bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic growth inhibitory and differentiating effects of trimidox and tiazofurin in human promyelocytic leukemia HL-60 cells
Authors: Szekeres T , Fritzer M , Strobl H , Gharehbaghi K , Findenig G , Elford HL , Lhotka C , Schoen HJ , Jayaram HN
Published: October 2019

Increased ribonucleotide reductase (RR) activity has been linked with malignant transformation and tumor cell growth. Therefore, this enzyme is considered to be an excellent target for cancer chemotherapy. We have examined the effects of a newly patented RR inhibitor, trimidox (3,4,5-trihydroxybenzohydroxamidoxime). Trimidox inhibited the growth of human promyelocytic leukemia HL-60 cells with an IC50 of 35 mumol/L. Incubation of HL-60 cells with 50 mumol/L trimidox for 24 hours decreased deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP) pools to 24% and 39% of control values, respectively. Incubation of HL-60 cells with 20 to 80 mumol/L trimidox even up to a period of 4 days did not alter the distribution of cells in different phases of cell cycle. Sequential incubation of HL-60 cells with trimidox (25 mumol/L) for 24 hours and then with 10 mumol/L tiazofurin (an inhibitor of inosine monophosphate dehydrogenase) for 4 days produced synergistic growth inhibitory activity, and the cell number decreased to 16% of untreated controls. When differentiation- linked cell surface marker expressions were determined in cells treated with trimidox and tiazofurin, a significantly increased fluorescence intensity was observed for the CD 11b (2.9-fold). CD 33 (1.9-fold), and HLA-D cell surface antigens. Expression of the transferrin receptor (CD71) increased 7.3-fold in cells treated with both agents, compared with untreated controls. Our results suggest that trimidox in combination with tiazofurin might be useful in the treatment of leukemia.
Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity
Authors: Rao LV , Nordfang O , Hoang AD , Pendurthi UR
Published: October 2019

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.
Clonality in juvenile chronic myelogenous leukemia
Authors: Busque L , Gilliland DG , Prchal JT , Sieff CA , Weinstein HJ , Sokol JM , Belickova M , Wayne AS , Zuckerman KS , Sokol L
Published: October 2019

Juvenile chronic myelogenous leukemia (JCML) is a myeloproliferative disease in which morbidity and mortality are primarily caused by nonhematopoietic organ failure from myelomonocytic infiltration or by failure of the normal bone marrow. Morphologic evidence of maturation arrest, karyotypic abnormalities, and progression to blast crisis are infrequent events. Viral infections and other reactive processes can initially mimic the clinical course of JCML, creating diagnostic problems. Because of the rarity of JCML and technical limitations, formal clonality studies have not been reported previously. Nine female JCML patients were identified by clinical criteria, characteristic ‘spontaneous’ in vitro cell growth, and negative cultures and titers for various viral agents. Peripheral blood and bone marrow samples were obtained at the time of diagnosis for cell separation and RNA and DNA isolation. To assess clonality, X-chromosome inactivation patterns were evaluated using three different, recently developed polymerase chain reaction-based clonality assays. All nine female JCML patients showed evidence for monoclonal origin of mononuclear cells at the time of diagnosis. Cell separation studies further traced the monoclonal origin back to at least the most primitive myeloid progenitor cell. Reversion to a polyclonal state was demonstrated after bone marrow transplant and also in one patient following treatment with 13-cis retinoic acid. This demonstration of clonality in JCML delineates it from the reactive processes and provides a basis for molecular genetic strategies to identify causally associated mutations.
Induction of differentiation of WEHI-3B D+ leukemic cells transfected with differentiation-stimulating factor/leukemia inhibitory factor receptor cDNA
Authors: Tomida M
Published: October 2019

