bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of endothelial and mesothelial cell function by interleukin- 13: selective induction of vascular cell adhesion molecule-1 and amplification of interleukin-6 production
Authors: Sironi M , Sciacca FL , Matteucci C , Conni M , Vecchi A , Bernasconi S , Minty A , Caput D , Ferrara P , Colotta F
Published: October 2019

The present study was designed to explore the interaction of interleukin-13 (IL-13) with vascular endothelial cells (EC). In vitro exposure to IL-13 of human umbilical vein EC induced surface expression of vascular cell adhesion molecule-1 (VCAM-1). At optimal concentrations (10 to 50 ng/mL) and exposure times (24 hours), IL-13 was a twofold to threefold less effective inducer of VCAM-1 than IL-1, which was used as reference EC activator. When IL-13 was combined with IL-1, an almost additive induction of VCAM-1 was observed. Induction of VCAM-1 by IL-13 was selective in that E-selectin and intercellular adhesion molecule-1 (ICAM-1) were unaffected. IL-13 caused a modest reduction of IL-1 induction of E-selectin and ICAM-1. Surface expression of VCAM-1 on IL-13-treated cells was associated with mRNA induction (as assessed by Northern analysis and reverse transcriptase- polymerase chain reaction), with predominance of transcripts encoding the 7 Ig domain form of this molecule. In agreement with previous reports, IL-13 inhibited cytokine production in human monocytes. In contrast, IL-13 was a weak inducer and an amplifier (in concert with IL- 1) of IL-6 expression in EC. Mesothelial cells, which share properties with EC and regulate the traffic and function of leukocytes in serosal cavities, were stimulated to express VCAM-1 and IL-6 by IL-13. Thus, IL- 13 elicits a spectrum of responses in vascular endothelium remarkably similar to that of IL-4 and IL-10. Interaction of these cytokines with vascular endothelium may play an important role in the induction and expression of Th2-dependent responses.
Interleukin-2 may induce prolonged remissions in advanced acute myelogenous leukemia
Authors: Meloni G , Foa R , Vignetti M , Guarini A , Fenu S , Tosti S , Tos AG , Mandelli F
Published: October 2019

The administration of interleukin-2 (IL-2) may induce complete remissions in acute myelogenous leukemia (AML) patients with a low proportion of residual bone marrow (BM) blasts. To confirm this preliminary observation, we treated 14 AML patients with advanced disease and with a residual BM blastosis that ranged between 7% and 24% with repeated 5-day cycles of high-dose recombinant IL-2 administered by daily continuous intravenous infusion. Patients who responded have been subsequently submitted to a monthly maintenance scheme with subcutaneous IL-2 at lower doses. While using this schedule and closely monitoring clinical and laboratory conditions, side effects were acceptable and no toxic deaths recorded. Eight of the 14 patients treated with high-dose IL-2 obtained a complete remission (CR). Five remain in persistent CR (four in third CR and one in fourth CR) after a median follow-up time of 32 months (14, 30, 32, 33, and 68 months, respectively). In all five patients, the IL-2-induced remission is the longest in the natural history of the disease. These findings show that IL-2 displays an antileukemic effect in AML with limited residual disease, and suggest that IL-2 should be considered a therapeutic option for resistant or relapsed AML patients.
Cloning and expression of a new mammalian chaperonin gene from a multipotent hematopoietic progenitor clone
Authors: Xie X , Palacios R
Published: October 2019

Molecular chaperones assist in the folding and assembly of proteins in cells. Although chaperonins have been shown in prokaryotes, mitochondria, and chloroplasts long-ago, a cytoplasmic heteromeric chaperonin complex was isolated only recently and found to contain at least five to six polypeptides, one of which was identified as the product of the T complex polypeptide-1 (TCP-1) gene. We have isolated and cloned a novel gene called A45 from a cDNA library constructed from poly (A)+ RNA of a multipotent hematopoietic progenitor clone. The A45 cDNA encodes a predicted polypeptide of M(r) 58,118 that exhibits 32% overall amino acid sequence identity to TCP-1 and contains the putative adenosine triphosphate-binding domain and two characteristic consensus regions that are conserved in all chaperonins. The A45 gene is expressed in hematopoietic precursors cells at a much higher level than in nonhematopoietic cells and tissues. We conclude that A45 represents a new member of the mammalian chaperonins that is involved in the folding and assembly of polypeptides.
Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface
Authors: Diquelou A , Lemozy S , Dupouy D , Boneu B , Sakariassen K , Cadroy Y
Published: October 2019

We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and 2,600 s-1. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring prothrombin fragments 1 + 2 (F 1 + 2), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5- minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (81%, 21%, and 2% at 50, 650, and 2,600 s-1, respectively) and plasma levels of F 1 + 2, T-AT and FPA were shear rate dependent and highest at 50 s-1. There was a positive correlation between F 1 + 2 and T-AT and the fibrin deposition (P < .01). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F 1 + 2 and T-AT were also dependent on the shear rate, but highest at 650 and 2,600 s-1. F 1 + 2 and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F 1 + 2 and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.
Detection of minimal residual disease in acute myelomonocytic leukemia with abnormal marrow eosinophils by nested polymerase chain reaction with allele specific amplification
Authors: Hebert J , Cayuela JM , Daniel MT , Berger R , Sigaux F
Published: October 2019

