bluuebunny/crossref_metadata_2025 huggingface dataset:
Uncoupling fibrin from integrin receptors hastens fibrinolysis at the platelet-fibrin interface
Authors: Braaten JV , Jerome WG , Hantgan RR
Published: October 2019

A well-characterized in vitro model system composed of thrombin- stimulated gel-filtered human platelets, fibrin-(ogen), plasminogen, and recombinant tissue plasminogen activator (rt-PA) was used to examine the relationship between platelet-fibrin adhesive interactions and the lytic resistance of a platelet-rich thrombus. Laser light scattering kinetic experiments demonstrated that the ligand-mimetic peptide D-RGDW and an anti-alpha IIb beta 3 monoclonal antibody both inhibited clot retraction, but neither integrin-targeted reagent affected the overall delay in lysis of “bulk” fibrin caused by thrombin- stimulated platelets. However, lysis of the model platelet-rich thrombus did proceed some 30% more quickly when treated with a plasminogen activator inhibitor (PAI)-resistant t-PA variant. Taken together, these results confirm that platelet-released PAI-1 is a major determinant of global lytic resistance. Next events occurring during fibrinolysis in the unique microenvironment near the platelet surface were monitored by scanning electron microscopy and quantitative fluorescence microscopy. Scanning electron micrographs of the partially lysed model thrombus in the presence of 200 mumol/L of D-RGDW showed no platelet aggregates, and fibrin was attached by fewer strands to the platelets. Quantitative fluorescence microscopy, using fluorescein- labeled fibrin, showed that fibrin adherent to the surface of thrombin- stimulated platelets lysed 20% to 50% more slowly than bulk fibrin (monitored in parallel by laser light scattering). Furthermore, this microspectroscopic technique showed that D-RGDW reduced the quantity of platelet-bound fibrin, and accelerated lysis near the platelet surface with both native rt-PA and the PAI-resistant variant. These observations suggest that the dense network of fibrin bound to the platelet surface is protected from fibrinolysis by tissue-type plasminogen activators. Further, uncoupling fibrin from its platelet receptors uniquely hastens fibrinolysis at the cell/fibrin interface.
The SCL/TAL-1 gene is expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis
Authors: Kallianpur AR , Jordan JE , Brandt SJ
Published: October 2019

Activation of the SCL (or TAL-1) gene as a result of chromosomal translocation or deletion is a frequent molecular lesion in acute T- cell leukemia. By virtue of its membership in the basic helix-loop- helix family of transcription factors, the SCL gene is a candidate to regulate events in hematopoietic differentiation. We have used polyclonal antibody raised against a bacterial expressed malE-SCL fusion protein to characterize SCL protein expression in postimplantation embryos and in neonatal and adult mice. SCL protein was detected at day 7.5 post coitum at both embryonic and extraembryonic sites, approximately 24 hours before the formation of recognizable hematopoietic elements. Expression then localized to blood islands of the yolk sac followed by localization to fetal liver and spleen, paralleling the hematopoietic activity of these tissues during development. SCL protein was detected in erythroblasts in fetal and adult spleen, myeloid cells and megakaryocytes in spleen and bone marrow, mast cells in skin, and in rare cells in fetal and adult thymus. In addition, SCL protein was noted in endothelial progenitors in blood islands and in endothelial cells and angioblasts in a number of organs at times coincident with their vascularization. SCL expression was also observed in other nonhematopoietic cell types in the developing skeletal and nervous systems. These results show that SCL expression is one of the earliest markers of mammalian hematopoietic development and are compatible with a role for this transcription factor in terminal differentiation of the erythroid and megakaryocytic lineages. SCL expression by cells in the thymus suggests that the gene may be active at some stage of T-cell differentiation and may be relevant to its involvement by chromosomal rearrangements in T- lymphoid leukemias. Finally, expression of the gene in developing brain, cartilage, and vascular endothelium indicates SCL may have actions in neural development, osteogenesis, and vasculogenesis, as well as in hematopoietic differentiation.
A polymerase chain reaction study of the stability of Ig heavy-chain and T-cell receptor delta gene rearrangements between presentation and relapse of childhood B-lineage acute lymphoblastic leukemia
Authors: Steward CG , Goulden NJ , Katz F , Baines D , Martin PG , Langlands K , Potter MN , Chessells JM , Oakhill A
Published: October 2019