Differentiation-stimulating factor (D-factor)/leukemia inhibitory factor can induce the differentiation of mouse myeloid leukemia M1 cells and also stimulate proliferation of the interleukin-3 (IL-3)- dependent cell line, DA-1a. To determine whether D-factor can induce the differentiation of leukemia cells other than M1 cells, WEHI-3B D+ mouse myelomonocytic leukemia cells were transfected with a plasmid containing mouse D-factor receptor cDNA. Expression of D-factor receptor in transfected cells was determined by binding of [125]D- factor and analyzed by Scatchard's method. The transfected cells had high-affinity D-factor receptors with a dissociation constant of 100 to 200 pmol/L and binding sites per cell varied from 67 to 1,500 among several clones. The cells expressing a high level of D-factor receptor were induced to differentiate by D-factor; about 60% of the cells exhibited the ability to reduce nitroblue tetrazolium and expression of the differentiation antigen Mac-1 (CD11b) on the cell surface increased. The effect of cytokines, which induce the differentiation of M1 cells, on the transfected WEHI-3B cells was examined. The sensitivity to oncostatin M was identical to that against D-factor in the cells of each clone. Expression of D-factor receptor in WEHI-3B cells promoted sensitivity to IL-6 and granulocyte colony-stimulating factor (G-CSF). Induction of differentiation of the cells accompanied the suppression of proliferation. Treatment of the cells with D-factor for longer than 5 days resulted in 50% inhibition of growth. These results indicate that the stimulating effect of D-factor on the differentiation of malignant myeloid cells is not unique to M1 cells.
Hematopoietic cell phosphatase associates with erythropoietin (Epo) receptor after Epo-induced receptor tyrosine phosphorylation: identification of potential binding sites
Authors: Yi T , Zhang J , Miura O , Ihle JN
Published: October 2019

Erythropoietin (Epo) binding to its receptor (EpoR) induces tyrosine phosphorylation in responsive cells and this ability is required for a mitogenic response. One of the substrates of tyrosine phosphorylation is the Epo receptor (EpoR). The carboxyl region of EpoR cytoplasmic domain is required for EpoR phosphorylation and has been shown to negatively affect the response to Epo both in vivo and in cell lines. Hematopoietic cell phosphatase (HCP) has also been hypothesized to negatively regulate erythropoiesis, based on the hypersensitivity to Epo of erythroid lineage cells in moth-eaten mice that genetically lack HCP. In the studies presented here, we show that HCP binds the tyrosine phosphorylated Epo receptor through the amino-terminal src-homology 2 (SH2) domain of HCP. Using a series of phosphotyrosine-containing peptides, potential HCP binding sites in the cytoplasmic domain of the EpoR are identified. The results support the concept that, after Epo stimulation, phosphorylation of EpoR provides a docking site for HCP in the receptor complex. Recruitment of HCP to the complex and its subsequent dephosphorylation of substrates and/or associated kinases may be important to mitigate the ligand-induced mitogenic response.
The cloning and characterization of the human transcobalamin II gene
Authors: Regec A , Quadros EV , Platica O , Rothenberg SP
Published: October 2019

Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by receptor-mediated endocytosis. In genetic disorders that are characterized by congenital deficiency of TCII, intracellular Cbl deficiency occurs, resulting in an early onset of megaloblastic anemia that is sometimes accompanied by a neurologic disorder. To define the genetic basis for TCII deficiency, we have cloned and characterized the human gene that encodes this protein. The gene spans a minimum of 18 kbp and contains nine exons and eight introns, with a polyadenylation signal sequence located 509 bp downstream from the termination codon and a transcription initiation site beginning 158 bp upstream from the ATG translation start site. The 5′ flanking DNA does not have a TATA or CCAAT regulatory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5′-CCCC-3′ tetramers. This sequence is a motif for a trans-active transcription factor (ETF) that regulates expression of the epidermal growth factor receptor gene (EGFR), which also lacks TATA and CCAAT regulatory elements. A GC-rich sequence that binds the SP1 protein is located 356 nucleotides upstream from the first of the series of CCCC tetramers. Although this GC sequence is at an unusual location with respect to the CAP site, a 507-bp fragment containing this GC box drives the chloramphenicol acetyltransferase (CAT) reporter gene after transient transfection into NIH 3T3 cells. No CAT activity was observed when a 420-bp fragment lacking this GC box but containing the ETF-binding domains was similarly transfected into this cell line. One consensus and two atypical motifs for the c-myc ligand are located downstream and upstream, respectively, of the GC box, and this could explain the elevated plasma TCII observed in some patients with multiple myeloma, as the c-myc product is overexpressed in some myeloma cells. Restriction endonuclease digestion of genomic DNA from eight normal subjects with Taq I, Hinfl, Msp I, and Bgl I identified three patterns of restriction fragment length polymorphism (RFLP). A number of the exon/intron splice junctions of human TCII, TCI, and IF genes are located in homologous regions of these proteins, providing evidence that these genes have evolved by duplication of an ancestral gene. This characterization of the TCII gene and the RFLP should facilitate the identification of the mutation(s) responsible for the genetic abnormalities of TCII expression.
Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow
Authors: Galy AH , Cen D , Travis M , Chen S , Chen BP
Published: October 2019

