1994). Considering this fact, the present study was designed to test the antiproliferative effect of a wide range of PIH analogues to identify the most active compounds. A total of 36 ligands have been examined that were synthesized by condensation of three types of aromatic aldehydes (pyridoxal, salicylaldehyde, and 2-hydroxy-1- naphthyladehyde) with a range of acid hydrazides. The effects of these chelators were assessed using the human NB cell line, SK-N-MC. Although PIH was far more effective than DFO at preventing Fe uptake from transferrin, it was less effective than DFO at preventing cellular proliferation (DFO ID50 = 22 mumol/L; PIH ID50 = 75 mumol/L). In contrast, 14 PIH analogues were far more efficient than DFO at preventing proliferation (ID50 = 1 to 7 mumol/L) and may have potential as antitumor agents. The most effective compounds were those hydrazones derived from 2-hydroxy-1-naphthylaldehyde. Most of the PIH analogues were considerably more effective than DFO at both preventing 59Fe uptake from 59Fe-transferrin and in mobilizing 59Fe from prelabeled NB cells. In addition, a linear relationship between Fe chelation efficacy and antiproliferative activity was found only for hydrazones derived from salicylaldehyde. Apart from gallium (Ga) nitrate having an antiproliferative effect by itself, this metal potentiated the antiproliferative effect of PIH but not that of DFO. Spectrophotometric studies showed that PIH could chelate Ga, and it can be suggested that, like the PIH-Fe complex that donates Fe to reticulocytes (Ponka et al, Biochim Biophys Acta 718:151, 1982), the PIH-Ga complex may efficiently bestow Ga to NB cells. The results suggest that analogues of PIH deserve further vigorous investigation because they may be useful therapeutic agents for the treatment of cancer.
CD8-depleted donor lymphocyte infusion as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation
Authors: Giralt S , Hester J , Huh Y , Hirsch-Ginsberg C , Rondon G , Seong D , Lee M , Gajewski J , Van Besien K , Khouri I
Published: October 2019

Donor lymphocyte infusions can reinduce complete remission in the majority of patients with chronic myelogenous leukemia (CML) who relapse into chronic phase after allogeneic bone marrow transplantation (BMT). Such infusions are associated with a high incidence of graft- versus-host disease (GVHD) and marrow aplasia. BMT using selective depletion of CD8+ lymphocytes from donor cells reduces the incidence of GVHD without an increase in leukemia relapse. We hypothesized that infusion of CD8-depleted donor peripheral blood lymphocytes could also reinduce complete remissions with a lesser potential to produce symptomatic GVHD in patients with CML who relapsed after allogeneic BMT. Ten patients with Ph(+) CML who relapsed a median of 353 days after BMT (range, 82 to 1,096 days) received donor lymphocyte infusions depleted of CD8+ cells. Nine patients received a single infusion and 1 received two infusions. Four patients were treated while in chronic phase with clonal evolution, 2 during accelerated phase, 3 during blast crisis, and 1 in a cytogenetic relapse. A mean of 0.9 +/- 0.3 x 10(8) mononuclear cells/kg were infused, containing 0.6 +/- 0.4 x 10(6) CD3+CD8+ cells/kg. Six patients achieved hematologic and cytogenetic remission at 4, 8, 11, 15, 39, and 54 weeks after lymphocyte infusion. Two patients developed > or = grade II acute GVHD, and 1 patient developed mild chronic GVHD. We conclude that donor lymphocyte infusions depleted of CD8+ cells can induce remissions with a low rate of severe acute GVHD in patients with CML who relapse after allogeneic BMT, supporting the hypothesis that CD8+ lymphocytes are important effectors of GVHD, but may not be essential for the graft-versus- leukemia effect against this disease. Further controlled studies are required to confirm these preliminary observations.
Chloroquine treatment affects T-cell priming to minor histocompatibility antigens and graft-versus-host disease
Authors: Schultz KR , Bader S , Paquet J , Li W
Published: October 2019