Ig heavy-chain (IgH) and partial V delta 2-D delta 3 T-cell receptor (TCR) gene rearrangements were investigated, by polymerase chain reaction (PCR) amplification and sequence analysis, in 52 patients at presentation and first relapse and in 14 at both first and second relapse of B-lineage acute lymphoblastic leukemia. In combination, these techniques amplified one or more clonal markers at presentation in 90% of patients (IgH-PCR, 75%; V delta 2-D delta 3-PCR, 46%; both, 33%). Changes in the pattern of amplification between presentation and first relapse were seen in 31% of patients positive by IgH-PCR at presentation and in 25% of those positive by TCR delta-PCR. Only 3 patients showed complete change in their rearrangements, which is suggestive of relapse with a new clone. Furthermore, despite the high reported rates of oligoclonality and clonal evolution at the IgH locus, the results presented show that false-negative minimal residual disease (MRD) detection can be avoided by designing D-N-J probes to all presentation rearrangements. Using a PCR approach for both gene markers, false-negative testing because of clonal evolution would have only occurred in 3 (8%) of the IgH-positive patients, in contrast to 5 (21%) of V delta 2-D delta 3-positive patients. Combining these two systems increases the proportion of patients open to study to 90%, allows comparative studies of the sensitive of the two methods, and reduces the rate of false-negative assessment of MRD caused by clonal evolution to less than 10%. We conclude that large prospective PCR studies of MRD detection should examine gene rearrangements at multiple loci to maximize their applicability and to minimize false-negative relapse prediction.
Granulocyte colony-stimulating factor (G-CSF) production and G-CSF receptor structure in patients with congenital neutropenia
Authors: Guba SC , Sartor CA , Hutchinson R , Boxer LA , Emerson SG
Published: October 2019

Congenital neutropenia (Kostmann's syndrome [KS]) is an autosomal recessive syndrome that is characterized by profound neutropenia, resulting in major clinical infections and death. Since the neutropenia and symptoms in KS improve in response to exogenous administration of granulocyte colony-stimulating factor (G-CSF), we studied bone marrow cytokine (G-CSF, granulocyte-macrophage CSF [GM-CSF], and interleukin- 6) production under both basal and stimulated conditions. No differences in G-CSF, GM-CSF, or IL-6 gene expression were found in bone marrow stromal cells between normal controls and KS patients, and all three cytokines were detected by enzyme-linked immunosorbent assay (ELISA) in medium conditioned by bone marrow stromal cells from normal donors and patients with KS. Each KS patient tested had detectable, functional G-CSF in their own serum before exogenous G-CSF administration. Since G-CSF production appeared normal in KS patients, we then asked whether we could detect structural defects in the signaling portion of G-CSF receptor genes. Polymerase chain reaction (PCR) amplification of the G-CSF receptor transmembrane region alone, and of the transmembrane plus cytosolic portions of the receptor, yielded the size products predicted from the sequences of the normal G- CSF receptor. Single-strand conformational polymorphism (SSCP) analysis of G-CSF receptor PCR products demonstrated no variance in structural conformation between KS patients and normal subjects. These results demonstrate that bone marrow stromal cells in patients with KS secrete normal concentrations of functional G-CSF and suggest that the neutropenia in KS patients is caused by an inability of neutrophilic progenitor and precursor cells to respond to normal, physiologic levels of G-CSF. Such a defect, clinically responsive to pharmacologic doses of G-CSF, might be caused by defects in the post-G-CSF receptor signal transduction pathway.
Effects of interleukin-3 and c-kit ligand on the survival of various classes of human hematopoietic progenitor cells
Authors: Brandt JE , Bhalla K , Hoffman R
Published: October 2019

We examined the effects of interleukin-3 (IL-3) and c-kit ligand (KL) on the survival of differentiated hematopoietic progenitor cells (HPC), the burst-forming unit-erythroid (BFU-E); colony-forming unit- granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM); and CFU- granulocyte-monocyte (CFU-GM) and more primitive hematopoietic cells that give rise to these progenitor cells (pre-colony-forming cells [pre- CFC]). CD34+ HLA-DR+ cells, which are highly enriched for committed HPC, and CD34+ HLA-DR- c-kit+ cells, which contain the most primitive assayable hematopoietic cells, including long-term bone marrow culture- initiating cells, high proliferative potential-CFC, the CFU-blast, and the BFU-megakaryocyte, were suspended in serum-free medium in the presence or absence of IL-3 or KL. CD34+ HLA-DR+ cells incubated under serum-free conditions or in the presence of KL for 96 hours lost greater than 90% of assayable unilineage or multilineage HPC, whereas those cells incubated in the presence of IL-3 retained 40% of the number of HPC present at time 0. The effect of IL-3 on HPC survival was most pronounced on the BFU-E and CFU-GEMM present within CD34+ HLA-DR+ cells. Addition of IL-3, but not of KL, to CD34+ HLA-DR+ cells delayed the appearance of morphologic changes and DNA fragmentation patterns associated with cell death occurring by apoptosis. CD34+ HLA-DR-c-kit+ cells were incubated under similar serum-free conditions in the presence or absence of IL-3 or KL, and the frequency of pre-CFC was determined by limiting dilution analysis. The frequency of pre-CFC in cells incubated for 48 hours in the absence of serum was similar to that of cells incubated in the presence of IL-3 and approximately doubled when CD34+ HLA-DR- c-kit+ cells were incubated in the presence of KL. Addition of KL to serum-free suspension cultures of CD34+ HLA-DR- c-kit+ cells delayed the appearance of DNA fragmentation patterns associated with apoptosis to a greater extent than did the addition of IL-3. These studies suggests that IL-3, but not KL, promotes HPC survival, whereas KL plays a greater role than IL-3 in sustaining more primitive HPC, such as pre-CFC. The effects of both cytokines in mediating HPC and primitive hematopoietic cell survival appear to be related, in part, to their ability to suppress apoptosis.
Differential effect of growth hormone and insulin-like growth factor-I, insulin-like growth factor-II, and insulin on Ig production and growth in human plasma cells
Authors: Kimata H , Yoshida A
Published: October 2019

