bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells
Authors: Mayani H , Dragowska W , Lansdorp PM
Published: October 2019

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells, a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells, purified from umbilical cord blood by flow cytometry and cell sorting, were selected for this study because of their high content of clonogenic cells (34%), particularly multipotent progenitors (CFU-MIX, 12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF, IL-6, IL-3, and erythropoietin (Epo); (3) MGF, IL-6, granulocyte-macrophage colony- stimulating factor (GM-CSF)/IL-3 fusion protein (FP), macrophage colony- stimulating factor (M-CSF), and granulocyte-CSF (G-CSF); and (4) MGF, IL-6, FP, M-CSF, G-CSF, and Epo. Maximum numbers of erythroid progenitors (BFU-E, up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF, IL-6, IL-3, and Epo, whereas maximum levels of myeloid progenitors (CFU-C, up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF, IL-6, FP, M-CSF, and G-CSF. When MGF, IL-6, FP, M-CSF, G-CSF, and Epo were present, maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore, the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors, but rather had a permissive effect on the development of lineage-restricted clonogenic cells.
Neonatal alloimmune thrombocytopenia due to a new platelet-specific alloantibody
Authors: McFarland JG , Blanchette V , Collins J , Newman PJ , Wang R , Aster RH
Published: October 2019

An infant with severe neonatal alloimmune thrombocytopenia is described in whom an antibody directed at a new platelet-specific alloantigen, Ca (HPA-6b), is implicated. The new alloantigen is of low frequency in the population and was localized to platelet glycoprotein (GP) IIIa. Immunoprecipitation studies using murine monoclonal antibodies specific for the GP complex IIb-IIIa and GPIIIa alone (AP2 and AP3) suggest that the location of the Ca epitope on GPIIIa may be near the binding site for AP3. Neonatal alloimmune thrombocytopenia associated with Ca is likely to be as severe as that seen in cases due to incompatibilities for the HPA-1 (PIA) and HPA-4 (Pen) platelet alloantigen systems, because each is located on GPIIIa, a densely represented molecule on the platelet surface.
Production and characterization of a bispecific IgG capable of inducing T-cell-mediated lysis of malignant B cells
Authors: Link BK , Weiner GJ
Published: October 2019

Bispecific monoclonal antibodies (bsabs) recognizing both CD3 and a tumor antigen can redirect T-cell-mediated cytotoxicity toward cells bearing that antigen. Such bsabs have been shown to be more effective than monospecific monoclonal antibodies (MoAbs) at preventing tumor growth in animal models of B-cell malignancy. The current studies describe the production and preliminary evaluation of a bsab designed to induce the lysis of malignant human B cells by human T cells. The bsab was obtained from a hybrid-hybridoma cell line produced by fusing OKT3-secreting hybridoma cells with hybridoma cells that secrete 1D10. 1D10 is an MoAb that recognizes an antigen found on a majority of malignant human B cells that has not been detected to a significant degree on normal resting or activated lymphocytes. High performance liquid chromatography (HPLC) was used to separate bsab from monospecific antibodies that were also present in the hybrid-hybridoma antibody product. The bsab was then evaluated in vitro for its ability to induce lysis of malignant B cells by activated T cells. The bsab consistently induced extensive lysis in vitro of 1D10 (+) cells, including both cell lines and cells obtained from patients with a variety of B-cell malignancies. No such effect was seen with activated T cells alone or activated T cells with monospecific antibody. No increased lysis was seen with 1D10 (-) cell lines. The bsab also mediated lysis of malignant B cells by autologous T cells. We conclude bsab containing an OKT3 arm and a 1D10 arm can induce T-cell-mediated lysis in a manner that is both potent and specific. This supports further evaluation of this bsab as a potential immunotherapy of B-cell malignancy.
Epstein-Barr virus-related oral T-cell lymphoma associated with human immunodeficiency virus immunosuppression
Authors: Thomas JA , Cotter F , Hanby AM , Long LQ , Morgan PR , Bramble B , Bailey BM
Published: October 2019

Epstein-Barr virus (EBV) is generally held to infect B cells and epithelial cells, although there are now reports of EBV infection in normal T cells and neoplastic T-cell diseases. In patients with human immunodeficiency virus (HIV) infection, EBV is associated with the benign epithelial lesion, hairy leukoplakia, and has been reported in up to 80% of acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma. This study shows the presence of EBV in malignant oral T-cell lymphoma in three AIDS patients, two of whom had concurrent manifestation of hairy leukoplakia. The T-cell lineage of the tumor cells was determined by positive immunophenotyping for T-cell markers and lack of B-cell or nonhematopoietic (cytokeratin) determinants. All tumors contained monoclonal T-cell populations shown by polymerase chain reaction, which showed amplification of T-cell receptor gamma chain DNA without evidence of Ig heavy chain gene rearrangement. Furthermore, these lesions showed the presence of EBV DNA and expression of EBV latent gene products in the tumor cells. EBV involvement in AIDS-related T-cell lymphoma has not been widely reported and may represent a further manifestation of opportunistic EBV infection arising in the HIV-immunocompromised host.
Interleukin-6 gene expression in multiple myeloma: a characteristic of immature tumor cells
Authors: Hata H , Xiao H , Petrucci MT , Woodliff J , Chang R , Epstein J
Published: October 2019