Graft-versus-host disease (GVHD) caused by T-cell recognition of minor histocompatibility (MiHC) antigens is a major complication of bone marrow transplantation. GVHD therapy has focused on removal or suppression of donor T cells, but modulation of MiHC antigen presentation to CD4+ T cells may represent an alternative approach. Chloroquine is known to inhibit major histocompatibility complex (MHC) class II presentation of antigen in vitro by affecting invariant chain dissociation from MHC class II. The goal of this study was to evaluate the role of chloroquine in abrogating T-cell priming to MiHC and GVHD in mice after transplantation of an MiHC incompatible donor. C57BL/6 mice were treated with phosphate-buffered saline or chloroquine at 400 micrograms intraperitoneally every day for 5 days before priming with BALB.B cells (MiHC-incompatible) followed by weekly injections of chloroquine at 400 micrograms for 4 to 8 weeks. Chloroquine treatment decreased the proliferative T-cell response to MiHC by 67% and the cytolytic T-cell activation by greater than 50%. After bone marrow transplantation (LP/J into C57BL/6; MiHC-incompatible), GVHD was significantly decreased in chloroquine-treated mice (17% with GVHD) as compared with that in controls (92% with GVHD). Chloroquine treatment did not have other effects in vivo on the normal T- and B-cell mitogenic responses, T-cell allogeneic responses, and MHC class II and I surface expression. Chloroquine treatment does decrease the ability of C57BL/6 antigen-presenting cells to stimulate C3H.SW T cells reactive with MiHC expressed on C57BL/6 cells, suggesting an effect on MHC class II presentation of MiHC in vivo. Treatment with chloroquine in vivo appears to result in decreased CD4+ T-cell priming to MiHC and GVHD by decreased class II MHC antigen presentation. Thus, chloroquine treatment may represent an alternative approach to control GVHD.
Optimizing dose and scheduling of filgrastim (granulocyte colony- stimulating factor) for mobilization and collection of peripheral blood progenitor cells in normal volunteers [see comments]
Authors: Grigg AP , Roberts AW , Raunow H , Houghton S , Layton JE , Boyd AW , McGrath KM , Maher D
Published: October 2019

To define an optimal regimen for mobilizing and collecting peripheral blood progenitor cells (PBPC) for use in allogeneic transplantation, we evaluated the kinetics of mobilization by filgrastim (recombinant met- human granulocyte colony-stimulating factor [r-metHuG-CSF]) in normal volunteers. Filgrastim was injected subcutaneously for up to 10 days at a dose of 3 (n = 10), 5 (n = 5), or 10 micrograms/kg/d (n = 15). A subset of volunteers from each dose cohort underwent a 7L leukapheresis on study day 6 (after 5 days of filgrastim). Granulocyte-macrophage colony-forming cell (GM-CFC) numbers in the blood were maximal after 5 days of filgrastim; a broader peak was evident for CD34+ cells between days 4 and 6. The 95% confidence intervals (CI) for mean number of PBPC per milliliter of blood in the three dose cohorts overlapped on each study day. However, on the peak day, CD34+ cells were significantly higher in the 10 micrograms/kg/d cohort than in a pool of the 3 and 5 micrograms/kg/d cohorts. Mobilization was not significantly influenced by volunteer age or sex. Leukapheresis products obtained at the 10 micrograms/kg/d dose level contained a median GM-CFC number of 93 x 10(4)/kg (range, 50 x 10(4)/kg to 172 x 10(4)/kg). Collections from volunteers receiving lower doses of filgrastim contained a median GM- CFC number of 36 x 10(4)/kg (range, 5 x 10(4)/kg to 204 x 10(4)/kg). The measurement of CD34+ cells per milliliter of blood on the day of leukapheresis predicted the total yield of PBPC in the leukapheresis product (r = .87, P < .0001). Assuming a minimum GM-CFC requirement of 50 x 10(4)/kg (based on our experience with autologous PBPC transplantation), all seven leukapheresis products obtained at the 10 micrograms/kg/d dose level were potentially sufficient for allogeneic transplantation purposes. We conclude that in normal donors, filgrastim 10 micrograms/kg/d for 5 days with a single leukapheresis on the following day is a highly effective regimen for PBPC mobilization and collection. Further studies are required to determine whether PBPC collected with this regimen reliably produce rapid and sustained engraftment in allogeneic recipients.
Effect of interleukin-1 beta converting enzyme inhibitor on acute myelogenous leukemia progenitor proliferation
Authors: Estrov Z , Black RA , Sleath PR , Harris D , Van Q , LaPushin R , Estey EH , Talpaz M
Published: October 2019