The effect of human growth hormone (GH) and insulin-like growth factor- I (IGF-I), IGF-II, and insulin on human plasma cell responses was studied. GH enhanced Ig production and thymidine uptake in the human plasma cell lines, IM-9 and AF-10. IGF-I, but not IGF-II or insulin, also enhanced Ig production and proliferation in them. However, enhancement by GH was not mediated by IGF-I, because enhancement was blocked by anti-GH antibody (Ab), but not by Ab to IGF-I or IGF-I receptor. Conversely, the enhancement by IGF-I was blocked by either Ab to IGF-I or IGF-I receptor, but not by anti-GH Ab. GH and IGF-I also enhanced production of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM and thymidine uptake in PCA-1+ plasma cells generated in vitro. Again, enhancement by GH was specifically blocked by anti-GH Ab, whereas enhancement by IGF-I was specifically blocked by either Ab to IGF-I or IGF-I receptor. These results indicate that GH and IGF-I may play important roles in plasma cell responses.
Epstein-Barr virus DNA is abundant and monoclonal in the Reed-Sternberg cells of Hodgkin's disease: association with mixed cellularity subtype and Hispanic American ethnicity
Authors: Gulley ML , Eagan PA , Quintanilla-Martinez L , Picado AL , Smir BN , Childs C , Dunn CD , Craig FE , Williams JW Jr , Banks PM
Published: October 2019

One hundred twenty-five cases of Hodgkin's disease from the United States (79), Mexico City (31), and Costa Rica (15) were analyzed for the presence of Epstein-Barr virus (EBV) by in situ hybridization to EBER1 transcripts. EBV was more frequently detected in the Reed- Sternberg (RS) cells of mixed cellularity Hodgkin's disease (37 of 48 [77%]) compared with the nodular sclerosis subtype (19 of 71 [27%], P < .001). The presence of EBV was also associated with Hispanic ethnicity (P < .001). In a multivariate analysis, patient age, gender, and geographic location were less predictive of EBV positivity than were mixed cellularity histology (odds ratio = 8.3) and Hispanic ethnicity (odds ratio = 4.3). Southern blot analysis of EBV terminal repeat fragments using the Xho1a probe showed that the viral DNA was monoclonal in 17 of 17 cases having EBER1-positive RS cells. By comparison, EBV DNA was not detected by Southern analysis in 20 cases lacking EBER1 in RS cells, even when occasional background lymphocytes expressed EBER1. Because clonal viral DNA was so readily detected in EBER1-positive cases, the EBV genome is probably amplified at least 50- fold in the infected RS cells. Monoclonality of EBV DNA implies that the RS cells were infected before malignant transformation.
Fc gamma receptor II (CD32) on malignant B cells influences modulation induced by anti-CD19 monoclonal antibody
Authors: Vervoordeldonk SF , Merle PA , van Leeuwen EF , van der Schoot CE , von dem Borne AE , Slaper-Cortenbach IC
Published: October 2019

Antigenic modulation is one of many factors determining the effectiveness of monoclonal antibody (MoAb)-mediated therapy. To select the isotype of a CD19 MoAb most suitable for radioimmunotherapy of patients with B-cell malignancies, we studied the influence of MoAb isotype on modulation, after binding of the MoAb to different cell-line cells. The CD19-IgG1 MoAb was found to induce modulation of CD19 antigens on Daudi cell line cells more rapidly than did its IgG2a switch variant. We provide evidence that this difference in modulation rate is caused by the expression of Fc gamma receptor II (Fc gamma RII) on these cells. Experiments aimed at elucidating the mechanism of Fc gamma RII involvement in modulation induction by CD19-IgG1 showed that Fc gamma RII did not comodulate with CD19 MoAbs. However, cocrosslinking of CD19 and Fc gamma RII with CD19-IgG1 MoAb resulted in enhanced calcium mobilization in Daudi cells. This increased signal induction accompanies the enhanced capping and subsequent modulation of CD19 antigens. Because Fc gamma RII is expressed in varying densities on malignant B cells in all differentiation stages, our results have implications for the MoAb isotype most suitable for use in MoAb-based therapy of patients with B-cell malignancies.
Isolation and characterization of gelatinase granules from human neutrophils
Authors: Kjeldsen L , Sengelov H , Lollike K , Nielsen MH , Borregaard N
Published: October 2019