Acute myelomonocytic leukemia with bone marrow eosinophilia (AML-M4Eo in the French-American-British FAB] classification) is frequently associated with pericentric inversion of chromosome 16, inv(16)(p13q22). Recently, the molecular cloning of teh breakpoints has led to the identification of the two fused genes, CBFB on 16q and MYH11 on 16p. We have analyzed 24 patients with AML-M4Eo at diagnosis and 47 patients with AML of other FAB subtypes, by a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the CBFB/MYH11 fusion mRNAs. Three types of fusion mRNAs were detected in 22 samples of AML- M4Eo (type A, n = 20; type C, n = 1; and type D, n = 1). Among these 22 positive samples, inv(16) was found in the 20 cytogenetically studied cases. No fusion transcript was detected in two patients with AML-M4Eo and in patients with other types of AML. These results confirm that CBFB/MYH11 transcripts (with a predominant type A form) are present in most cases of inv(16) AML. Moreover, detection of the hybrid transcript is closely associated with the finding of abnormal bone marrow (BM) eosinophils in AML-M4Eo as it is not found in other, FAB subtypes of AML, including AML-M4. To assess the presence of type A CBFB/MYH11 fusion transcripts in five AML-M4Eo patients in remission, we designed a sensitive assay combining nested PCR and allele-specific amplification (NPASA). Residual leukemia cells were detected in four patients who were in remission from 4 to 22 months, but not in one patient in long-term remission (5 years). The clinical relevance of persistent CBFB/MYH11 fusion transcripts in remission remains to be established by studying a large prospective series of patients. NPASA provides a useful and sensitive tool for the detection of minimal residual disease in inv(16) AML and, potentially, in other leukemias associated with translocations that result in a predominant fusion transcript.
Anti-M monoclonal antibodies cross-reacting with variant Mg antigen: an example of modulation of antigenic properties of peptide by its glycosylation
Authors: Jaskiewicz E , Czerwinski M , Syper D , Lisowska E
Published: October 2019

Some monoclonal antibodies (MoAbs) directed against blood group M- related epitope of glycophorin A (GPA) were found to agglutinate rare variant erythrocytes carrying GPA of Mg type. In contradistinction to normal GPA-M or -N, the N-terminal portion of GPA-Mg is not glycosylated. Therefore, the multipin peptide synthesis was used for testing the specificity of the cross-reacting MoAbs. Among several anti- M and anti-N MoAbs tested, only three anti-M (E3, E6, 425/2B) agglutinated Mg erythrocytes and showed binding to the synthetic octapeptides corresponding to N-terminal sequences of GPA-M (SSTTGVAM), GPA-N (LSTTEVAM), and GPA-Mg (LSTNEVAM). Testing multiple peptide analogs (window and replacement analysis) showed that these MoAbs were specific for peptidic epitope in which Met8 and Val6 were the most essential amino acid residues. The amino acid replacements Ser<-->Leu1 or Gly<-->Glu5 (M v N) and Thr<-->Asn4 (M and N v Mg) had no or negligible effect on the reaction of synthetic peptides with the MoAbs. However, when Ser2, Thr3, and Thr4 carry O-linked sialooligosaccharides (normal GPA-M or -N), the MoAbs recognize Gly5- and sialic acid- dependent blood group M-related epitope. An interesting finding concerning anti-M/Mg MoAbs described here is the fact that glycosylation of amino acid residues adjacent to the most important part of peptidic epitope not only differentially modulates the proper exposure of peptidic epitope, but also alters the requirement for some amino acid residues present within the epitope. Pathologic conditions, including hematologic disorders, are often accompanied by alterations in protein glycosylation, resulting not only from differences in the structure of antigen polypeptide chain, but also from changes in specificity or expression of enzymes involved in glycosylation. Our present findings draw attention to possibility of the bidirectional modulation of protein antigenicity by glycosylation and may be helpful in interpretation of some results obtained with MoAb used for diagnostic or other purposes.
Inhibition of nitric oxide production is associated with enhanced weight loss, decreased survival, and impaired alloengraftment in mice undergoing graft-versus-host disease after bone marrow transplantation
Authors: Drobyski WR , Keever CA , Hanson GA , McAuliffe T , Griffith OW
Published: October 2019