Interleukin-1 beta (IL-1 beta) converting enzyme (ICE) is a cysteine protease that specifically cleaves precursor IL-1 beta to its biologically active form. Recent studies have also implicated ICE in the induction of apoptosis in vertebrate cells. Because IL-1 plays a major role in acute myelogenous leukemia (AML) blast proliferation, we sought to investigate the effect of ICE inhibition on AML progenitors. To do this, we used bocaspartyl (benzyl) chloromethylketone (BACMK) an inhibitor designed to penetrate cells and bind covalently to the active site of ICE. Our preliminary experiments showed that incubation of activated peripheral blood cells with 2.5 mumol/L of BAMCK downregulated production of mature IL-1 beta but had no effect on tumor necrosis factor-alpha. To test the effects of the inhibitor on AML cells, we first used the OCI/AML3 cell line. We found that these cells produce IL-1 beta and bind the biotinylated cytokine and that IL-1 inhibitors, such as IL-1 neutralizing antibodies, IL-1 receptor antagonist, and soluble IL-1 receptors, specifically inhibit OCI/AML3 proliferation, indicating that IL-1 beta is an autocrine growth factor for OCI/AML3 cells. The ICE inhibitor suppressed OCI/AML3 growth in a dose-dependent manner (at 0.4 to 4 mumol/L) and downregulated mature IL- 1 beta production, as assessed by Western immunoblotting. Similar results were obtained with marrow aspirates from 16 AML patients. The ICE inhibitor suppressed proliferation of AML precursors (by up to 78%; mean, 44%) in a dose-dependent fashion at concentrations ranging from 0.4 to 5 mumol/L but not proliferation of normal marrow progenitors; the suppressive effect was reversed by IL-1 beta. Furthermore, incubation of AML cells with 4 mumol/L BAMCK downregulated the production of mature IL-1 beta, suggesting that the growth-inhibitory effect is mediated through suppression of the biologically active cytokine. Our data indicate that inhibition of ICE suppresses AML blast proliferation and suggest that ICE inhibitors may have a role in future therapies for AML.
Analysis of signaling events associated with activation of neutrophil superoxide anion production by eosinophil granule major basic protein
Authors: Haskell MD , Moy JN , Gleich GJ , Thomas LL
Published: October 2019

This study was undertaken to identify the signaling events involved in activation of neutrophil superoxide anion (O2-) production by eosinophil granule major basic protein (MBP). MBP did not produce an immediate increase in the cytosolic free calcium concentration ([Ca2+]i), characteristic of phospholipase C activation, but did cause a gradual increase in [Ca2+]i in cytochalasin B-treated cells. Preincubation with 0.01 to 3 micrograms/mL pertussis toxin did not inhibit MBP-stimulated O2- production, and MBP did not stimulate an increase in diradylglycerol levels. MBP did stimulate a low level of phospholipase D activity, as measured by a time-dependent increase in phosphatidic acid and, in the presence of 0.5% ethanol, phosphatidylethanol. Inhibition of MBP-stimulated O2- production by genistein and Western blot analysis using an antiphosphotyrosine antibody showed tyrosine kinase activation by MBP. Calmodulin antagonists (calmidazolium and W-7) caused up to 80% inhibition of MBP- stimulated O2- production. In agreement with the pharmacologic sensitivity, MBP did not stimulate any 51Cr release. These data indicate that tyrosine kinase and calmodulin-dependent steps are involved in the noncytotoxic stimulation of neutrophil O2- production by MBP.
A randomized placebo-controlled phase III study of granulocyte- macrophage colony-stimulating factor in adult patients (> 55 to 70 years of age) with acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490)
Authors: Rowe JM , Andersen JW , Mazza JJ , Bennett JM , Paietta E , Hayes FA , Oette D , Cassileth PA , Stadtmauer EA , Wiernik PH
Published: October 2019