Interleukin-6 (IL-6) has been suggested to play a major role in multiple myeloma. To investigate the source and target cells of IL-6 activity in multiple myeloma, expression of the cytokine and its receptor genes by myeloma plasma cells was studied. Tumor cells were sorted from bone marrow aspirates of myeloma patients using 4-parameter gating. Myeloma cells were identified as CD38high CD45negative- intermediate and by their light-scatter characteristics. Sorted cells contained only myeloma plasma cells. No contaminating cells were present as determined morphologically, by monoclonal cytoplasmic Ig analysis, and by polymerase chain reaction (PCR) amplification of marker genes. Myeloma cells from 45% of patients expressed IL-6. IL-6 receptor transcripts were found in 68% of the specimens. IL-6 gene expression correlated with expression of the IL-6 receptor gene (P < .005). Correlations observed between the expression of CD45, a protein tyrosine phosphatase expressed by B lymphocytes but not by plasma cells, and the expression of the IL-6 and IL-6-receptor genes (P < .0002 and P < .005, respectively) suggest that an autocrine IL-6 loop is functioning in myeloma in preplasma cells.
Induction of functional lipoxin A4 receptors in HL-60 cells
Authors: Fiore S , Romano M , Reardon EM , Serhan CN
Published: October 2019

The appearance of [11,12–3H]lipoxin A4 (LXA4) specific binding sites was examined with human acute promyelocytic leukemic cell line 60 (HL- 60) cells exposed to either retinoic acid, phorbol 12-myristate 13- acetate (PMA), or dimethyl sulfoxide (DMSO). All three agents induced a threefold to fivefold increase in the expression of specific [11,12- 3H]LXA4 binding. Similar results were obtained in parallel with [14,15- 3H]leukotriene (LT) B4. For both 3H-ligands, homologous displacement curves were similar and independent of the agent used to induce differentiation. Specific binding of [11,12–3H]LXA4 to differentiated HL-60 cells gave a kd = 0.6 +/- 0.3 nmol/L. The appearance of both [11,12–3H]LXA4 and [14,15–3H]LTB4-specific binding sites was inhibited by actinomycin D, and LXA4 binding was sensitive to protease treatment. Specific binding of [11,12–3H]LXA4 was not evident with human platelets, red blood cells (RBCs) or the cultured B-cell (Raji), T-cell (Jurkat) lines save human endothelial cells (kd = 11.0 +/- 0.3 nmol/L). The structural specificity of induced [11,12–3H]-LXA4 recognition sites was assessed with LXB4, LTC4, LTB4, and trihydroxyhepatanoic methyl ester. Only LTC4, at 3-log molar excess, competed for 3H-LXA4-specific binding with HL-60 cells and gave a 30% reduction. The leukotriene D4 receptor antagonist SKF 104353 was ineffective in blocking [11,12- 3H]LXA4-specific binding with HL-60 cells while it competed for specific [11,12–3H]LXA4 binding with endothelial cells where LTD4 binding appears to be virtually identical to that of LXA4 binding. In addition, the LTB4 receptor antagonist ONO 4057 was ineffective at competing for [11,12–3H]LXA4 binding. When phospholipase D activation was monitored in human polymorphonuclear leukocytes (PMN) and HL-60 cells, a correlation was shown between activation and specific 3H-LXA4 binding. LXA4-induced phospholipase D (PLD) activation gave a biphasic concentration-dependent response comprised of at least two components: one phase being islet-activating protein (IAP)-sensitive (LXA4 10(-9) mol/L peak activity) and the other was staurosporine-sensitive (LXA4 10(-7) mol/L peak activity). Results indicate that HL-60 cells exposed to differentiating agents express [11,12–3H]LXA4 recognition sites also present in PMN. In addition, specific LXA4 recognition sites of myeloid cells can be distinguished by competition binding with SKF 104353 and 3H-LXA4 cross-reactivity with putative LTD4 receptors present on human endothelial cells. Moreover, they provide evidence indicating that binding of LXA4 to its recognition sites confers functional responses.
Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma
Authors: Gribben JG , Neuberg D , Freedman AS , Gimmi CD , Pesek KW , Barber M , Saporito L , Woo SD , Coral F , Spector N
Published: October 2019