We recently confirmed the existence of gelatinase granules as a subpopulation of peroxidase-negative granules by double-labeling immunogold electron microscopy on intact cells and by subcellular fractionation. Further characterization of gelatinase granules has been hampered by poor separation of specific and gelatinase granules on both two-layer Percoll gradients and sucrose gradients. We have developed a three-layer Percoll density gradient that allows separation of the different granules and vesicles from human neutrophils; in particular, it allows separation of specific and gelatinase granules. This allows us to characterize these two granule populations with regard to their content of membrane proteins, which become incorporated into the plasma membrane during exocytosis. We found that gelatinase granules, defined as peroxidase-negative granules containing gelatinase but lacking lactoferrin, contain 50% of total cell gelatinase, with the remaining residing in specific granules. Furthermore, we found that 20% to 25% of both the adhesion protein Mac-1 and the NADPH-oxidase component cytochrome b558 is localized in gelatinase granules. Although no qualitative difference was observed between specific granules and gelatinase granules with respect to cytochrome b558 and Mac-1, stimulation of the neutrophil with FMLP resulted in a selective mobilization of the least dense peroxidase-negative granules, ie, gelatinase granules, which, in concert with secretory vesicles, furnish the plasma membrane with Mac-1 and cytochrome b558. This shows that gelatinase granules are functionally important relative to specific granules in mediating early inflammatory responses.
Effect of gamma irradiation of red blood cell units on T-cell inactivation as assessed by limiting dilution analysis: implications for preventing transfusion-associated graft-versus-host disease
Authors: Pelszynski MM , Moroff G , Luban NL , Taylor BJ , Quinones RR
Published: October 2019

Transfusion-associated graft-versus-host disease can be prevented by treating cellular blood products with gamma irradiation. A wide range of gamma irradiation dose levels has been used in routine practice. We used limiting dilution analysis, which measures clonable T cells, to assess the influence of 500 to 3,000 cGy of gamma irradiation delivered from a 137Cs source on T cells when delivered in situ to ADSOL- preserved red blood cell (RBC) units in blood bags. In a series of experiments using RBC units irradiated within 24 hours after collection, 1,500 cGy inactivated > 4 log10 of T cells; however, viable T cells were detected in all experiments. With 2,000 cGy, a > or = 4.7 log10 decrement in T-cell growth occurred in 7 of 8 experiments. With 2,500 or 3,000 cGy, no T-cell growth (> 5 log10 depletion) was detected. Comparable effects were observed with ADSOL-preserved RBC units in the standard PL 146 plastic container and in the recently developed PL 2209 plastic container. T-cell inactivation, as a function of gamma irradiation dose, was similar when either a 137Cs or a linear accelerator source was used. T cells isolated from ADSOL-preserved RBC units after storage for 7 and 21 days, although reduced in number as compared with a fresh unit stored for 24 hours, were viable, capable of proliferation, and susceptible to inactivation by gamma irradiation. Using a sensitive in vitro assay for T-cell proliferation, we found that a gamma irradiation dose of 2,500 cGy may be required to completely inactivate T cells in RBC units.
Potential use of interleukin-6 in bone marrow transplantation: effects of recombinant human interleukin-6 after syngeneic and semiallogeneic bone marrow transplantation in mice
Authors: Givon T , Revel M , Slavin S
Published: October 2019

The potential of recombinant glycosylated human interleukin-6 (rhIL-6) for enhancing immunohematopoietic reconstitution and survival after syngeneic and semiallogeneic bone marrow transplantation (BMT) in BALB/c mice subjected to total body irradiation (TBI) was investigated. rhIL-6 produced enhanced reconstitution of white blood cells as assessed on days 8 and 14 after syngeneic BMT and of platelets as assessed on day 10. Moreover, rhIL-6 treatment produced significant improvement of survival in lethally irradiated mice receiving either syngeneic or semiallogeneic BMT with limiting number of BM cells. This effect of IL-6 was not seen with large BM cell inocula producing high survival by themselves. rhIL-6 showed no toxic effects and did not affect the survival of mice that were lethally irradiated but not reconstituted by BM cells. However, the sensitivity of mice to sublethal irradiation was increased by rhIL-6 in the absence of BM cell transplantation. In experimental conditions inducing graft-versus-host disease (GVHD), in which lethally irradiated (BALB/c x C57BL/6)F1 mice received mixtures of BM and spleen cells from C57BL/6 donors, rhIL-6 was found to enhance GVHD manifestations. No consistent enhancement of T-cell in vitro proliferative responses to allogeneic spleen cells or T- and B-cell-dependent mitogens were seen in the splenocytes obtained from recipients of syngeneic or semiallogeneic BMT. Our data suggest that rhIL-6 may be useful in BMT procedures to enhance thrombopoiesis and hematologic recovery, as well as to increase overall survival rates. In addition, the potentiation of GVHD, which is considered to correlate with graft-versus-leukemia effects, may be of interest in enhancing GVHD-dependent antitumor effects in protocols combining radiochemotherapy with BMT.
Variable transcription of BCR-ABL by Ph+ cells arising from hematopoietic progenitors in chronic myeloid leukemia [see comments]
Authors: Keating A , Wang XH , Laraya P
Published: October 2019