The treatment of adult patients greater than 55 to 70 years of age with acute myelogenous leukemia (AML) is associated with a treatment-related mortality of approximately 25%. This prospective, double-blind randomized study was designed to see if the use of granulocyte- macrophage colony stimulating factor (GM-CSF; yeast-derived) could shorten the period of neutropenia and to determine any effect this would have on therapy-related morbidity and mortality. A total of 124 patients entered this study. Induction consisted of standard daunorubicin and cytarabine. A day-10 bone marrow was examined; if this was aplastic without leukemia, patients received blinded placebo or GM- CSF from day 11 until neutrophil recovery. Patients who entered complete remission received the identical study medication (blinded GM- CSF or placebo) in consolidation that they had received during induction. The overall complete remission rate was 52%; 60% for the GM- CSF arm and 44% for the placebo arm (P = .08). Median times to neutrophil recovery were significantly shortened on the GM-CSF arm. The overall treatment-related toxicity from start of GM-CSF/placebo was reduced on the GM-CSF arm (P = .049). Similarly, the infectious toxicity was significantly reduced on the GM-CSF arm (P = .015). The median survival for all patients was 10.6 months in the GM-CSF group and 4.8 months in the placebo arm (P = .048). It appears that GM-CSF is safe and efficacious for adult patients greater than 55 to 70 years of age with AML; its major impact is in reducing the duration of neutropenia and therapy-related mortality and morbidity. This may result in a better response rate.
Apoptosis of human hematopoietic progenitor cells induced by crosslinking of surface CD43, the major sialoglycoprotein of leukocytes
Authors: Bazil V , Brandt J , Tsukamoto A , Hoffman R
Published: October 2019

Interactions of hematopoietic progenitor cells (HPC) with bone marrow stroma, mediated by adhesion molecules, are assumed to be critically important to the regulation of hematopoiesis. However, the specific roles of individual adhesion molecules involved in these interactions are poorly understood. Here, a monoclonal antibody, MEM-59, recognizing CD43, an adhesion molecule highly expressed on HPC, is shown to induce apoptosis in this cell population. This process operates at the single- cell level, and its initiation requires crosslinking of surface CD43 and the presence of cytokines. In contrast to HPC, more differentiated cells originating from this primitive cell population, as well as peripheral lymphocytes, do not undergo apoptosis in response to the CD43-mediated stimulation. Thus, CD43 may function as a negative regulator of early hematopoietic events, delivering a signal for apoptosis of HPC.
Long-term human hematopoiesis in SCID-hu mice bearing transplanted fragments of adult bone and bone marrow cells
Authors: Heike Y , Ohira T , Takahashi M , Saijo N
Published: October 2019

An attempt was made to establish an SCID-hu murine model of long-term human hematopoiesis by coimplantation of a bone fragment and bone marrow (BM) cells from an adult human. The SCID-hu mice were treated with a cytokine mixture (recombinant human stem cell factor, interleukin-3, granulocyte/macrophage colony-stimulating factor, and granulocyte colony-stimulating factor) for 4 months and were then maintained for further 8 months under cytokine-free conditions. In the peripheral blood, spleen, and implanted bone fragments in the SCID-hu mice that had received both a bone fragment and BM cells, human CD59+ cells were detected 1 year after transplantation; however, they were not detectable in SCID-hu mice that had received either a bone fragment or BM cells only. Thus, implantation of both a bone fragment and BM cells appears to provide a model of long-term adult human hematopoiesis in SCID mice.
Differential expression of receptors for interleukin-3 on subsets of CD34-expressing hematopoietic cells of rhesus monkeys
Authors: Wognum AW , Visser TP , de Jong MO , Egeland T , Wagemaker G
Published: October 2019