Although molecular biologic techniques can now detect minimal numbers of residual cancer cells in patients in complete clinical remission, the clinical significance of minimal residual disease has never been conclusively established. If the detection of minimal residual disease predicts which patients will relapse, then therapy could be altered based upon the detection of these cells. The t(14;18) can be detected by polymerase chain reaction (PCR) amplification in 50% of patients with B-cell non-Hodgkin's lymphoma and allows detection of one lymphoma cell in up to 1 million normal cells. To determine the clinical significance of the detection of minimal residual lymphoma cells in the bone marrow (BM) PCR amplification was used to detect the presence of residual lymphoma cells after autologous BM transplantation (ABMT) in serial BM samples from 134 patients with B-cell lymphoma in whom a bcl- 2 translocation could be detected. PCR analysis was performed on a total of 542 BM samples obtained while these patients were in complete remission. Disease-free survival was markedly increased in patients with no PCR-detectable lymphoma cells in the marrow compared with those in whom residual lymphoma cells were detected (P < .00001), and the presence of detectable lymphoma cells was associated with a 48-fold increase in the risk of relapse. Of the 77 patients (57%) with no PCR- detectable lymphoma cells in their most recent BM sample, none have relapsed. In contrast, all 33 patients (25%) who have relapsed had PCR- detectable lymphoma cells detected in their BM before clinical relapse occurred. In 19 patients (14%), residual lymphoma cells in the BM were detected early following transplantation and subsequently were no longer detectable, although these patients received no further therapy. In these patients, residual lymphoma cells may already have been irreversibly damaged by the high-dose therapy or an endogenous immune mechanism may be capable of eliminating residual lymphoma cells in some patients. Therefore, although the detection of minimal residual disease by PCR following ABMT in patients with lymphoma identifies those patients at high risk of relapse, the presence of residual minimal disease early after transplantation may not be associated with poor prognosis in a small subset of patients. Confirmatory studies will be required to determine more definitively the role of minimal disease detection to identify which patients require additional therapy.
Granulocyte-macrophage colony-stimulating factor-induced antibody- dependent cellular cytotoxicity in bone marrow macrophages: application in bone marrow transplantation
Authors: Charak BS , Agah R , Mazumder A
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to induce antitumor activity in peripheral blood monocytes. We examined the role of GM-CSF on bone marrow (BM) macrophages in inducing antibody-dependent cellular cytotoxicity (ADCC) against murine and human tumor cells in vitro and in vivo with the aim of applying this approach in an autologous bone marrow transplantation (BMT) setting. GM- CSF induced a potent ADCC in BM macrophages against a murine melanoma in vitro. Treatment with GM-CSF alone or with antibody alone had no effect, whereas therapy with combination of both these agents resulted in a significant reduction in dissemination of melanoma both in a nontransplant as well as in BMT settings, with results being more optimal in the latter setting. Adoptive transfer of BM macrophages harvested from mice undergoing therapy with GM-CSF plus antibody significantly reduced the dissemination of melanoma in secondary recipients but only after irradiation, not in intact mice. GM-CSF also induced significant ADCC in human BM macrophages against a melanoma and a lymphoma in vitro and against a lymphoma implanted in nude mice in vivo. Again, these effects were more optimal after chemotherapy. These data suggest that treatment with GM-CSF plus tumor-specific monoclonal antibodies after BMT may induce an antitumor effect and help eradicate the minimal residual disease.
High serum and ascitic soluble interleukin-2 receptor alpha levels in advanced epithelial ovarian cancer [see comments]
Authors: Barton DP , Blanchard DK , Michelini-Norris B , Nicosia SV , Cavanagh D , Djeu JY
Published: October 2019

This study was undertaken to determine if advanced epithelial ovarian cancer was associated with increased serum and ascitic levels of soluble interleukin-2 receptor alpha (sIL-2R alpha). Serum and ascitic fluid samples from 23 ovarian cancer patients were analyzed for sIL-2R alpha using an enzyme-linked immunosorbent assay and compared with the serum and peritoneal levels in 18 normal females. The samples were analyzed for CA-125 levels using a radioimmunoassay and the total protein was also measured. Normal individuals had low serum levels of sIL-2R alpha (367.5 +/- 44.6 U/mL), with similar levels of sIL-2R alpha in the normal peritoneal fluid (438.6 +/- 48.8 U/mL). In contrast, the serum and ascitic fluid levels in ovarian cancer patients were significantly higher (746.7 +/- 82.9 U/mL, P = .0006; 2,656.7 +/- 373.7 U/mL, P = .00002, respectively). The results for sIL-2R alpha were also significant when the levels were expressed per milligram of total protein. More importantly, in almost every ovarian cancer patient the ascitic sIL-2R alpha level far exceeded the serum level, a pattern also observed for CA-125. There was no correlation between the serum and ascitic sIL-2R alpha levels, or between the serum and ascitic CA-125 levels. Although the serum levels of sIL-2R alpha and CA-125 were elevated in the same patient, overall there was no correlation between the serum sIL-2R alpha and serum CA-125 levels, either when the levels were expressed in absolute units or per milligram of total protein. Similarly, there was no correlation between sIL-2R alpha and CA-125 levels in individual ascitic samples. While CA-125 levels may reflect an independent index of tumor burden, these results suggest that selective accumulation of sIL-2R alpha in the ascites may be one of the factors associated with the known nonresponsiveness of the infiltrating lymphocytes against ovarian carcinoma cells.
Subtractive cDNA cloning of a novel member of the Ig gene superfamily expressed at high levels in activated B lymphocytes
Authors: Kozlow EJ , Wilson GL , Fox CH , Kehrl JH
Published: October 2019