The pathophysiologic role of nitric oxide (NO) in graft-versus-host disease (GVHD) was investigated in a murine bone marrow (BM) transplantation model where donor and recipient were H-2-matched but differed at multiple minor histocompatibility antigens. Host AKR/J (H- 2K) mice received lethal total body irradiation as pretransplant conditioning followed by transplantation of donor B10.BR (H-2K) BM cells with or without spleen cells as a source of GVH-reactive T cells. NO production, as assessed by serum nitrate and nitrite levels, was increased for up to 3 weeks posttransplant in animals undergoing both moderate and severe GVHD. Administration of NG-methyl-L-arginine (L- NMA), an inhibitor of nitric oxide synthase, to animals undergoing GVHD resulted in effective suppression of NO production when compared with saline-treated GVHD control animals. Suppression of NO production by L- NMA in GVHD animals was associated with enhanced weight loss early posttransplant and decreased overall survival. Histologic analysis of tissues from L-NMA-treated and saline-treated GVHD animals showed that early weight loss was not because of an exacerbation of GVHD, indicating that NO did not appear to play an immunosuppressive role in this experimental model. L-NMA-treated animals with enhanced weight loss were observed to have splenic atrophy, decreased extramedullary hematopoiesis, and a reduction in BM cellularity when compared with GVHD control mice that were weight-matched before transplant. Analysis of T-cell chimerism in the spleen showed that L-NMA treatment impaired donor T-cell repopulation. In vitro colony-forming unit (CFU) assays were performed to further assess the role of NO on BM progenitor cell growth. L-NMA added directly into culture had no effect on CFU- granulocyte/macrophage (CFU-GM) formation in normal murine BM. In contrast, total CFU-GM from L-NMA-treated animals were significantly reduced when compared with GVHD controls or BM control animals who did not develop GVHD. Collectively, these data indicate that inhibition of NO impairs hematopoietic reconstitution and support the premise that NO appears to play a novel role in the facilitation of alloengraftment posttransplant.
Expression of the interleukin-6 (IL-6), IL-6 receptor, and gp130 genes in acute leukemia
Authors: Inoue K , Sugiyama H , Ogawa H , Yamagami T , Azuma T , Oka Y , Miwa H , Kita K , Hiraoka A , Masaoka T
Published: October 2019

Expression patterns of interleukin-6 (IL-6), IL-6 receptor (IL-6R), and gp130 genes in 39 patients with acute myeloid leukemia (AML), in 23 patients with acute lymphoblastic leukemia (ALL), and in 7 patients with acute mixed lineage leukemia (AMLL) were studied by quantitative reverse transcriptase-polymerase chain reaction. Significant levels of IL-6 were expressed in 8 (21%) of 39 AML patients and in 2 (29%) of 7 AMLL patients, whereas in ALL, the expression of IL-6 was almost negligible. IL-6R was expressed in all patients with AML and AMLL, whereas only half of ALL patients expressed low levels of IL-6R as compared with those with AML and AMLL. However, gp130 was ubiquitously expressed in all the leukemia patients, and there was no significant difference in gp130 expression among AML, ALL, and AMLL. Significant correlation was observed between the expression of IL-6R and gp130 in AML. When tested for in vitro response to IL-6, the leukemic cells from 3 of 7 AML, none of 3 ALL, and both of 2 AMLL patients significantly responded to IL-6, showing the correlation between the expression levels of IL-6R and gp130 and the responsiveness of leukemic cells to IL-6. These results showed that quantitation of IL-6R and gp130 expression by reverse transcriptase-polymerase chain reaction is useful for the rapid prediction of the responsiveness of leukemic cells to IL- 6, especially in cases of administration of IL-6.
Synergy between dexamethasone and interleukin-5 for the induction of major histocompatibility complex class II expression by human peripheral blood eosinophils
Authors: Guida L , O'Hehir RE , Hawrylowicz CM
Published: October 2019

The hematopoietic growth factors interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) regulate the survival, maturation, and activation of eosinophils. Corticosteroids in contrast have a negative effect both on the hematopoietic process and the function of eosinophils. We have unexpectedly observed synergy between IL-5 and glucocorticoids such as dexamethasone and hydrocortisone for induction of the MHC class II antigens HLA-DR and HLA-DP on eosinophils isolated from human blood. Similarly glucocorticoids enhanced GM-CSF and IL-3, but not interferon gamma (IFN gamma), induced expression of these antigens. Expression of a third MHC class II molecule, HLA-DQ, was not induced on eosinophils by any of the cytokines alone, but in one of three donors tested, IL-3 plus dexamethasone induced high levels of expression. Although cytokine- induced expression of the accessory molecule intercellular adhesion molecule 1 (ICAM-1) was partially inhibited by glucocorticoids, cytokine- and dexamethasone-treated eosinophils presented antigen more efficiently to a hemagglutinin peptide-specific T-cell clone than eosinophils treated with cytokine alone. These results highlight a potential new role for endogenous or exogenous glucocorticoid hormones in enhancing MHC class II expression by eosinophils.
p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox
Authors: Porter CD , Parkar MH , Verhoeven AJ , Levinsky RJ , Collins MK , Kinnon C
Published: October 2019

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91- phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91- phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.
Targeting of human immunodeficiency virus-infected cells by CD8+ T lymphocytes armed with universal T-cell receptors
Authors: Roberts MR , Qin L , Zhang D , Smith DH , Tran AC , Dull TJ , Groopman JE , Capon DJ , Byrn RA , Finer MH
Published: October 2019