The target cell specificity of interleukin-3 (IL-3) was examined by flow cytometric analysis of IL-3 receptor (IL-3R) expression on rhesus monkey bone marrow (BM) cells using biotinylated IL-3. Only 2% to 5% of unfractionated cells stained specifically with the biotinylated IL-3 and most of these cells were present within the CD34+ subset. IL-3Rs were detected on small CD34dull/RhLA-DRbright/CD10+/CD27+/CD2-/++ +CD20- cells, which probably represent B-cell precursors. IL-3R+ CD34- BM cells, which were detected at low frequencies, consisted of small CD20dull/surface-IgM+/RhLA-DR+ cells. These cells represented immature B lymphocytes, whereas CD20bright mature B cells were IL-3R-. The highest IL-3R levels were detected on CD34dull/RhLA-DRbright blast-like cells. These cells differentiated into monocytes, neutrophils, and basophils after IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in vitro. The CD34bright/IL-3R- subset contained all clonogenic erythroid and myeloid progenitors (burst- forming unit-erythroid and colony-forming unit-culture), whereas CD34bright/IL-3Rdull cells differentiated into monocytes, neutrophils, and erythroid cells after shorter culture periods. This finding showed that IL-3R expression increases during monocyte and granulocyte differentiation. Results of three-color experiments indicated that IL- 3Rs are expressed on CD34bright/RhLA-DRbright cells as well as on CD34bright/RhLA-DRdull cells, with the latter population expression approximately twofold to threefold lower IL-3R levels. A large fraction (> 30%) of single-cell/well-sorted CD34bright/RhLA-DRdull cells formed multilineage colonies after 2 to 4 weeks of stimulation with IL-3, GM- CSF, Kit ligand, and IL-6. Individual colonies contained cells that still expressed CD34 as well as differentiated monocytes, granulocytes, and erythroid cells. These results confirmed that the CD34bright/RhLA- DRdull subset was enriched for immature, multipotent progenitor cells, whereas the CD34bright/RhLA-DRbright population mainly contained lineage-committed precursors. The results are consistent with the concept that IL-3Rs are induced at very early stages of hematopoiesis, as identified by high expression of CD34 and low expression of RhLA-DR. IL-3R expression continues to be low during differentiation into lineage-committed progenitors; gradually increases on differentiating progenitor cells for B cells, granulocytes, monocytes, and, possibly also, erythrocytes; but finally declines to undetectable levels during terminal differentiation into mature cells of all lineages in peripheral blood, with the exception of basophils.(ABSTRACT TRUNCATED AT 400 WORDS)
Kaolin clotting time and dilute Russell's viper venom time distinguish between prothrombin-dependent and beta 2-glycoprotein I-dependent antiphospholipid antibodies
Authors: Galli M , Finazzi G , Bevers EM , Barbui T
Published: October 2019

Antiphospholipid (aPL) antibodies include anticardiolipin (aCL) and lupus anticoagulant (LA) antibodies. LA antibodies recognize the complex of lipid-bound (human) prothrombin, in this way inhibiting the phospholipid-dependent coagulation reactions, whereas aCL antibodies are directed towards beta 2-glycoprotein I (beta 2-GPI) bound to an anionic lipid surface. According to their behavior in coagulation reactions, we have divided aCL antibodies into two groups: aCL-type A, which inhibit the phospholipid-dependent coagulation reactions because they enhance the binding of beta 2-GPI to the procoagulant phospholipid surface; and aCL-type B antibodies, which are devoid of anticoagulant properties. We report the distinctive laboratory and clinical profiles of 25 patients with well-characterized, phospholipid-dependent inhibitor of coagulation. Fourteen patients had LA antibodies (aCL-type B were concomitantly present in 10 cases, while in the other four, aCL titer was normal), and the other 11 had aCL-type A antibodies. The laboratory evaluation of the two groups showed the dilute Russell viper venom time (dRVVT) to be the most abnormal coagulation test in the aCL- type A-positive group, whereas the kaolin clotting time (KCT) was the most abnormal assay in the LA-positive group. In fact, the ratios of the coagulation times of patient plasma over normal pooled plasma (mean +/- standard deviation) for LA versus aCL-type A antibodies were 1.48 +/- 0.27 versus 2.20 +/- 0.42, P = .0001, and 2.22 +/- 0.42 versus 1.50 +/- 0.42, P = .0003, for the dRVVT and KCT, respectively. No differences were observed either in the ratios of the activated partial thromboplastin times and the prothrombin times or the plasma levels of beta 2-GPI and prothrombin. Conversely, aCL titers were significantly higher in aCL-type A-positive patients (147 +/- 44 U) than in the LA- positive group (61 +/- 55 U; P = .0003). We ruled out the possibility that platelet contamination of plasma could account for the observed coagulation profiles, as the two patterns were reproduced in platelet- free plasma. In addition, we performed clotting tests in plasma in the presence of phospholipids and calcium after addition of factor IXa or factor Xa. The assay performed with factor Xa was more sensitive to the presence of aCL-type A antibodies, while the assay performed with factor IXa was preferentially sensitive to LA-containing plasmas, supporting the earlier findings with the dRVVT and KCT assays.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulation of interleukin-12 expression in human monocytes: selective priming by interferon-gamma of lipopolysaccharide-inducible p35 and p40 genes
Authors: Hayes MP , Wang J , Norcross MA
Published: October 2019