Using subtractive cDNA cloning we have isolated a series of cDNA clones that are exclusively or selectively expressed in B lymphocytes. mRNA transcripts from one such cDNA clone, referred to as BL11, were found to be expressed at low levels in RNA from normal B lymphocytes, but at very high levels in RNA from in vitro activated B lymphocytes. One major 2.5-kb BL11 mRNA transcript was detected, while low levels of 4.8- , 1.8-, and 1.6-kb transcripts were also found. BL11 mRNA transcripts were absent or present at low levels in RNA prepared from resting or mitogen activated T cells, a variety of lymphoid cell lines including several B-cell lines, and several different tissues. Low levels of BL11 transcripts were found in poly(A) RNA purified from brain and lung. A study of the kinetics of BL11 mRNA accumulation in B lymphocytes stimulated in vitro with Staphylococcus aureus Cowan strain I showed a rapid induction of BL11 mRNA within 2 hours of stimulation with peak expression by 16 hours and a mild decrease with time following the peak levels. Consistent with the in vitro data, in situ hybridization using antisense BL11 RNA probes and human tonsillar tissue localized BL11 transcripts in B-cell-enriched areas. Multiple BL11 cDNA and genomic clones were isolated and sequenced to complete and verify the BL11 cDNA sequence (2,404 bp). A 615-nucleotide open reading frame predicted to encode for a 205-amino acid protein with a molecular weight of 23 Kd was identified. Search of protein data bases with the predicted BL11 protein showed homologies to several members of the Ig superfamily. Analysis of the predicted protein showed a likely signal peptide, a single membrane spanning region, and one V-like Ig domain with three predicted n-glycosylation sites. Southern blot analysis of human genomic DNA suggested that BL11 is a single copy gene without evidence of rearrangement. Primer extension and S1 nuclease mapping identified four tightly clustered transcriptional start sites approximately 40 bp upstream of the predicted translation start site. The first 270 bp of the promoter region were sequenced and found to contain a CATAA box rather than a TATAA box and several DNA motifs found in activation genes. BL11 should prove to be an interesting gene that likely encodes for a protein involved in B-cell activation.
Regulation of myeloblastin messenger RNA expression in myeloid leukemia cells treated with all-trans retinoic acid
Authors: Labbaye C , Zhang J , Casanova JL , Lanotte M , Teng J , Miller WH Jr , Cayre YE
Published: October 2019

Retinoic acid is known to induce differentiation of human myeloid leukemia cells in vitro. Recently, all-trans retinoic acid has been used to induce remissions in patients with acute promyelocytic leukemia, probably through differentiation of the leukemia cells. Myeloblastin (mbn) is a protease that has been identified in the human leukemia cell line HL-60. Downregulation of this protease can inhibit proliferation and induce differentiation of HL-60-derived leukemia cells. Here we have investigated the regulation of mbn messenger RNA (mRNA) expression in two human leukemia cell lines, HL-60 and NB4, treated with all-trans retinoic acid. Under this treatment, downregulation of mbn mRNA was observed in both cell lines, but was considerably delayed in NB4 cells that carry the t(15;17) translocation characteristic of acute promyelocytic leukemia. We have found that multiple mechanisms were involved in the control of mbn mRNA expression. These mechanisms were different in HL-60 and NB4 cells. Our results show that in HL-60 cells, all-trans retinoic acid rapidly decreased transcription of mbn. In contrast, in the t(15;17)-positive NB4 cells treated with all-trans retinoic acid, upregulation of mbn mRNA expression was followed by a late downregulation, both achieved via posttranscriptional mechanisms.
Activation of factor XI in plasma is dependent on factor XII [see comments]
Authors: Brunnee T , La Porta C , Reddigari SR , Salerno VM , Kaplan AP , Silverberg M
Published: October 2019

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.
Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization
Authors: Fibi MR , Aslan M , Hintz-Obertreis P , Pauly JU , Gerken M , Luben G , Lauffer L , Siebold B , Stuber W , Nau G
Published: October 2019

Recombinant human erythropoietin (rhuEpo)-specific mouse monoclonal antibodies (MoAbs) have been produced and characterized. All antibodies were specifically reactive with rhuEpo in enzyme-linked immunosorbent assay (ELISA). Epitope exclusion studies showed three distinct epitope regions, A, B, and C, recognized by neutralizing MoAbs. An additional epitope region D was recognized by non-neutralizing MoAbs. Antibodies defining an epitope region competed with each other for binding sites, but did not compete with antibodies defining a different epitope region. Group B antibodies were able to compete for the receptor binding site on rhuEpo with a soluble human Epo-receptor-lg fusion protein. No single peptide sequences were found to specifically interact either with group B MoAbs or with the rhuEpo-receptor. Therefore, it is suggested that epitope region B and the receptor binding site share binding determinants that are primarily composed of conformational epitopes. Because group A and group C antibodies did not compete with the receptor for binding to the receptor binding site of the rhuEpo molecule, it is suggested that neutralization via epitope regions A and C is mediated through binding inhibition caused by conformational changes, transmuting the binding site(s) for the receptor. Conversely, binding to the receptor seems to induce conformational changes in the hormone molecule, eliminating epitopes for group A and C antibodies.
Biologically active thrombomodulin is synthesized by adherent synovial fluid cells and is elevated in synovial fluid of patients with rheumatoid arthritis
Authors: Conway EM , Nowakowski B
Published: October 2019