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.
Functional expression of adhesion receptors and costimulatory molecules by fresh and immortalized B-cell non-Hodgkin's lymphoma cells
Authors: Vyth-Dreese FA , Dellemijn TA , van Oostveen JW , Feltkamp CA , Hekman A
Published: October 2019

Peripheral blood lymphocytes of a patient with follicular B-cell non- Hodgkin's lymphoma (B-NHL) were immortalized in vitro by Epstein-Barr virus (EBV). Eight cell lines were obtained (termed BNS1, BNS2–1 through BNS2–7), which showed a pattern of idiotypic (id) Ig surface expression and Ig JH and kappa gene rearrangement, identical to that of the parent cells (termed NS), confirming their neoplastic origin. Induction of allogeneic T-cell proliferation by NS cells was mediated by HLA-DR, leukocyte function-associated antigen-1 (LFA-1), LFA-3, B7–1/CD80, and CTLA4 and resulted in the upregulation (LFA-3, intercellular adhesion molecule-1 [ICAM-1], CD40) and induction (B7–1/CD80, B7–2/CD86, L16/activated LFA-1) of accessory molecules on NS cells. In turn, responder T lymphocytes were induced to express B7–1/CD80, B7–2/CD86, CD40 ligand (CD40L), ICAM-1, L16/activated LFA-1, and HLA-DR, reflecting bidirectional signaling between T lymphocytes and B-NHL cells. Preactivation of NS cells by EBV transformation or CD40 engagement resulted in enhanced expression of accessory molecules and abolished the requirement for accessory cells during allostimulation. These resting and activated clonal B cells will be useful in further dissecting the requirements for B-NHL costimulation.
Hairy cell identification by immunohistochemistry of tartrate-resistant acid phosphatase
Authors: Janckila AJ , Cardwell EM , Yam LT , Li CY
Published: October 2019

Tartrate-resistant acid phosphatase (TRAcP) has been an indispensible marker for hairy cell leukemia (HCL) for over two decades. However, the traditional TRAcP cytochemical stain cannot be performed effectively on sections of paraffin-embedded tissues that are important resources for histopathologic evaluation in diagnosis and treatment of HCL. Wide variation in expression of TRAcP activity by hairy cells (HCs) within and among patients is an interesting biologic phenomenon that has not been explained and can cause some diagnostic uncertainty as well. To solve the problem of staining TRAcP in paraffin sections and to begin to address the questions of variable TRAcP expression in HCL, we developed a monoclonal antibody to TRAcP, 9C5, for immunohistochemical identification of HCs. In smears of blood and bone marrow, immunocytochemistry of TRAcP using 9C5 was as specific but slightly less sensitive than direct cytochemical staining of enzymatic activity. In paraffin sections of spleen and bone marrow from HCL patients, immunohistochemistry with 9C5 stained the HCs with high sensitivity and specificity and clearly showed the characteristic diffuse infiltration by HCs. Other cells noted to stain strongly with 9C5 were occasional macrophages in bone marrow smears and osteoclasts and occasional tissue macrophages in paraffin sections. These are cells known to express abundant TRAcP activity. Immunohistochemistry with anti-TRAcP monoclonal antibody 9C5 may have utility as an added option in the diagnosis of HCL, as a means to evaluate residual disease in HCL patients undergoing new treatments, and as a way to address questions regarding variable expression of TRAcP activity by HCs within and among patients with HCL. Also, 9C5 has potential as a reagent for the immunoassay of bone-derived serum TRAcP in patients with certain bone diseases and cancers with bone metastasis.
Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA
Authors: Simsek S , Faas BH , Bleeker PM , Overbeeke MA , Cuijpers HT , van der Schoot CE , von dem Borne AE
Published: October 2019

Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3′ noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.
Lymphohematopoietic progenitors of normal mice
Authors: Ball TC , Hirayama F , Ogawa M
Published: October 2019

We have identified and characterized the lymphohematopoietic progenitors in the bone marrow of normal mice using a single-step methylcellulose culture assay. Lineage-negative Ly-6A/E (Sca-1)+ progenitors isolated from normal mice were plated in methylcellulose culture containing steel factor (SF), interleukin-7 (IL-7), erythropoietin (Ep), and IL-11. After 16 to 17 days of culture, pre-B-cell-containing multilineage myeloid colonies can be microscopically identified; however, flow-cytometric analysis of individual colonies for B220-positive cells proved superior to in situ microscopic identification of lymphomyeloid colonies. Approximately 10% (6/66) of the mixed colonies without a conspicuous B-cell component had B220- positive cells. The single cell origin of the lymphomyeloid colonies was confirmed by micromanipulation. Although the combination of SF, IL-7, and Ep was sufficient to support formation of lymphomyeloid colonies, addition of IL-11, granulocyte colony-stimulating factor or IL-12 to the combination of SF, IL-7, and Ep increased the number of lymphomyeloid colonies. IL-1 alpha and IL-3 independently inhibited the expression of the B-lymphoid lineage when added to the combination of SF, IL-7, Ep, and IL-11. Approximately four times more lymphohematopoietic progenitors are present in normal mice than in mice treated with 5-fluorouracil.
Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro
Authors: Tsuchiyama J , Mori M , Okada S
Published: October 2019

The hematopoietic microenvironment (HIM) of mouse spleen predominantly induces the differentiation of hematopoietic progenitors into erythroid lineage in vivo. However, the mechanisms of this phenomenon have not been fully explored because of the lack of an adequate in vitro system mimicking the spleen hematopoiesis. To reconstruct the HIM of mouse spleen in vitro, we established spleen stromal cell lines from a three-dimensional (3D) spleen primary culture in collagen gel matrix. Of these, SPY3–2 cells were negative for preadipocytic and endothelial markers, had a fibroblastoid morphology, and were not converted to adipocytes in the presence of 1 mumol/L hydrocortisone. They supported the maintenance and multilineal differentiation of hematopoietic progenitor cells for more than 8 weeks in vitro. The differentiated hematopoietic cells in the coculture medium were predominantly monocytes rather than granulocytes. Furthermore, erythropoiesis was predominantly induced in the presence of 2 U/mL erythropoietin and continued for more than 12 weeks. The number of burst-forming units-erythroid (BFU-E) was increased 10 times after 3 weeks of coculture, which was followed by pronounced production of erythroid cells in the coculture after week 4. SPY3–2 expressed high levels of c-kit ligand and low levels of granulocyte macrophage colony-stimulating factor and interleukin-3, and these molecules were all involved in this long-term erythropoiesis. Thus, the clonal SPY3–2 cell line will provide a novel HIM in vitro analogous to that of mouse spleen in vivo. These results suggest that 3D collagen gel culture may facilitate the establishment of functioning stromal cell lines of hematopoietic organ.
Generation of hematopoietic colony-forming cells from embryonic stem cells: synergy between a soluble factor from NIH-3T3 cells and hematopoietic growth factors
Authors: Bigas A , Martin DI , Bernstein ID
Published: October 2019