Interleukin-12 (IL-12) is a monocyte/macrophage-derived cytokine that is critical for T lymphocyte and natural killer cell activities and functions. In this study, we examined the regulation of IL-12 expression by human monocytes in response to bacterial lipopolysaccharide (LPS). Several novel aspects of IL-12 induction from monocytes were shown. Optimal expression of IL-12 mRNA and bioactivity required specific priming of monocytes by interferon-gamma (IFN-gamma) before LPS stimulation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) provided an equivalent priming stimulus for LPS-induced tumor necrosis factor (TNF) and IL-12 p40 mRNA, but primed poorly for LPS-inducible p35 message and secreted IL-12 activity. Macrophage colony-stimulating factor (M-CSF), although a potent survival factor for monocytes, showed no priming activity for IL-12 production. Time course experiments demonstrated independent regulation of p40 and p35 by IFN-gamma and LPS. LPS inducibility of p40 expression required only a brief exposure to IFN-gamma (2 hours), while prolonged exposure (+/- 24 hours) to IFN-gamma resulted in diminishing levels of p40 mRNA. p35 inducibility (by LPS) required a longer exposure to IFN-gamma (8 to 16 hours), and continued to be inducible up to 40 hours following IFN- gamma priming. Both mRNAs were rapidly induced (1 to 2 hours) in IFN- gamma-primed monocytes; p35 message reached a plateau by 2 hours, while p40 continued to accumulate. Finally, both p40 and p35 were directly induced by LPS in the presence of cycloheximide. These results indicated that both p40 and p35 are LPS-inducible in monocytes following IFN-gamma pretreatment, and that the regulated expression of p35 controls the level of active IL-12 protein in purified human monocytes. The selectivity of priming by IFN-gamma is in accord with a putative role for IL-12 in the initiation and amplification of TH1-type responses.
Heterogeneous expression of Epstein-Barr virus latent proteins in endemic Burkitt's lymphoma
Authors: Niedobitek G , Agathanggelou A , Rowe M , Jones EL , Jones DB , Turyaguma P , Oryema J , Wright DH , Young LS
Published: October 2019

Epstein-Barr virus (EBV)-infected cells may sustain three distinct forms of virus latency. In lymphoblastoid cell lines, six EBV-encoded nuclear antigens (EBNA1, 2, 3A, 3B, 3C, -LP), three latent membrane proteins (LMP1, 2A, 2B), and two nuclear RNAs (EBERs) are expressed. This form of latency, termed latency III, is also encountered in some posttransplant lymphoproliferative disorders. In EBV-positive cases of Hodgkin's disease, the EBERs, EBNA1, and the LMPs are expressed (latency II), whereas in Burkitt's lymphoma (BL) only the EBERs and EBNA1 have been detected (latency I). We have studied the expression of EBV proteins in 17 cases of EBV-positive endemic BL by immunohistology. Expression of LMP1 was seen in variable proportions of tumor cells in two cases and EBNA2 was detected in some tumor cells in three other cases. Also, the BZLF1 trans-activator protein was expressed in a few tumor cells in 6 cases, indicating entry into the lytic cycle. A phenotypic drift from latency I to latency III has been observed previously in some BL cell lines. Our results suggest that a similar phenomenon may occur in BL in vivo and indicate that the operational definition of EBV latencies is not easily applied to human tumors.
Changes in the growth properties of CD34+, CD38- bone marrow progenitors during human fetal development
Authors: Waller EK , Huang S , Terstappen L
Published: October 2019