Thrombomodulin (TM) is a transmembrane glycoprotein that interacts with thrombin, thereby serving as a cofactor in the activation of protein C, a major physiologically relevant natural anticoagulant. Although initially described as a vascular endothelial cell receptor, TM has also been reported to be synthesized by several cells, including megakaryocytes, platelets, monocytes, neutrophils (PMN), mesothelial cells, and synovial lining cells. A prominent feature of rheumatoid arthritis (RA) is infiltration of PMN into the joint space. To determine whether TM might play a role in the inflammatory process, we examined synovial fluid for the presence of TM in 10 patients with RA and five patients with osteoarthritis (OA). We determined that the mean synovial fluid and plasma TM levels in the OA group were 23.5 ng/mL and 24.2 ng/mL, respectively, whereas those with RA had a significantly elevated mean synovial fluid TM level of 136.2 ng/mL as compared with the plasma TM concentration of 43.9 ng/mL (P < .05). Synovial fluid TM levels did not correlate with PMN counts (r = .261). Purified TM from synovial fluid was identical in molecular weight to plasma-derived TM and was biologically functional with respect to protein C cofactor activity. Using direct immunofluorescence, we determined that adherent cultured synovial fluid cells that are not monocytoid in origin express surface and cytoplasmic TM, thereby providing an alternative source of the protein. Biologic activity of the cell-surface TM was confirmed by acceleration of thrombin-dependent protein C activation. Northern analysis of RNA extracted from the cultured cells indicated that TM messenger RNA was present, suggesting local synthesis. Our results indicate that in RA-associated synovial effusions, biologically active TM is increased, the source of which may be from plasma, PMN, and/or synovial lining cells. TM may play a regulatory role either in fibrin deposition in the inflamed joint and/or in the progression of the inflammatory process.
Human immunodeficiency virus-related conditions in children and adults with hemophilia: rates, relationship to CD4 counts, and predictive value
Authors: Eyster ME , Rabkin CS , Hilgartner MW , Aledort LM , Ragni MV , Sprandio J , White GC , Eichinger S , de Moerloose P , Andes WA
Published: October 2019

To further elucidate the natural history of human immunodeficiency virus (HIV) infection, we studied intermediate HIV-related conditions occurring before acquired immunodeficiency syndrome (AIDS) in a prospectively observed multicenter cohort of 738 HIV-infected persons with hemophilia. We analyzed the frequency in adults and children of common HIV-related conditions and the relative risk of AIDS after occurrence of these conditions, controlling for age at seroconversion and the percentage of CD4+ lymphocytes. Thrombocytopenia was the most frequently observed condition with cumulative incidences of 43% +/- 7% in adults and 27% +/- 6% in children and adolescents by 10 years after seroconversion. Oral candidiasis, fever, weight loss, and non-AIDS pneumonia were two to four times more common in adults than children, whereas herpes zoster risk was similar in the two age groups. HIV- related conditions were infrequent during the first 4 years of infection, particularly in children. With the exception of thrombocytopenia, mean CD4 counts were less than 350 cells/microL at the onset of the conditions. The relative hazard of AIDS after oral candidiasis was 18 in children and 3.8 in adults. Relative hazard in adults was also increased after persistent fever (10), weight loss (3.2), and non-AIDS pneumonia (2.2). Herpes zoster and thrombocytopenia were not significantly associated with AIDS in either age group. We conclude that intermediate HIV-related conditions occur more frequently in adults than in children with hemophilia. Persistent fever is the strongest predictor of AIDS in adults, whereas oral candidiasis is the strongest predictor in children. These findings should facilitate the design and conduct of clinical trials as well as the management of HIV- infected children and adults.
Prolonged survival of B-lineage acute lymphoblastic leukemia cells is accompanied by overexpression of bcl-2 protein
Authors: Campana D , Coustan-Smith E , Manabe A , Buschle M , Raimondi SC , Behm FG , Ashmun R , Arico M , Biondi A , Pui CH
Published: October 2019