We have developed an immunotherapeutic approach with potential application in the treatment of viral and malignant disease. We show that primary CD8+ T cells isolated from peripheral blood can be genetically modified by retroviral transduction to express high levels of universal (major histocompatibility complex-unrestricted) chimeric T- cell receptors specific for human immunodeficiency virus (HIV) antigens. Two classes of HIV-specific URs in which the antigen-binding domain is comprised of either CD4 or a single-chain antibody are capable of activating a number of T-cell effector functions in response to target cells, including cytolysis, in a highly sensitive and specific manner. Importantly, we have addressed a number of issues which, although particularly relevant to the clinical application of this approach in the treatment of HIV infection, may also impact on the potential of UR immunotherapy for other disease targets. The UR immunotherapeutic system is particularly suited for evaluation in the clinical setting.
Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells
Authors: Nichols GL , Raines MA , Vera JC , Lacomis L , Tempst P , Golde DW
Published: October 2019

Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in clonally derived hematopoietic precursors and their progeny. The Ph chromosome arises from a translocation that deregulates the c-ABL protein tyrosine kinase, giving it transforming potential and increased kinase activity. We observed a unique 39-kD tyrosine phosphoprotein (pp39), previously reported in blastic CML cell lines, in neutrophils from 50 cases of chronic phase CML. This protein was prominently and constitutively tyrosine-phosphorylated in CML neutrophils and was not phosphorylated in normal neutrophils. Stimulation of normal neutrophils with cytokines and agonists did not induce tyrosine phosphorylation of proteins migrating in the region of pp39, and the phosphorylation state of pp39 in CML neutrophils was not affected by kinase inhibitors known to downregulate the ABL kinase. The pp39 was not phosphorylated in hematopoietic cells from healthy donors or from patients with Ph chromosome-negative myeloproliferative disorders. Using micro amino acid sequencing of purified preparations of pp39, we identified pp39 as CRKL protein, which is consistent with recent immunologic studies in the blastic K562 cell line. Immunoblotting with anti-CRKL antibodies showed the presence of CRKL protein in CML cells and cell lines as well as in antiphosphotyrosine immunoprecipitates from CML cells. Our results suggest that pp39 CRKL in CML neutrophils may be stably tyrosine-phosphorylated by the BCR/ABL kinase at an early stage of myeloid differentiation when the ABL kinase is active. CRK, CRKL, and other SH2 (SRC homology domain)/SH3-containing proteins function as adaptor molecules in nonreceptor tyrosine kinase signalling pathways. Although the CRKL protein is present in normal neutrophils, it is not tyrosine-phosphorylated, and the inability to induce such phosphorylation in normal neutrophils suggests a special role of this phosphoprotein in the pathogenesis of CML. Constitutive phosphorylation of CRKL is unique to CML, indicating that it may be a useful target for therapeutic intervention.
Anti-CD40 antibody binding modulates human multiple myeloma clonogenicity in vitro
Authors: Tong AW , Zhang BQ , Mues G , Solano M , Hanson T , Stone MJ
Published: October 2019

Ligand binding of the B-cell lineage antigen CD40 enhances growth and interleukin-6 (IL-6) secretion in human B cells (the CD40/IL-6 loop). IL-6 has an autocrine and paracrine role in human multiple myeloma (MM) cell growth. With the use of the CD40 monoclonal antibody (MoAb) G28–5, we examined CD40 expression and the effect of CD40 binding on MM clonogenic colony (MCC) formation to characterize the IL-6/CD40 loop activity in MM. CD40 was expressed on plasmacytoid cells in 21 of 28 plasma cell dyscrasia (PCD) bone marrow (BM) biopsies tested (10 of 14 MM, 2 of 2 Waldenstrom's macroglobulinemia [WM], 2 of 2 plasma cell leukemia [PCL], 6 of 8 monoclonal gammopathy of undetermined significance [MGUS], and 1 of 2 primary amyloidosis [AL]). G28–5 binding increased MCCs by 35% to 150% in 11 of 17 CD40+ PCD BM cultures, but did not affect MCC formation in CD40- specimens or normal BM colony forming units (CFU-GEMM, CFU-GM, BFU-E). Responsive cultures originated from BM of patients with MM (2 of 5 cases tested), WM (2 of 2), PCL (2 of 2), and MGUS (5 of 6). CD40-responsiveness was not significantly inhibited by the presence of an anti-IL-6 MoAb (2 of 2 MGUS cultures tested), and did not correlate with the capacity to respond to IL-6 stimulation (n = 17, P > .05) or a detectable level of endogenous IL-6 (n = 15, P > .05). Additional studies were performed with PCD cell lines to characterize the interrelationship of CD40 activation and IL-6 production. Fifty percent to greater than 95% of cells from the RPMI 8226 and ARH77 lines expressed CD40, whereas 6% of U266 cells were CD40+. For RPMI 8226, ARH-77, and U266 cells, the increased MCC formation after anti-CD40 stimulation was not affected by the presence of an anti-IL-6 neutralizing MoAb and was not accompanied by detectable IL-6 secretion. There was no apparent increase in IL-6 mRNA transcription following G28–5 treatment of U266 or RPMI 8226 cells. Our observations indicate that CD40 is expressed in a subset of human myeloma cells present in various PCDs. Cell-line studies suggest that the CD40+ myeloma cell may regulate MM clonogenic colony formation without activating the IL-6 pathway.
Molecular mapping of the Cromer blood group Cra and Tca epitopes of decay accelerating factor: toward the use of recombinant antigens in immunohematology
Authors: Telen MJ , Rao N , Udani M , Thompson ES , Kaufman RM , Lublin DM
Published: October 2019