Recent studies suggest that the BCR-ABL gene plays a critical role in the pathogenesis of Ph+ chronic myeloid leukemia (CML). We investigated the hematopoietic colonies derived from the marrows of 12 patients with Ph+ CML in chronic phase by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of BCR-ABL mRNA and by cytogenetics. Colonies were individually harvested and each colony divided into two portions, one for cytogenetics and the other for isolation of total RNA for PCR of BCR-ABL transcripts and for an RNA internal control. We found that 23% +/- 18% (mean +/- SD, range 0% to 60%) of Ph+ colonies did not transcribe the aberrant gene. In each case when BCR-ABL transcription was not detected, normal ABL mRNA was present. The data suggest that hitherto unknown mechanisms may regulate BCR-ABL expression in some Ph+ cells and indicate that caution should be exercised in the interpretation of results using RT-PCR analysis of hematopoietic colonies from clinical specimens and from experiments with antisense oligonucleotides directed at the BCR-ABL gene. These data also raise the notion of a transitional Ph+ precursor cell in which BCR-ABL may become upregulated and lead to a fully expressed phenotype. We conclude that further studies correlating the frequency of Ph+ PCR- progenitors with prognostic clinical variables are warranted.
Transcriptional regulation and chromatin structure of the human CD34 gene promoter region
Authors: He XY , Cockerill PN , Cen D , Davis BR
Published: October 2019

The human CD34 surface antigen is selectively expressed on stem/progenitor cells within the hematopoietic system. Because CD34 expression is tightly linked to the immature status of hematopoietic cells, with expression being rapidly lost as hematopoietic cells mature and differentiate, an examination of its regulation may provide important insights into the molecular control of blood cell development. A comparison of the CD34 nuclear transcription rate in CD34+ and CD34- cells indicated that the CD34 gene is transcriptionally regulated in hematopoietic cell lines. In a previous report, we had identified two major clusters of CD34 transcription initiation sites by 5′ RACE (rapid amplification of cDNA ends) analysis. In transient transfection experiments, we now demonstrate the ability of sequences encompassing each of these clusters to function as promoters of transcription in CD34+ cells. These promoters functioned at equivalent levels in CD34+ and CD34- cells, and the addition of 5′ flanking sequences, extending as far as 3.7 kb upstream, to the core promoters did not differentially modify the level of expression in CD34+ versus the CD34- cell lines. An examination of DNase I hypersensitivity sites within an 18-kb segment of DNA, extending 9 kb either side of the proximal promoter, showed six sites that were primarily associated with CD34- expressing cells. Taken together, these data indicate that the CD34 promoter sequences alone do not confer tissue-or stage-specific expression. Appropriate transcriptional regulation of the CD34 gene must be controlled by chromatin structure, as identified by DNase I hypersensitivity, and/or by other tissue- and stage-specific elements present outside of the promoter region.
Inverse expression of bcl-2 protein and Fas antigen in lymphoblasts in peripheral lymph nodes and activated peripheral blood T and B lymphocytes
Authors: Yoshino T , Kondo E , Cao L , Takahashi K , Hayashi K , Nomura S , Akagi T
Published: October 2019

To examine the regulatory mechanism of apoptosis in lymphoid cells, expression of both bcl-2 protein and Fas antigen was investigated in reactive lymph nodes, in resting lymphocytes from peripheral blood (PBLs), and in PBLs stimulated with pokeweed mitogen, interleukin-4 (IL- 4) + anti-IgM antibody, IL-2 + anti-CD3 antibody, phytohemagglutinin + phorbol myristate acetate using immunohistochemistry and flow cytometry. Germinal center cells expressed a large amount of Fas antigen, which is associated with the induction of apoptosis in lymphoid cell lines, in contrast to the lack of bcl-2 protein as an apoptosis inhibitor. On the other hand, mantle zone lymphocytes expressed a high level of bcl-2 protein and less Fas antigen. This inverse expression of bcl-2 protein and Fas antigen was also shown in activated T and B lymphocytes from peripheral blood. These lymphoblasts fell into apoptosis dose-dependently in the presence of anti-Fas monoclonal antibody, but resting lymphocytes that expressed both bcl-2 protein and Fas antigen did not undergo apoptosis. These findings suggest that bcl-2 expression prevents the apoptosis of lymphoid cells induced by the Fas antigen-dependent mechanism and that apoptosis of lymphocytes is exquisitely controlled, at least in part, by regulation of the bcl-2 and Fas genes.
High levels of interleukin-6 are associated with low tumor burden and low growth fraction in multiple myeloma
Authors: Ballester OF , Moscinski LC , Lyman GH , Chaney JV , Saba HI , Spiers AS , Klein C
Published: October 2019