Overexpression of bcl-2 delays the onset of apoptosis in lymphohematopoietic cells. We measured levels of bcl-2 protein in normal and leukemic human B-cell progenitors with a specific monoclonal antibody and flow cytometry. Normal immature B cells had low levels of bcl-2 protein; the intensity of fluorescence, expressed as molecules of soluble fluorochrome per cell, within CD10+ cells was 3,460 +/- 1,050 (mean +/- SD; 5 samples). In 16 cases of B-lineage acute lymphoblastic leukemia (ALL), cells had levels of bcl-2 that were strikingly higher than those of their normal counterparts (33,560 +/- 14,570; P < .001 by t-test analysis). We next investigated whether the intensity of bcl-2 expression correlated with the resistance of immature B cells to in vitro culture. In 12 cases of B-lineage ALL, the cells recovered after 7 days of culture on allogeneic bone marrow stromal layers were 69% to 178% (median, 95.5%) of those originally seeded. Prolonged survival of leukemic cells in vitro was observed even in the absence of stromal layers in 6 of these 12 cases; the intensity of bcl-2 protein expression in these cases was 45,000 +/- 13,270, compared with 21,500 +/- 7,260 in the 6 cases in which greater than 99.5% of cells rapidly died by apoptosis under the same culture conditions (P = .003). Five immature B-cell lines, continuously growing in the absence of stroma, had the highest bcl-2 expression (79,400 +/- 20,330). By contrast, most normal CD19+, sIg-immature B cells died despite the presence of bone marrow stromal layers; 9.7% to 28.2% were recovered after 7 days of culture in three experiments. We conclude that abnormal bcl-2 gene expression influences the survival ability of B-cell progenitors. This may contribute to leukemogenesis and explain the aptitude of leukemic lymphoblasts to expand outside the bone marrow microenvironment.
The 5q- syndrome: a single-institution study of 43 consecutive patients
Authors: Mathew P , Tefferi A , Dewald GW , Goldberg SL , Su J , Hoagland HC , Noel P
Published: October 2019

A favorable prognosis and a low rate of leukemic transformation has been attributed to the 5q- syndrome, a myelodysplastic syndrome (MDS) characterized by macrocytic anemia, hypolobulated micromegakaryocytic hyperplasia, and an interstitial deletion of chromosome 5. We examined the characteristics and outcome of 43 consecutive patients in our institution strictly defined by morphologic criteria and a solitary 5q- cytogenetic defect. The median age at diagnosis was 68 years, with a clear female predominance (7:3). Eighty percent of the patients were red blood cell transfusion-dependent at diagnosis and all untransfused patients had macrocytic indexes. In contrast, significant neutropenia or thrombocytopenia was rare. The French-American-British (FAB) class distributions were RA (72%), RARS (7%), RAEB (16%), and RAEB-IT (5%). At a median follow-up of 31 months, 56% of the patients survive, with a projected median survival of 63 months. The incidence of acute leukemia was 16% and was uniformly fatal. Clinical hemosiderosis occurred in 28% of the patients, resulting in two deaths. Neither survival nor the risk of leukemic transformation was predictable from initial clinical parameters, including FAB classification, Bournemouth score, and degree of aneuploidy. The lack of significant neutropenia and thrombocytopenia seemed to account for a very low incidence of infection and bleeding resulting in a prognosis equal or superior to historical patients with MDS. Therapeutic endeavors, including the use of corticosteroids, androgens, cis-retinoic acid, pyridoxine, and danazol, were largely unsuccessful.
The distribution of erythrocyte phospholipids in hereditary spherocytosis demonstrates a minimal role for erythrocyte spectrin on phospholipid diffusion and asymmetry
Authors: Kuypers FA , Lubin BH , Yee M , Agre P , Devaux PF , Geldwerth D
Published: October 2019

In the human erythrocyte membrane phosphatidylcholine and sphingomyelin reside mainly in the outer leaflet, whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, are mainly found in the inner leaflet. Maintenance of phospholipid asymmetry has been assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin. To investigate whether spectrin contributes to maintaining the phospholipid transbilayer distribution and kinetics of redistribution, we studied erythrocytes from hereditary spherocytosis patients whose spectrin levels ranged from 34% to 82% of normal. The phospholipid composition and the accessibility of membrane phospholipids to hydrolysis by phospholipases were in the normal range. Spin-labeled phosphatidylserine and phosphatidylethanolamine analogues that had been introduced into the outer leaflet were rapidly transported at 37 degrees C to the inner leaflet, whereas the redistribution of spin-labeled phosphatidylcholine was slower. The kinetics of transbilayer movement of these spin-labeled phospholipid in all samples was in the normal range and was not affected by the level of spectrin. Although these erythrocyte membranes contained as little as 34% of the normal level of spectrin and were characterized by several physical abnormalities, the composition, distribution, and transbilayer kinetics of the phospholipids were found to be normal. We therefore conclude that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.
Methotrexate, cyclosporine, or both to prevent graft-versus-host disease after HLA-identical sibling bone marrow transplants for early leukemia?
Authors: Ringden O , Horowitz MM , Sondel P , Gale RP , Biggs JC , Champlin RE , Deeg HJ , Dicke K , Masaoka T , Powles RL
Published: October 2019