Cromer blood group antigens reside on the complement regulatory protein decay accelerating factor (DAF, CD55). This glycosyl- phosphatidylinositol-anchored glycoprotein is widely distributed, especially among cell types in contact with plasma. Numerous Cromer blood group antigens have been defined using alloantibodies induced by transfusion or pregnancy. However, few pairs of antithetical antigens have been described in this system, presumably because of the rarity of the low-frequency alleles. Analysis of polymerase chain reaction- amplified genomic DNA showed that the Cr(a-) phenotype has a Ala193-- >Pro substitution in short consensus repeat 4 (SCR4) of DAF, and the Tc(a-b+) phenotype has a Arg18-->Leu substitution in SCR1 of DAF. The locations of Cra and Tca epitopes were confirmed by analysis of Chinese hamster ovary cell transfectants expressing a Cr(a-) allele-specific transfectant and a chimeric protein containing only SCR1 of DAF, respectively. Overall, these studies further show the usefulness of an approach based on recombinant proteins in mapping blood group antigen epitopes and identifying blood group antibodies.
The proto-oncogene c-fgr is expressed in normal mantle zone B lymphocytes and is developmentally regulated during myelomonocytic differentiation in vivo
Authors: Link DC , Zutter M
Published: October 2019

The proto-oncogene c-fgr is a member of the c-src gene family of cytoplasmic tyrosine kinases. Previous studies have suggested that it is normally expressed in neutrophils, monocytes, macrophages, and natural killer cells. c-fgr is also expressed in the B cells of certain lymphoproliferative disorders, namely, Epstein-Barr virus-associated lymphoproliferative disease, and in chronic lymphocytic leukemia, but it has not previously been detected in normal or reactive human lymphoid tissue. In this study we have determined the pattern of p55c- fgr protein expression in normal human hematopoietic and lymphoid tissues at the single-cell level using immunohistochemical and immunofluorescent techniques. We show that p55c-fgr expression is developmentally regulated with high-level expression first evident at the myelocyte stage of myeloid differentiation. In addition, we show that p55c-fgr is expressed in circulating B lymphocytes isolated from chronic lymphocytic leukemia patients but is not expressed in normal circulating B lymphocytes. Surprisingly, p55c-fgr is also expressed in a subpopulation of normal B lymphocytes, the mantle zone B lymphocytes. This demonstration that p55c-fgr is expressed in a normal B-lymphocyte subpopulation suggests that its expression in certain B-cell lymphoproliferative disorders may be an indirect consequence of, rather than a primary cause of, the neoplastic transformation process.
Elevated dihydrofolate reductase and impaired methotrexate transport as elements in methotrexate resistance in childhood acute lymphoblastic leukemia
Authors: Matherly LH , Taub JW , Ravindranath Y , Proefke SA , Wong SC , Gimotty P , Buck S , Wright JE , Rosowsky A
Published: October 2019

A retrospective study of clinical resistance to methotrexate (MTX) was performed on 29 archival specimens of frozen lymphoblasts obtained from children with acute lymphoblastic leukemia (ALL), including 19 at initial presentation and 10 at first relapse. Blasts were assayed for dihydrofolate reductase and MTX transport by flow cytometry using the fluorescent methotrexate analog, PT430 (Rosowsky et al, J Biol Chem 257:14162, 1982). In contrast to tissue culture cells, patient blasts were often heterogeneous for dihydrofolate reductase content. Of the 19 specimens at initial diagnosis, 7 exhibited dual blast populations, characterized by threefold to 10-fold differences in relative dihydrofolate reductase; the dihydrofolate reductase-overproducing populations comprised 12% to 68% of the total blasts for these specimens. Remission duration intervals for patients exhibiting dual blast populations were notably shorter than for patients expressing a single blast population with lower dihydrofolate reductase ( < or = 9 months v > or = 15 months, respectively), a difference that was statistically significant (P = .045). There was no apparent correlation between expression of increased dihydrofolate reductase at diagnosis and known patient and disease prognostic features (immunophenotype, age, sex, and white blood count). For the relapsed patients, 4 of 10 exhibited dual lymphoblast populations with elevated dihydrofolate reductase. The majority of the patient lymphoblast specimens were entirely competent for MTX transport and, likewise, expressed immunoreactive reduced folate carriers by indirect immunofluorescence staining with specific antiserum to the transporter. Three patients (2 at relapse and 1 at diagnosis) exhibited heterogeneous expression of imparied MTX transport (14% to 73% of blasts). In only 1 of these patients did the majority of the lymphoblasts (73%) show impaired MTX transport and for this specimen, immunoreactive carrier proteins were virtually undetectable. These results suggest that heterogeneous expression of elevated dihydrofolate reductase and impaired MTX transport are important modes of resistance in childhood ALL patients undergoing chemotherapy with MTX and that these parameters may serve as predictive indices of clinical response to MTX.
Autologous bone marrow transplantation for first remission acute myeloblastic leukemia in patients older than 50 years: a retrospective analysis of the European Bone Marrow Transplant Group
Authors: Cahn JY , Labopin M , Mandelli F , Goldstone AH , Eberhardt K , Reiffers J , Ferrant A , Franklin I , Herve P , Gratwohl A
Published: October 2019