Interleukin-6 (IL-6) is a multifunctional cytokine postulated to play a central role as a growth factor for multiple myeloma (MM). We evaluated the spontaneous secretion of IL-6 in supernatants of Ficoll-Hypaque-- enriched bone marrow (BM) cultures from 35 patients with MM. The levels of IL-6 were correlated with biological and clinical characteristics of the disease. High levels of IL-6 production defined a subgroup of patients with low tumor burden as determined by lower serum beta 2- microglobulin (B2M) (P = .02) and lower percentage of myeloma cells infiltrating the bone marrow (P = .003), higher synthetic rates of monoclonal protein (P = .006), and low proliferative compartments as measured by the percentage of Ki-67--positive myeloma cells. Patients with high proliferative fractions (Ki-67--positive myeloma cells > 20%) had significantly lower levels of IL-6 when compared with patients with low proliferative fractions (P = .005). Our findings do not support IL- 6 as a major growth factor for MM, but demonstrate an association of high levels of IL-6 secretion with low tumor cell burden and low proliferative fraction.
Contact activation triggers stimulation of the monocyte 5-lipoxygenase pathway via plasmin
Authors: Weide I , Romisch J , Simmet T
Published: October 2019

The purpose of this study was to characterize the stimulus that activates the 5-lipoxygenase pathway in human peripheral monocytes (PM) during the process of contact activation. Incubation of PM, but not of polymorphonuclear leukocytes (PMN), in contact-activated, recalcified plasma induced a time-dependent release of leukotrienes (LT). The presence of platelets was required for the generation of cysteinyl-LT, but LTB4 formation also proceeded in their absence, although to a lesser extent. Plasmin, presumably generated via the intrinsic fibrinolytic pathway, was liable for the 5-lipoxygenase stimulation during contact activation inasmuch as (1) the 5-lipoxygenase pathway in PM was stimulated by contact-activated, recalcified, autologous or homologous plasma, but not by factor XII-deficient or prekallikrein- deficient plasma; (2) lysine analogs such as N alpha-acetyl-L-lysine, 6- aminohexanoic acid (6-AHA), or trans-4- (aminomethyl)cyclohexane-1- carboxylic acid (t-AMCA), which inhibit plasmin(ogen) binding to PM plasmin(ogen) binding sites, concentration-dependently reduced the cysteinyl-LT release; (3) plasminogen activators such as urokinase or streptokinase concentration-dependently enhanced the cysteinyl-LT release up to 10 and 1,000 IU/mL, respectively, while higher concentrations were less effective leading to bell-shaped concentration- response curves; (4) plasmin inhibitors such as aprotinin or alpha 2- antiplasmin concentration-dependently inhibited the cysteinyl-LT release; and (5) preincubation of plasma with monoclonal antibodies directed against plasminogen and capable of preventing plasminogen activation blocked the contact-mediated 5-lipoxygenase stimulation. Moreover, incubation of PM with plasmin, but not with plasma kallikrein, in Hanks' balanced salt solution (HBSS)-bovine serum albumin (BSA) 0.4% triggered a concentration-dependent release of LTB4 up to 0.1 caseinolytic units (CU)/mL, with higher concentrations being less effective. By contrast, release of cyclooxygenase metabolites such as thromboxane (TX) B2 and prostaglandin (PG) E2 was not stimulated by plasmin, indicating specificity for the 5-lipoxygenase pathway. With plasmin as a hitherto unknown stimulus of the 5-lipoxygenase pathway in PM, a novel link between contact activation and inflammation has been established.
A double-blind controlled study of granulocyte colony-stimulating factor started two days before induction chemotherapy in refractory acute myeloid leukemia. Kohseisho Leukemia Study Group [see comments]
Authors: Ohno R , Naoe T , Kanamaru A , Yoshida M , Hiraoka A , Kobayashi T , Ueda T , Minami S , Morishima Y , Saito Y
Published: October 2019

We conducted a prospective, double-blind controlled study to determine the efficacy of a recombinant granulocyte colony-stimulating factor (G- CSF, 200 microgram/m2) starting daily from 2 days before an induction therapy until neutrophils recovered to above 1,500/microL or until 35 days after the therapy in 58 patients with relapsed or refractory acute myeloid leukemia (AML). Twenty-eight patients in the G-CSF group showed significantly faster recovery of neutrophils (P < .001) than 30 patients in the placebo group. The incidence of febrile episodes and of documented infections was almost the same in both groups. However, among 39 patients who did not show any infectious episodes during the 2- week period after the start of chemotherapy, the incidence of documented infections after the third week tended to be lower in the G- CSF group, but not statistically significantly. There was no evidence that G-CSF stimulated the growth of AML cells in the bone marrow during the 2-day period before the chemotherapy, nor that G-CSF accelerated the regrowth of AML cells during the 5-week period after the therapy. Fifty percent of patients in the G-CSF group and 37% in the placebo group had complete remission (CR). Although the rate was higher in the G-CSF group, the difference was not statistically significant (P = .306). There was no difference between the two groups in event-free survival of all patients and in disease-free survival of patients who had achieved CR.
Modulation of spontaneous B-cell differentiation in macroglobulinemia by retinoic acid
Authors: Levy Y , Labaume S , Gendron MC , Brouet JC
Published: October 2019