Optimal prophylaxis of graft-versus-host disease (GVHD) is controversial. We compared efficacy of three posttransplant immune suppressive regimens in 2,286 recipients of HLA-identical sibling bone marrow transplants for acute lymphoblastic leukemia (ALL) in first remission, acute myelogenous leukemia (AML) in first remission, or chronic myelogenous leukemia (CML) in first chronic phase. Six hundred forty received methotrexate, 977 received cyclosporine, and 669 received combined cyclosporine and methotrexate. In children, the three regimens resulted in similar outcomes. In adults, cyclosporine and methotrexate had comparable risks of acute and chronic GVHD. Compared with methotrexate, cyclosporine was associated with less interstitial pneumonia (relative risk [RR] = 0.6; P < .001), less treatment-related mortality (RR = 0.6; P < .001), more relapses (RR = 1.6; P < .05), and less treatment failure (relapse or death from any cause; RR = 0.7; P < .001). Different effects were observed in different leukemias. In ALL, the rate of leukemia relapse was increased with cyclosporine versus methotrexate, with no effect on other outcomes. In AML and CML, interstitial pneumonia, treatment-related mortality, and treatment failures were decreased with cyclosporine, with no increase in relapse. Similar analyses comparing cyclosporine plus methotrexate with cyclosporine alone showed that adults receiving the combination had less acute GVHD (RR = 0.5; P < .001), less chronic GVHD (RR = 0.7; P < .01), and less interstitial pneumonia (RR = 0.7; P < .001). Treatment failure (RR = 0.8; P < .05) was marginally reduced. Separate analyses in ALL and AML showed less acute GVHD with combined therapy, but no significant effect on other outcomes. In CML, acute GVHD, interstitial pneumonia, treatment-related mortality, and treatment failure were decreased with combined therapy.
Detection of DNA rearrangements in the AML1 and ETO loci and of an AML1/ETO fusion mRNA in patients with t(8;21) acute myeloid leukemia
Authors: Nucifora G , Birn DJ , Erickson P , Gao J , LeBeau MM , Drabkin HA , Rowley JD
Published: October 2019

The (8;21)(q22;q22) translocation is a frequent karyotypic abnormality seen in approximately 40% of patients with acute myeloid leukemia subtype M2 (AML-M2) and an abnormal karyotype. The translocation interrupts two genes, AML1 on chromosome 21 and ETO on chromosome 8, that are consequently fused in the der(8) chromosome to produce a novel chimeric gene and message. Selected genomic DNA probes from chromosome 21 and from chromosome 8 near the breakpoint junction detect rearrangements in the DNA of about 80% of the patients with the rearrangement at diagnosis and in relapse. We analyzed the DNA of 20 patients with t(8;21) AML by standard Southern blot with probes originating from chromosomes 21 and 8 near the breakpoint junction, and we identified rearranged bands in 17 of the 20 patients at diagnosis and in relapse. We also used the polymerase chain reaction (PCR) with appropriate primers from the AML1 and ETO genes to amplify the cDNAs from a cell line with the t(8;21) and from seven AML patients with the t(8;21). We detected a fused transcript in the cell line and in all of the patients analyzed, including three patients who did not show any rearrangement by Southern blot analysis and one patient in hematologic remission, who later relapsed. Combining the results from Southern blot and PCR analysis, we could detect the t(8;21) in all of the patients tested. These results indicate that, whereas several DNA probes used as genetic markers do detect the t(8;21) in most, but not all Southern blots of patients with AML, PCR amplification with primers from AML1 and ETO can be used as a more sensitive and accurate means for detecting this chromosomal abnormality, and for observing the patients' response to therapy.
Recombinant human interleukin-11 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice
Authors: Neben TY , Loebelenz J , Hayes L , McCarthy K , Stoudemire J , Schaub R , Goldman SJ
Published: October 2019

The effects of recombinant human interleukin-11 (rhIL-11) on in vivo mouse megakaryocytopoeisis were examined. Normal C57Bl/6 mice and splenectomized C57Bl/6 mice were treated for 7 days with 150 micrograms/kg rhIL-11 administered subcutaneously. In normal mice, peripheral platelet counts were elevated compared with vehicle-treated controls after 3 days of rhIL-11 treatment and remained elevated until day 10. Splenectomized mice treated with rhIL-11 showed elevated peripheral platelet counts that were similar in magnitude to normal rhIL-11-treated mice. However, on day 10 the platelet counts in rhIL-11- treated, splenectomized mice were no longer elevated. Analysis of bone marrow megakaryocyte ploidy by two-color flow cytometry showed an increase, relative to controls, in the percentage of 32N megakaryocytes in both normal and splenectomized animals treated with rhIL-11. In normal mice, the number of spleen megakaryocyte colony-forming cells (MEG-CFC) were increased twofold to threefold relative to controls after 3 and 7 days of rhIL-11 treatment, whereas the number of bone marrow MEG-CFC were increased only on day 7. The number of MEG-CFC in the bone marrow of rhIL-11-treated, splenectomized mice was increased twofold compared with controls on both days 3 and 7 of the study. These data show that in vivo treatment of normal or splenectomized mice with rhIL-11 increased megakaryocyte progenitors, stimulated endoreplication of bone marrow megakaryocytes, and increased peripheral platelet counts. In addition, results in splenectomized mice showed that splenic hematopoiesis was not essential for the observed increases in peripheral platelets in response to rhIL-11 administration.
Basic fibroblast growth factor counteracts the suppressive effect of transforming growth factor-beta 1 on human myeloid progenitor cells
Authors: Gabrilove JL , Wong G , Bollenbacher E , White K , Kojima S , Wilson EL
Published: October 2019