High-dose chemotherapy, with or without radiotherapy, followed by autologous stem-cell rescue is used increasingly for the intensification of first remission in acute myeloblastic leukemia (AML). However, these treatments have been limited to young patients due to the increased risks of regimen-related toxicities and mortality with age. Several investigators have recently published the upper age limit for autologous bone marrow transplant (ABMT) in AML because of encouraging results. The results of ABMT for AML were studied in 111 patients > or = 50 years of age intensified in first remission. Median age at transplant was 53 years (range, 50 to 63 years). Fifty patients were conditioned with total body irradiation and 61 with polychemotherapy: 23 with busulfancyclophosphamide, 11 with the University College Hospital (UCH; London, UK) regimen, 6 with BAVC, and 21 with various other treatments. Marrow was purged in only 11 cases. Results were compared with 786 ABMTs performed for AML in patients between 16 and 49 years of age (median, 35 years). For AML in first remission, the probability of leukemia-free survival (LFS) at 4 years was 34% +/- 5% for patients aged 50 years or more and 43% +/- 2% for patients less than 50 years of age (P = .004), with a survival probability of 35% +/- 6% and 48% +/- 2%, respectively (P = .004). The probability of relapse was not significantly different between the two groups (52% +/- 7% v 50% +/- 2%), but transplant-related mortality was significantly higher in the older age group (28% +/- 5% v 14% +/- 2%; P < .0001) and mainly due to infectious complications. In a multivariate analysis, age less than 50 years was a favorable risk factor for LFS, treatment-related mortality (TRM), and survival but not for relapse incidence. These data suggest that ABMT should be considered in older AML patients.
True T-cell chronic lymphocytic leukemia: a morphologic and immunophenotypic study of 25 cases [see comments]
Authors: Hoyer JD , Ross CW , Li CY , Witzig TE , Gascoyne RD , Dewald GW , Hanson CA
Published: October 2019

We studied 25 T-cell chronic lymphocytic leukemia (T-CLL) cases collected over a 15-year period. Immunophenotypic analysis was performed in each case; 12 cases were evaluated by cytogenetics, and gene rearrangement studies were performed in 14 cases. The median age was 57 years with a male predominance (M:F, 15:10). The median presenting lymphocyte count was 36.3 x 10(9)/L (range, 3.9 to 438 x 10(9)/L). Fourteen patients (56%) had shotty adenopathy and ten (40%) had mild-to-moderate splenomegaly at presentation; four (16%) had erythematous skin lesions. The lymphocytes were predominantly small; some cases had a minor component of medium-sized cells (< 10%). The nuclear: cytoplasmic ratios were uniformly high with round to oval nuclei; however, a wide spectrum of nuclear outlines could be found, ranging from minimally to markedly convoluted. Nucleoli were either absent or small and inconspicuous. These lymphocytes did not have the morphology of prolymphocytes and did not contain cytoplasmic granules. Bone marrow infiltration was generally in an interstitial pattern; the degree of involvement ranged from 15% to 90%. Immunophenotyping showed that the lymphocytes were mature T-cells with a predominant CD4+ immunophenotype. Three cases displayed a CD8+ immunophenotype. The patients were treated with a variety of chemotherapeutic regimens with only a minimal response observed in two of 20 patients. We conclude that T-CLL is an uncommon chronic lymphoproliferative disorder (CLPD) that can be morphologically similar to B-CLL, is distinct from T- prolymphocytic leukemia, and has an aggressive clinical course that is refractory to therapy. It may also be difficult to distinguish T-CLL from other T-CLPD, especially the leukemic phase of peripheral T-cell lymphoma and some cases of Sezary syndrome.
Angiogenic factors stimulate mast-cell migration
Authors: Gruber BL , Marchese MJ , Kew R
Published: October 2019