Murine embryonic stem cells are able to differentiate into embryoid bodies (EBs) in vitro in the absence of leukemia-inhibitory factor with the formation of different types of hematopoietic precursors within these EBs. With the aim of determining the in vitro requirements for the continued development of hematopoietic colony-forming cells (CFCs) and their progeny from embryonic stem-derived cells, cells from EBs disrupted after 9 days of formation in the absence of leukemia-inhibitor factor were cultured under different conditions. Low numbers of day-9 EB cells (5 x 10(5) or less) cultured in the presence of several growth factors (interleukin-3 [IL-3], IL-1, c-kit ligand, basic fibroblast growth factor, insulin growth factor-1, IL-6, granulocyte colony-stimulating factor, fetal liver kinase-2 ligand) develop few or no CFCs after 1 week of culture. When these cells are plated on irradiated NIH-3T3 with IL-3 or c-kit ligand or combinations containing these and other growth factors, they are able to generate CFCs for at least 3 weeks. These cultures were found to include granulocytic, monocytic, erythrocytic, and megakaryocytic cells. Transwell cultures in which NIH-3T3 cells were separated from the EB cells and cultures in which cells were replaced by NIH-3T3 conditioned medium showed that the interaction between EB-derived cells and NIH-3T3 is via a soluble factor(s). These studies show that maximal generation of hematopoietic CFCs from precursors present in day-9 EBs is stimulated by a combination of known hematopoietic growth factors and a soluble factor(s) produced by NIH-3T3 cells.
The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor
Authors: Vlot AJ , Koppelman SJ , van den Berg MH , Bouma BN , Sixma JJ
Published: October 2019

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid-phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.
Role of extracellular adenosine triphosphate in the cytotoxic T- lymphocyte-mediated lysis of antigen presenting cells
Authors: Blanchard DK , Wei S , Duan C , Pericle F , Diaz JI , Djeu JY
Published: October 2019

The lysis of antigen presenting cells (APCs) by cytotoxic T lymphocytes (CTLs) may be one mechanism whereby an immune response is downregulated by Staphylococcus superantigens. Disappearance of monocytes/macrophages from staphylococcal enterotoxin A (SEA)-activated peripheral blood mononuclear cell (PBMC) cultures, but not from control PBMC cultures was seen by flow cytometry. Recently, adenosine triphosphate (ATP) has been described as an effector molecule in CTL-mediated lysis of some murine tumor target cells. We have also shown that ATP caused the lysis of human macrophages, and that treatment of cells with interferon gamma (IFN gamma) rendered macrophages significantly more sensitive to ATP than untreated cells. To show that this purine nucleotide may play a role in modulating the immune system, we generated human CTLs that were stimulated with SEA, and used them as effector cells against SEA-pulsed autologous macrophages. CTLs were found to specifically lyse SEA-pulsed macrophages, while control, unpulsed, macrophages were unaffected. The addition of hexokinase, an enzyme that hydrolyzes ATP, significantly abrogated the killing of SEA-pulsed cells during the assay. In examining the mechanism of cytotoxicity, electron microscopy showed that macrophages incubated with both ATP and CTLs underwent necrosis, rather than apoptosis. From these results, it is suggested that ATP is released from CTLs during antigen presentation, and that IFN gamma-activated macrophages, which are inherently more sensitive to this mediator, are readily lysed and therefore removed from circulation, thus downregulating an immune response.
Analysis of the human CD10/neutral endopeptidase 24.11 promoter region: two separate regulatory elements
Authors: Ishimaru F , Shipp MA
Published: October 2019

The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) is expressed on normal and malignant lymphoid progenitors, granulocytes, and a variety of epithelial cells. To further define the tissue-specific and developmentally related expression of CD10/NEP, we have characterized two separate regulatory regions that control the transcription of 5′ alternatively spliced CD10/NEP transcripts. These type 1 and 2 CD10/NEP regulatory regions are both characterized by the presence of multiple transcription initiation sites and the absence of classic TATA boxes and consensus initiator elements. The purine-rich type 1 regulatory region, which includes 5′ UTR exon 1 sequence, is characterized by multiple putative PU.1 binding sites and consensus ets-binding motifs. In marked contrast, the GC-rich type 2 regulatory region contains multiple putative Sp1 binding sites, a potential consensus retinoblastoma control element (RCE), and an inverted CCAAT box. In the majority of tissues examined to date, type 2 CD10/NEP transcripts were more abundant; the abundance of type 1 transcripts was more variable, with the highest type 1 levels in fetal thymus and certain lymphoblastic leukemia cell lines.
Interphase and metaphase detection of the breakpoint of 14q32 translocations in B-cell malignancies by double-color fluorescence in situ hybridization
Authors: Taniwaki M , Nishida K , Ueda Y , Misawa S , Nagai M , Tagawa S , Yamagami T , Sugiyama H , Abe M , Fukuhara S
Published: October 2019