We previously showed that clonal blood B cells from patients with macroglobulinemia spontaneously differentiate in vitro to plasma cells. This process is dependent on an interleukin (IL)-6 autocrine pathway. We investigate here whether all-trans-retinoic acid (RA) interferes with B-cell differentiation either in patients with IgM gammapathy of undetermined significance (MGUS) or Waldenstrom's macroglobulinemia (WM). RA at a concentration of 10(-5) to 10(-8) mol/L inhibited by 50% to 80% the in vitro differentiation of purified B cells from four of five patients with MGUS and from one of five patients with WM as assessed by the IgM content of day 7 culture supernatants. We next determined whether this effect could be related to an inhibition of IL- 6 secretion by cultured B cells and/or a downregulation of the IL-6 receptor (IL-6R), which was constitutively expressed on patients' blood B cells. A 50% to 100% (mean, 80%) inhibition of IL-6 production was found in seven of 10 patients (five with MGUS and two with WM). The IL- 6R was no more detectable on cells from patients with MGUS after 2 days of treatment with RA and slightly downregulated in patients with WM. It was of interest that B cells susceptible to the action of RA belonged mostly to patients with IgM MGUS, which reinforces our previous data showing distinct requirements for IL-6-dependent differentiation of blood B cells from patients with VM or IgM MGUS.
Wild-type p53 gene expression induces granulocytic differentiation of HL-60 cells
Authors: Soddu S , Blandino G , Citro G , Scardigli R , Piaggio G , Ferber A , Calabretta B , Sacchi A
Published: October 2019

Overexpression of wild-type p53 gene in malignant cell lines has been shown to inhibit cell proliferation in a number of cases. However, endogenous p53 protein seems to play little role in normal cell-cycle control as suggested by the normal development of p53 null mice, and by the low p53 protein levels expressed in most cell types. Recently, increased expression of endogenous p53 protein has been observed during the cellular response to DNA damage, as well as during differentiation of human hematopoietic cells. To study the role of the p53 gene in hematopoietic differentiation, we introduced the wild-type p53 gene or the temperature-sensitive p53(Val135) mutant into p53-deficient HL-60 promyelocytic leukemia cells. Morphological analysis, flow-cytometric determination of granulocytic or monocytic surface markers, and ability to reduce nitroblue tetrazolium (NBT) demonstrated that expression of exogenous wild-type p53 gene in HL-60 cells induces differentiation through the granulocytic pathway. Proliferation and cell-cycle analysis performed early after expression of wild-type p53 showed that induction of differentiation is not coupled with growth arrest, which suggests that p53 is involved in differentiation independently of its activity on the cell cycle.
Analysis of interphase cells for the Philadelphia translocation using painting probe made by inter-Alu-polymerase chain reaction from a radiation hybrid
Authors: Seong DC , Song MY , Henske EP , Zimmerman SO , Champlin RE , Deisseroth AB , Siciliano MJ
Published: October 2019

Fluorescence in situ hybridization (FISH) probe for the identification of the Philadelphia (Ph) translocation [t(9;22) (q34;q11)] in chronic myelogenous leukemia cells was developed by inter-Alu-polymerase chain reaction of DNA from an interspecific somatic cell hybrid containing approximately 5 Mb of human DNA covering the ABL gene region on human chromosome 9q34. This probe was large enough to be effective in identifying the genomic domains yet small enough to resolve them in more than 90% of bone marrow interphase cells. Combination of the probe with a cosmid contig probe for the BCR region of chromosome 22 in two- color FISH reduced the frequency of false-positive identification of the Ph chromosome to less than 1%. The procedure allows detection of as few as 1% Ph+ cells independent of the cycling status or BCR/ABL expression level of cells, and the quantitation of non-Ph chromosome- containing interphase nuclei in the marrow of patients judged 100% Ph+ by standard cytogenetics.
Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging
Authors: Zhong RK , Donnenberg AD , Rubin J , Ball ED
Published: October 2019

Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4- HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4- HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4- HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.
P-glycoprotein expression and function in circulating blood cells from normal volunteers
Authors: Klimecki WT , Futscher BW , Grogan TM , Dalton WS
Published: October 2019

In contrast to its clearly defined role as a multidrug efflux pump in neoplastic cells, the physiologic function of P-glycoprotein (P-gly) in normal cells is unclear. Recent reports identifying P-gly in normal blood and bone marrow suggest that hematopoietic development or function may be dependent on P-gly. To understand the normal function of P-gly in the blood, its level of expression and function must first be quantitated relative to a known standard. In this study, P-gly, MDR1 gene expression, and P-gly function were quantitated in normal leukocytes. P-gly and MDR1 expression were analyzed in individual leukocyte lineages (T-helper, T-suppressor, monocyte, granulocyte, B- lymphocyte, NK cell) from normal volunteers. P-gly on the cell surface was detected by fluorescent double-labeling for lineage (CD4, CD8, CD14, CD15, CD19, CD56, respectively) and P-gly (MRK16) with analysis by flow cytometry and in some cases immunoblot analysis. MDR1 mRNA analysis on purified lineages was performed using quantitative reverse transcription-polymerase chain reaction. P-gly function was determined for each lineage using dual-labeling for lineage and P-gly substrate (rhodamine 123). The P-gly expressing human myeloma cell line, 8226/Dox6, was used as a reference of comparison for levels of P-gly, MDR1 mRNA, and function. CD56+ cells expressed the highest levels of MDR1 mRNA followed by CD8+ > CD4+ approximately equal to CD15+ > CD19+ > CD14+, with percentage values relative to Dox6 of 49%, 17%, 8%, 8%, 4%, and 2%, respectively. The assays for P-gly immunofluorescence and function correlated well with mRNA analysis except for CD15+ cells (granulocytes), which showed a moderate MDR1 mRNA level with a lack of both function and surface P-gly staining. Granulocyte membranes did show P-gly on immunoblot analysis when probed with either C219 or JSB1. We conclude that (1) P-gly and the MDR1 mRNA are expressed in normal leukocytes, (2) this P-gly expression is lineage specific with relatively high levels among CD56+ cells, and (3) the expression of P- gly in granulocytes is not associated with transport of the P-gly substrate, rhodamine 123, out of the cell.
Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model)
Authors: Yeo EL , Sheppard JA , Feuerstein IA
Published: October 2019