We have previously described the isolation of separate populations of CD34+, CD38- stromal and hematopoietic progenitors cells within fetal bone marrow. The CD34+, CD38-, CD50+, HLA-DR+ population contained the majority of primitive hematopoietic progenitor cells, whereas stromal progenitors were contained within the CD34+, CD38-, CD50-, HLA-DR- population. In this study, we compared the frequencies and total numbers of clonogenic CD34+, CD38- stromal and hematopoietic cells as a function of fetal gestational age using single-cell fluorescent- activated cell sorting (FACS). At 14 weeks of gestation, 1/500 fetal bone marrow mononuclear cells were primitive hematopoietic CD34+, CD38- , HLA-DR+ progenitor cells, whereas 1/1,000 were stromal progenitors with the CD34+, CD38-, HLA-DR- phenotype. During fetal ontogeny there was a continuous, age-dependent decrease in the frequency of stromal progenitors, such that, at 24 weeks of gestation, only 1/100,000 of bone marrow cells had the CD34+, CD38-, HLA-DR- phenotype and were clonogenic stromal cells when isolated by FACS. In contrast, 1/250 bone marrow cells in a 24-week fetus had the CD34+, CD38-, HLA-DR+ phenotype and were clonogenic hematopoietic progenitors. The decrease in the frequency of stromal progenitors was a function of both a decreased frequency of cells with the CD34+, CD38-, HLA-DR- phenotype and a decrease in the growth potential of individual with this phenotype. The total numbers of mononuclear cells and the total numbers of hematopoietic progenitors in two fetal femurs increased in parallel, 100-fold, between 14 and 24 weeks of gestation. In contrast, the total numbers of clonogenic CD34+, CD38-, HLA-DR- stromal progenitor cells remained constant during this period. Although adult bone marrow samples contained stromal progenitor cells at a frequency of approximately 1/7,000 mononuclear cells, clonogenic stromal cells with the CD34+, CD38-, HLA-DR- phenotype could not be isolated by single- cell FACS from these samples. Thus, there are significant differences between the frequencies and biologic characteristics of stromal and hematopoietic stem cells during fetal and postnatal ontogeny.
Role of busulfan and total body irradiation on growth of prepubertal children receiving bone marrow transplantation and results of treatment with recombinant human growth hormone
Authors: Giorgiani G , Bozzola M , Locatelli F , Picco P , Zecca M , Cisternino M , Dallorso S , Bonetti F , Dini G , Borrone C
Published: October 2019

Seventy-six prepubertal children receiving autologous or allogeneic bone marrow transplantation (BMT) were enrolled in a prospective study on the impact of different pretransplant preparative regimens on growth. Patients were divided into three groups: group I, consisting of 37 children who had received total body irradiation (TBI) and cytotoxic drugs as preparative regimen; group II, including 17 children receiving prophylactic cranial irradiation before being conditioned with TBI and cytotoxic drugs; and group III, composed of 22 patients transplanted after a busulfan (BU)-containing myeloablative therapy. All patients have a minimum follow-up of 2 years, whereas 48 and 34 patients have been studied until 3 and 4 years after transplant, respectively. Height and growth rate were expressed as standard deviation score (SDS). Growth hormone (GH) secretion in response to pharmacologic stimuli was evaluated after documented growth failure. Patients with GH deficiency were treated with recombinant human GH, and response to therapy was evaluated. The main impairment of growth rate in patients belonging to group II was observed in the first year after TBI (growth rate SDS changing from -0.12 +/- 0.23 to -1.23 +/- 0.25, P < .005), with only a slight loss in the following years, whereas in group I children growth failure occurred in the third year after TBI (-1.36 +/- 0.28 SDS in comparison to a pre-BMT SDS of 0.10 +/- 0.15, P < .005). Therefore, growth velocity between these two groups differed significantly in the first 2 years (P < .01) but subsequently equalized. On the contrary, all BU-treated children but 2 grew normally. GH deficiency was shown in the vast majority of children with growth impairment. Twenty-three children treated with recombinant human GH are evaluable; a successful response was observed in all but 1, with the mean growth rate increasing from -2.29 +/- 0.27 before treatment to 0.86 +/- 0.38 and to 1.66 +/- 0.56 SDS at 1 and 2 years after treatment, respectively (P < .001). In conclusion, growth rate impairment was common in patients receiving TBI, with the speed of onset of both decreased growth velocity and GH deficiency depending mainly on the total dose of radiation. On the contrary, patients receiving BU did not experience significant problems in terms of growth velocity. The timely start of appropriate hormonal replacement therapy may ameliorate the final growth of children undergoing BMT.
Homozygous deletions of p16/MTS1 gene are frequent but mutations are infrequent in childhood T-cell acute lymphoblastic leukemia
Authors: Ohnishi H , Kawamura M , Ida K , Sheng XM , Hanada R , Nobori T , Yamamori S , Hayashi Y
Published: October 2019