The breakpoint of 14q32 translocations found in B-cell malignancies was delineated specifically in both metaphase spreads and interphase nuclei by double-color fluorescence in situ hybridization (FISH) using bacteriophage clones containing the human immunoglobulin gamma chain gene locus (Ig gamma) and a cosmid clone, CY24–68, containing VH segments. CY24–68 is more telomeric than Ig gamma, separated by approximately 1 megabase (Mb). FISH studies were performed on four patients with non-Hodgkin's lymphoma (NHL), one with acute lymphoblastic leukemia (ALL), one with plasma cell leukemia (PCL), and three cell lines. In each patient with t(8;14), t(14;18), and t(3;14), the signal of Ig gamma gene was observed on der(14) and that of CY24–68 at respective partner sites of these translocations, 8q24.1, 18q21.3, and 3q27. Interphase nuclei with a signal of Ig gamma clearly separated from that of CY24–68 were more frequently encountered in all of the patients (45% to 74%) than those in normal controls (4% to 5%). Even in cases where only interphase nuclei were available for FISH studies, 14q32 translocations are detected as shown in two patients each with NHL and t(11;14)-carrying PCL. In two cell lines, HS-1 derived from ALL carrying t(8;14) and FR4 derived from a plasmacytoma carrying a complex form of t(8;14), the signal of Ig gamma was observed at the breakpoint region 8q24.1 of the der(8) in addition to the der(14), indicating that translocation event occurred within the Ig gamma locus. Intense Ig gamma signal was found at the breakpoint region on the der(14)t(11;14) in HBL-2 derived from NHL, indicating amplification of the Ig gamma gene, and presumably the resultant chimeric DNA between Ig gamma and DNA sequences at 11q13. The present approach allowed us to unequivocally detect tumor-specific breakpoints of 14q32 translocations. Furthermore, interphase FISH provides a rapid diagnostic procedure to detect 14q32 translocations in B-cell malignancies.
Prolonged impairment of hematopoiesis after high-dose therapy followed by autologous bone marrow transplantation
Authors: Domenech J , Linassier C , Gihana E , Dayan A , Truglio D , Bout M , Petitdidier C , Delain M , Petit A , Bremond JL
Published: October 2019

Hematopoietic reconstitution has been studied in 180 patients after autologous bone marrow transplantation based on peripheral blood cell (PBC) recovery time and marrow progenitor counts sequentially tested for up to 4 years. Several factors that could influence hematopoietic reconstitution have been analyzed including sex, age, diagnosis, disease status, conditioning regimen, graft progenitor content, graft in vitro purging, and postgrafting administration of growth factors. Before transplantation, marrow progenitor values were normal only for colony-forming unit granulocyte macrophage (CFU-GM) in contrast to colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and colony-forming unit-megakaryocyte (CFU-Meg). After transplantation, as described with allogenic grafts, these values remained low for several years, although PBC counts were nearly normalized within a few weeks. Pregraft values were reached after 2 years for CFU-GM and BFU-E, and after 4 years for CFU-E, while CFU-Meg failed to reach pregraft values after this time. Normal levels were reached after 4 years only by CFU-GM. On univariate and multivariate analysis, the following factors appeared to delay both PBC and marrow progenitor reconstitution: underlying disease (particularly acute myeloid leukemias), graft characteristics such as low stem cell content and in vitro purging, conditioning regimens with total body irradiation or busulfan, and lack of postgraft administration of growth factors. In conclusion, high-dose therapy followed by bone marrow transplantation induces a deep and prolonged impairment of hematopoiesis irrespective of any alloimmune reaction or postgraft immunosuppressive therapy.(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular detection of the (2;5) translocation of non-Hodgkin's lymphoma by reverse transcriptase-polymerase chain reaction
Authors: Downing JR , Shurtleff SA , Zielenska M , Curcio-Brint AM , Behm FG , Head DR , Sandlund JT , Weisenburger DD , Kossakowska AE , Thorner P
Published: October 2019