We have previously shown that basic fibroblast growth factor (bFGF) is mitogenic for human bone marrow stromal cells and enhances myelopoiesis in human long-term bone marrow culture. In the present study, we examined the mechanism by which bFGF enhances granulopoiesis. We observed that bFGF significantly abrogated the inhibitory effect of transforming growth factor-beta 1 (TGF-beta 1) on granulocyte- macrophage colony-stimulating factor (GM-CSF)-supported progenitor cell growth (P = .009). The partial reversal of TGF-beta 1-mediated suppression was dependent on the dose of bFGF used. In addition, we noted that the inclusion of neutralizing antibody to TGF-beta 1 significantly augmented the clonogenic response to GM-CSF. We have also shown that 10 ng/mL or 100 ng/mL of bFGF resulted in a 30% to 100% increase in GM-CSF-mediated progenitor cell growth (P = .0001). These data suggest that bFGF may enhance myelopoiesis by modulating the inhibitory response to TGF-beta 1.
Endotoxin-induced tissue factor messenger RNA in human monocytes is negatively regulated by a cyclic AMP-dependent mechanism
Authors: Ollivier V , Houssaye S , Ternisien C , Leon A , de Verneuil H , Elbim C , Mackman N , Edgington TS , de Prost D
Published: October 2019

Tissue factor (TF) is a transmembrane receptor that serves as the major cofactor for factor VIIa-catalyzed proteolytic activation of factors IX and X. In response to bacterial lipopolysaccharide (LPS), monocytes transcribe, synthesize, and express TF on their surface, thereby conveying to activated monocytes the ability to initiate the blood coagulation protease cascades. Agents that elevate cellular cyclic AMP (cAMP) inhibit the functional expression of TF by LPS-stimulated monocytes. In this study, we investigated the mechanism of this suppression. Northern blot analysis of total RNA from LPS-stimulated monocytes showed a concentration-dependent decrease in TF messenger RNA (mRNA) levels in response to dibutyryl-cAMP (dBt-cAMP). TF mRNA and procoagulant activity were inhibited as early as 1 hour after the addition of dBt-cAMP and the inhibition persisted through 4 hours. Suppression of specific mRNA abundance was also observed with agents, including forskolin and iso-butyl-methyl-xanthine (IBMX), that increase cAMP levels by independent mechanisms. Flow immunocytometric analysis confirmed that cell-surface TF protein levels declined in parallel with TF functional activity. The rate of decay of TF mRNA after the arrest of transcription by actinomycin D was not altered by the addition of dBt-cAMP, IBMX, or forskolin, thus excluding effects on TF mRNA stability. We conclude that elevated cAMP levels suppress TF mRNA by reducing the rate of TF gene transcription.
The kinetics of plasminogen activation by thrombin-cleaved pro- urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator
Authors: Liu JN , Gurewich V
Published: October 2019

Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A- chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90- fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro- UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb- UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.
Interferon-alpha in mixed cryoglobulinemia patients: a randomized, crossover-controlled trial
Authors: Ferri C , Marzo E , Longombardo G , Lombardini F , La Civita L , Vanacore R , Liberati AM , Gerli R , Greco F , Moretti A
Published: October 2019

The effects of interferon-alpha (IFN-alpha) on clinical and serologic manifestations of mixed cryoglobulinemia (MC) were investigated by randomized, crossover-controlled trial in 26 patients. The trial alternated 6 months with and 6 months without IFN-alpha therapy (2 x 10(6) IU daily for a month, then every other day for 5 months). In 22 patients, pretreatment steroid dosage remained unchanged during the study. Six patients dropped out (three because of side effects), whereas another 20 patients experienced a significant improvement of purpura (P < .02) and serum transaminases (P < .005) during IFN-alpha treatment. The presence of clinical improvement was supported by the outcome measurements of several immunologic parameters. In particular, serum cryoglobulins were significantly reduced (P < .04) during IFN- alpha therapy. A rebound phenomenon of clinical and serologic parameters was observed after IFN-alpha discontinuation. In addition, no variations were recorded during 6 months without therapy. Hepatitis C virus (HCV) RNA was detected in 91% (20/22) of our MC patients; in 2/13 cases HCV RNA was no longer detectable in serum samples after IFN- alpha therapy. Thus, IFN-alpha could be considered as treatment for MC in patients with HCV seropositivity.