Mast cells accumulate at sites of angiogenesis. The factor(s) that control mast-cell recruitment at these sites have yet to be defined. We sought to determine if angiogenic factors result in mast-cell chemotaxis. In this study, we observed that platelet-derived growth factor-AB (PDGF-AB), vascular endothelial cell growth factor (VEGF), and basic fibroblast growth factor (bFGF) each cause directed migration of murine mast cells at picomolar concentrations, with a typical bell-shaped dose-response curve. Another potent angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), appears to promote chemokinesis of mast cells, whereas tumor necrosis factor-alpha, a weak angiogenic factor, is less robust but still functions as a mast cell chemotactic factor. Epidermal growth factor (EGF), a growth factor with minimal angiogenic properties, was ineffective as a mast cell chemotactic factor. A checkerboard analysis confirmed the directional chemotactic response of PDGF-AB, VEGF, and bFGF, while indicating the chemokinetic response induced by PD-ECGF. Cross-desensitization of growth-factor-induced directed migration was observed between PDGF-AB and bFGF, and also between PDGF-AB and PD-ECGF. Tyrosine kinase-inhibitor genistein effectively dampened the chemotactic responses, whereas pertussis toxin had no effect. In summary, our findings suggest that factors known to act on endothelial cells and stimulate neovascularization may simultaneously serve to recruit mast cells to these sites. The local accumulation of mast cells is believed to facilitate new vessel formation through complex cell:cell interactions.
Identification and conditions for selective expression of megakaryocytic markers in Friend erythroleukemia cells
Authors: Paoletti F , Vannucchi AM , Mocali A , Caporale R , Burstein SA
Published: October 2019

Friend murine erythroleukemia cells (MELCs) have been reevaluated in terms of their nature and potential pathways of differentiation. MELC induced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also found to exhibit traits of the megakaryocytic lineage. Erythroid differentiation was shown by the typical synthesis and accumulation of hemoglobin (Hb); megakaryoblastoid differentiation of MELCs upon induction was shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% fetal calf serum (FCS) (instead of the usual 5%), induced cells to selectively express high levels of AChE (up to approximately 170 mU/mg protein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the whole cell population and approached its maximum within 3 days of incubation with the inducer. Subsequently, MELCs become committed to terminal division, undergoing growth arrest and expression of the megakaryocytic phenotype even after the removal of HMBA. There were no appreciable changes of basal AChE levels in MELCs that were either made resistant to HMBA or treated with 0.1 mmol/L hemin that activated differentiated erythroid function without commitment. Phorbol 12-myristate 13-acetate (PMA), known to repress induced Hb synthesis in these cells, did not prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increase that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permitted only when MELCs were transferred in the late phase or at the end of commitment from low to high serum media. Variations of FCS content in culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either megakaryocytic or mixed erythromegakaryocytic phenotype. These findings suggested that MELC might be considered, at least, as a bipotential model of differentiation to be used for studies on regulation of either megakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE levels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-antimouse platelet monoclonal antibody 4A5.(ABSTRACT TRUNCATED AT 400 WORDS)
A murine cytokine fusion toxin specifically targeting the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on normal committed bone marrow progenitor cells and GM-CSF-dependent tumor cells
Authors: Chan CH , Blazar BR , Eide CR , Kreitman RJ , Vallera DA
Published: October 2019

A fusion protein was synthesized consisting of the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mGM-CSF hybrid gene was cloned into a vector under the control of an inducible promoter and the protein expressed in Escherichia coli. After induction, a protein was purified from inclusion bodies in accord with the deduced molecular weight of DT390 mGM-CSF. Cell-free studies of the adenosine diphosphate-ribosylating activity of DT390 mGM-CSF showed results that were similar to those of native DT. The DT390 mGM-CSF immunotoxin inhibited FDCP2.1d, a murine myelomonocytic tumor line expressing the GM-CSF receptor with an IC50 (concentration inhibiting 50% activity) of 5 x 10(-11) mol/L. The fusion toxin was specifically cytotoxic and directed by the GM-CSF portion of the molecule because addition of a monoclonal antibody directed against GM-CSF inhibited its ability to kill the cell line. Cell lines that do not express GM-CSF receptor were not inhibited by the fusion toxin. DT390 mGM-CSF was also able to specifically inhibit normal committed bone marrow (BM) progenitor cells as measured in clonal colony-forming unit granulocyte-macrophage assays. Together, these findings indicate that DT390 mGM-CSF may be useful as a novel tool for purging BM of contaminating leukemia cells or in vivo for eliminating residual leukemia cells. Also, it can be used to determine whether committed and/or noncommitted BM progenitor cells express the GM-CSF receptor.
Bone marrow transplantation for Fanconi anemia
Authors: Gluckman E , Auerbach AD , Horowitz MM , Sobocinski KA , Ash RC , Bortin MM , Butturini A , Camitta BM , Champlin RE , Friedrich W
Published: October 2019