The t(2;5)(p23;q35) translocation was initially identified in cases of anaplastic large-cell lymphoma (ALCL) that expressed the Ki-1 (CD30) antigen. We have recently cloned this translocation and shown it to encode a chimeric product consisting of the N-terminal portion of a nonribosomal nucleolar phosphoprotein, nucleophosmin (NPM), from chromosome 5, fused to the kinase domain of a novel transmembrane tyrosine-specific protein kinase, anaplastic lymphoma kinase (ALK), from chromosome 2. To better define the spectrum of lymphomas that contain this translocation, we have analyzed 70 cases of non-Hodgkin's lymphoma (NHL) for expression of the t(2;5)-derived NPM/ALK chimeric message by reverse transcriptase-polymerase chain reaction (RT-PCR). Using a previously described set of oligonucleotide primers, NPM/ALK chimeric transcripts were detected in 21 of 22 cases that contained the t(2;5) by cytogenetic analysis and in 10 of 48 cases that either lacked evidence of the t(2;5) or had unsuccessful cytogenetics. In all but 1 case, the NPM/ALK PCR products were of identical size and sequence, suggesting that the genomic chromosome breaks are clustered in a single intron in both NPM and ALK. The NPM/ALK-expressing cases were not confined to NHLs with anaplastic morphology and included 15 ALCLs, 6 immunoblastic lymphomas, and 10 diffuse large-cell lymphomas. Moreover, only slightly greater than half of the cases with anaplastic morphology and 59% of CD30-expressing cases were NPM/ALK positive. Thus, neither anaplastic morphology nor the expression of CD30 accurately predicted the presence of this molecular genetic subtype of lymphoma.
Cloning and expression of unique murine macrophage colony-stimulating factor transcripts
Authors: Pogue-Geile K , Sakakeeny MA , Panza JL , Sell SL , Greenberger JS
Published: October 2019

Cocultivation of cells from the gamma-irradiated D2XRII murine bone marrow stromal cell line with an interleukin-3/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent hematopoietic progenitor cell line FDC-P1JL26 stimulates the emergence of factor-independent hematopoietic cell sublines. Several lines of evidence suggested that M-CSF or a protein antigenically related to M-CSF, termed leukemogenic stromal factor (LSF), that was expressed by D2XRII cells may have played a role in the emergence of the factor-independent sublines. In an effort to isolate a factor antigenically related to M-CSF, molecular clones were isolated from a D2XRII cDNA library that hybridized to a mouse M-CSF genetic probe. Two of these molecular clones, designated 60.8.2 and 6452, contained an 885-bp deletion in the M-CSF coding region. Such a cDNA clone has not been previously described in the mouse, but a cDNA clone homologous to it has been isolated from a human pancreatic tumor cell line, MIA-PaCa-2. Three transcripts (4.8, 3.4, and 1.8 kb) were detected that hybridized to an oligonucleotide probe that was specific to RNA transcripts containing the 60.8.2 deletion. The level of the 1.8-kb transcript was not detectably induced by ionizing irradiation; however, the levels of the 4.8-kb and 3.4-kb transcripts and two other M-CSF transcripts of sizes to 4.4 kb and 2.3 kb showed a 1.4- to 2.2-fold increase after gamma irradiation. Reverse transcription-polymerase chain reaction showed that the deletion-specific transcript(s) was detected in multiple mouse bone marrow stromal cell lines and in normal mouse tissues. The present studies establish the existence of an increased spectrum of murine M-CSF transcripts in bone marrow stromal cells and other tissues. This complexity of transcripts along with their increased accumulation after irradiation provides additional evidence for a role of proteins encoded by M-CSF transcripts in the response of bone marrow stromal cells to ionizing irradiation.
Resistance to activated protein C as an additional genetic risk factor in hereditary deficiency of protein S
Authors: Zoller B , Berntsdotter A , Garcia de Frutos P , Dahlback B
Published: October 2019