Fifty-six primary childhood T-cell acute lymphoblastic leukemia (T-ALL) samples and 17 T-ALL cell lines were examined for mutations and homozygous deletions of the p16/MTS1 gene using polymerase chain reaction single-strand conformation polymorphism and Southern blot analysis. Homozygous deletions were found in 22 primary samples (39%) and in 10 cell lines (59%). In contrast, mutations including small deletions and/or insertions were identified in only 4 primary samples (7%) and in 2 cell lines (12%). Mutations included samples (7%) and in 2 cell lines (12%). Mutations included one nonsense mutation at codon 72, one missense mutation at codon 58, one deletion (29 bp from codon 52–61), one insertion (7 bp into codon 50), and two deletion/insertions (codon 63 and intron 1). Four of the six mutations caused subsequent stop codon and presumably produced truncated p16 protein. Our results suggest that p16 gene alterations are involved in the development of T- ALLs and that the inactivation of the p16 gene occurs mainly through homozygous deletions rather than mutations.
T-cell death by apoptosis in vertically human immunodeficiency virus- infected children coincides with expansion of CD8+/interleukin-2 receptor-/HLA-DR+ T cells: sign of a possible role for herpes viruses as cofactors?
Authors: Lauener RP , Huttner S , Buisson M , Hossle JP , Albisetti M , Seigneurin JM , Seger RA , Nadal D
Published: October 2019

One mechanism proposed to play a role in T-cell depletion in human immunodeficiency virus (HIV) infection is apoptosis (activation-induced cell death). We assessed whether apoptosis is related to activation of T cells in vivo and its possible triggers. DNA was extracted from peripheral blood mononuclear cells (PBMC) taken from 16 vertically HIV- infected children and 9 HIV-negative children born to HIV-positive mothers (controls) and tested by agarose gel electrophoresis for the presence of DNA fragments specific for apoptosis. Signs of apoptosis were found on in vitro culture of PBMC from 12 of 16 HIV-infected children, but not in PBMC from the nine controls. Eleven of the 12 HIV- infected children with apoptosis showed an elevated (> 15%) proportion of CD3+/HLA-DR+ cells. This was due to an increased proportion of CD8+/HLA-DR+ cells, as shown in 7 of 7 further tested patients. In none of the probands an increased (> 5%) proportion of IL-2 receptor expressing CD3+ cells was found. T cells undergoing apoptosis were preferentially of the CD8+ phenotype. Expansion of circulating CD8+/interleukin-2 receptor (IL-2R)-/HLA-DR+ T cells is known to occur during active infection with herpes viruses. To investigate the possible role of herpes viral coinfections for apoptosis in HIV infection, we focused on Epstein-Barr virus (EBV) as an example for a herpes virus usually acquired during childhood. In 10 of 12 patients with apoptosis, we found increased levels of EBV genome in PBMC and/or tissues, indicating active EBV replication. By contrast, no increased burden of EBV was found in the four HIV-infected patients without apoptosis or in the controls. Our data indicate that in children the occurrence of apoptosis in HIV infection is closely related to activation of CD8+ T cells. Furthermore, primoinfection with or reactivation of herpes viruses, such as EBV, may substantially contribute to such T-cell activation and the ensuing apoptosis. Additional studies are warranted to evaluate the contribution of herpes virus-triggered apoptosis to the T-cell loss leading to the acquired immunodeficiency syndrome.