Fanconi anemia is a genetic disorder associated with diverse congenital abnormalities, progressive bone marrow failure, and increased risk of leukemia and other cancers. Affected persons often die before 30 years of age. Bone marrow transplantation is an effective treatment, but there are few data regarding factors associated with transplant outcome. We analyzed outcomes of HLA-identical sibling (N = 151) or alternative related or unrelated donor (N = 48) bone marrow transplants for Fanconi anemia performed between 1978 and 1994 and reported to the International Bone Marrow Transplant Registry. Fanconi anemia was documented by cytogenetic studies in all cases. Patient, disease, and treatment factors associated with survival were determined using Cox proportional hazards regression. Two-year probabilities (95% confidence interval) of survival were 66% (58% to 73%) after HLA-identical siblings transplants and 29% (18% to 43%) after alternative donor transplants. Younger patient age (P .0001), higher pretransplant platelet counts (P = .04), use of antithymocyte globulin (P = .005), and use of low-dose (15 to 25 mg/kg) cyclophosphamide plus limited field irradiation (P = .009) for pretransplant conditioning and cyclosporine for graft-versus-host disease prophylaxis (P = .002) were associated with increased survival. Bone marrow transplants are effective therapy for Fanconi anemia. The adverse impact of increasing age and lower pretransplant platelet count on transplant outcome favors earlier intervention, especially when there is an HLA-identical sibling donor.
Constitutive overexpression of the L-selectin gene in fresh leukemic cells of adult T-cell leukemia that can be transactivated by human T- cell lymphotropic virus type 1 Tax
Authors: Tatewaki M , Yamaguchi K , Matsuoka M , Ishii T , Miyasaka M , Mori S , Takatsuki K , Watanabe T
Published: October 2019

L-selectin is an adhesion molecule of the selectin family that mediates the initial step of leukocyte adhesion to vascular endothelium. Upon cellular activation, expression of the L-selectin gene is downregulated at both the protein and mRNA levels. To understand the mechanism of leukemic cell infiltration into organs, we studied the expression and regulation of L-selectin mRNA in fresh leukemic cells of adult T-cell leukemia (ATL) patients and investigated the response of the L-selectin promoter to human T-cell lymphotropic virus type 1 (HTLV-1) Tax, which is a viral transcriptional transactivator. Flow cytometry showed that L-selectin was expressed on fresh ATL cells along with other activation antigens. Northern blot analysis showed that ATL cells overexpressed that L-selectin mRNA and that the level was aberrantly upregulated after PMA stimulation. Studies using in situ hybridization showed expression of the L-selectin mRNA in the infiltrating leukemic cells in the liver of two ATL patients. Intravenous injection of a rat T-cell line that overexpresses L-selectin showed increased organ infiltration. The induction of Tax expression in JPX9 cells resulted in about a twofold increase in the mRNA expression levels compared with the basal level. Chloramphenicol acetyltransferase (CAT) assay after transient cotransfection showed about a fivefold transactivation of the L-selectin promoter by Tax. The serum level of the shed form of L-selectin was significantly increased in ATL patients (mean +/- SD, 4,215.4 +/- 4,111 ng/mL) compared with those of asymptomatic carriers and healthy blood donors (mean +/- SD, 1,148.0 +/- 269.0 ng/mL and 991.9 +/- 224 ng/mL, respectively). These results indicated that ATL cells constitutively overexpress the L-selectin gene that can be transactivated by HTLV-1 Tax. The overexpression of L-selectin, as well as of inflammatory cytokines, by ATL cells may provide a basis for ATL cells to attach the vascular endothelium, leading to transmigration and organ infitration.
The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells
Authors: Richardson DR , Neumannova V , Nagy E , Ponka P
Published: October 2019

The iron-responsive element-binding protein (IRE-BP) modulates both ferritin mRNA translation and transferrin receptor (TfR) mRNA stability by binding to specific mRNA sequences called iron-responsive elements (IREs). The regulation of IRE-BP in situ could possibly occur either through its Fe-S cluster and/or via free cysteine sulphydryl groups such as cysteine 437 (Philpott et al, J Biol Chem 268:17655, 1993; and Hirling et al, EMBO J 13:453, 1994). Recently, nitrogen monoxide (NO) has been shown to have markedly different biologic effects depending on its redox state (Lipton et al, Nature 364:626, 1993). Considering this fact, it is conceivable that the NO group, as either the nitrosonium ion (NO+) or nitric oxide (NO+), may regulate IRE-BP activity by S-nitrosylation of key sulphydryl groups or via ligation of NO. to the Fe-S cluster, respectively. This hypothesis has been examined using the NO+ generator, sodium nitroprusside (SNP); the NO. generator, S-nitroso-N-acetylpenicillamine (SNAP); and the NO./peroxynitrite (ONOO-) generator, 3-morpholinosydnonimine hydrochloride (SIN-1). Treatment of K562 cells for 18 hours with SNP (1 mmol/L) resulted in a pronounced decrease in both the RNA-binding activity of IRE-BP and the level of TfR mRNA. In addition, Scatchard analysis showed a marked decrease in the number of specific Tf-binding sites, from 590,000/cell (control) to 170,000/cell (test), and there was also a distinct decrease in Fe uptake. Furthermore, SNP did not decrease cellular viability or proliferation. In contrast, the NO. generator, SNAP (1 mmol/L), increased RNA-binding activity of IRE-BP, the level of TfR mRNA, and the number of TfRs in K562 cells. Moreover, both SNAP (1 mmol/L) and SIN-1 (0.5 mmol/L) reduced cellular proliferation. The results are discussed in context of the possible physiologic role of redox-related species of NO in regulating iron metabolism.