Carbohydrate moieties on leukocytes adhere to activated platelets via P- selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.
Interleukin-2 inhibits graft-versus-host disease-promoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects
Authors: Sykes M , Harty MW , Szot GL , Pearson DA
Published: October 2019

We have recently shown that a short course of high-dose interleukin-2 (IL-2) can markedly inhibit the graft-versus-host disease (GVHD)- promoting activity of donor CD4+ T cells. The difficulty in dissociating GVHD-promoting from graft-versus-leukemia (GVL) effects of alloreactive donor T cells currently prevents clinical bone marrow transplantation (BMT) from fulfilling its full potential. To test the capacity of IL-2 treatment to promote such a dissociation, we have developed a new murine transplantable acute myelogenous leukemia model using a class II major histocompatibility complex-positive BALB/c Moloney murine leukemia virus-induced promonocytic leukemia, 2B-4–2. BALB/c mice receiving 2.5 x 10(5) 2B-4–2 cells intravenously 1 week before irradiation and syngeneic BMT died from leukemia within 2 to 4 weeks after BMT. Administration of syngeneic spleen cells and/or a 2.5- day course of IL-2 treatment alone did not inhibit leukemic mortality. In contrast, administration of non-T-cell-depleted fully allogeneic B10 (H-2b) spleen cells and T-cell-depleted B10 marrow led to a significant delay in leukemic mortality in IL-2-treated mice. In these animals GVHD was inhibited by IL-2 treatment. GVL effects were mediated entirely by donor CD4+ and CD8+ T cells. Remarkably, IL-2 administration did not diminish the magnitude of the GVL effect of either T-cell subset. This was surprising, because CD4-mediated GVHD was inhibited in the same animals in which CD4-mediated GVL effects were not reduced by IL-2 treatment. These results suggest a novel mechanism by which GVHD and GVL effects of a single unprimed alloreactive T-cell subset can be dissociated; different CD4 activities promote GVHD and GVL effects, and the former, but not the latter activities are inhibited by treatment with IL-2.
Surface phenotype and Ig heavy-chain gene usage in chronic B-cell leukemias: expression of myelomonocytic surface markers in CD5- chronic B-cell leukemia
Authors: Ikematsu W , Ikematsu H , Okamura S , Otsuka T , Harada M , Niho Y
Published: October 2019

We investigated the surface expression of leukocyte differentiation antigens and the Ig heavy-chain variable region (VH) gene family use in leukemic cells from 26 Japanese patients with chronic B-cell leukemias with special reference to CD5 antigen expression. CD5 was expressed on leukemic cells in 21 of 26 cases (CD5+) but not in 5 cases (CD5-). Myelomonocytic marker, CD13 antigen was expressed on the leukemic cells in all 5 CD5- cases but in none of CD5+ cases. Leukemic cells in CD5- cases also expressed CD11b antigen more frequently than those in CD5+ cases (80% v 11%; P < .01). Another myeloid marker, CD33, was expressed neither on CD5+ nor CD5- leukemic cells. CD22, a restricted B-cell marker, was expressed more frequently on CD5- leukemic cells than on CD5+ leukemic cells (80% v 33%; P < .05). Another restricted B-cell activation marker, CD23, was expressed at similar frequency in both the CD5+ and CD5- groups (67% v 60%). Although CD45RA was expressed on the majority of leukemic B cells, the CD45RA expression level was significantly higher among CD5- cases than CD5+ cases (P < .01). In the analysis of VH gene expressed in chronic B-cell leukemias by polymerase chain reaction amplification, CD5+ cases preferentially used VH4 family members (48%; 10 of 21). CD5- cases, on the other hand, mainly used VH3 family (80%; 4 of 5). Thus, from our present observation of an albeit limited patient population, we have found an association between VH gene family use and CD5 antigen expression in chronic B-cell leukemias. We have also shown the differential expression of myelomonocytic markers in the CD5+ and CD5- chronic B-cell leukemias. These result are in agreement with previous suggestions that CD5 positivity is the hallmark for distinct clinical entity commonly referred to in the literature as chronic lymphocytic leukemia.