Inherited resistance to activated protein C (APC), which is caused by a single point mutation in the gene for factor V, is a common risk factor for thrombosis. In this study, the prevalence of APC resistance in 18 unrelated thrombosis-prone families with inherited protein S deficiency was investigated to determine its role as additional genetic risk factor for thrombosis. In addition, a detailed evaluation of the clinical manifestations in these families was performed. Venous thrombotic events had occurred in 47% of the protein S-deficient patients (64/136) and in 7% of relatives without protein S deficiency (14/191). As estimated from Kaplan-Meier analysis, 50% of protein S-deficient family members and 12% of those without protein S deficiency had had manifestation of venous thromboembolism at the age of 45 years. The age at the first thrombotic event ranged from 10 to 81 years (mean, 32.5 years) and a large intrafamilial and interfamilial variability in expression of thrombotic symptoms was seen. The factor V gene mutation related to APC resistance was present in 6 (38%) of 16 probands available for testing; in total, the mutation was found in 7 (39%) of the 18 families. In family members with combined defects, 72% (13/18) had had thrombosis as compared with 19% (4/21) of those with only protein S deficiency and 19% (4/21) of those with only the factor V mutation. In conclusion, APC resistance was found to be highly prevalent in thrombosis-prone families with protein S deficiency and was an additional genetic risk factor for thrombosis in these families. The results suggest thrombosis-prone families with protein S deficiency often to be affected by yet another genetic defect.
Retinoids downregulate both p60 and p80 forms of tumor necrosis factor receptors in human histiocytic lymphoma U-937 cells
Authors: Totpal K , Chaturvedi MM , LaPushin R , Aggarwal BB
Published: October 2019

Because retinoids are known to modulate the growth and differentiation effects of tumor necrosis factor (TNF), we investigated the effect of all-trans-retinoic acid (RA) on the cell surface expression of TNF receptors in human histiocytic lymphoma U-937 cells. RA decreased the specific binding of 125I-labeled TNF to these cells in a dose- and time-dependent manner. The maximal decrease occurred when cells were treated with 1 mumol/L RA for 24 hours at 37 degrees C. Scatchard analysis of the binding indicated that the decrease by RA was caused by a decrease in receptor number and not by a decrease in affinity. The downmodulation of TNF receptors was also confirmed by covalent receptor-ligand cross-linking studies. Receptor-mediated internalization of the ligand was also found to be decreased on treatment of cells with RA. Northern blot analysis also indicated a decrease in the transcript of the receptor. By using antibodies specific to either the p60 or p80 form of the TNF receptor, we found that both receptors were downregulated by RA. RA treatment also decreased TNF receptors on acute monocytic leukemia cell line THP-1. Other analogues of RA, specifically 9-cis-RA, (E)-4-[2-(5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl]-benzoic acid (TTNPB), and 3-methyl-TTNPB, which differ in their specificity towards different RA receptors, were also active in downregulating TNF receptors. 3-Methyl-TTNPB, which is more specific for the RXR form of the RA receptor, was found to be most potent. The downregulation of TNF receptors by RA correlated with the downmodulation of the antiproliferative effects of TNF against U-937 cells. Overall, our results indicate that RA downmodulates both the p60 and p80 form of the TNF receptor on cells of myeloid origin, which correlates with the cellular response.
The functional characterization of interleukin-10 receptor expression on human natural killer cells
Authors: Carson WE , Lindemann MJ , Baiocchi R , Linett M , Tan JC , Chou CC , Narula S , Caligiuri MA
Published: October 2019

Human natural killer (NK) cells are large granular lymphocytes that constitutively express functional forms of the interleukin-2 receptor (IL-2R) and lyse tumor and virally infected cells without prior sensitization. NK cells with high density expression of CD56 (CD56bright) express the high affinity IL-2R and proliferate in response to low (picomolar) concentrations of IL-2. CD56dim NK cells express the intermediate affinity IL-2R and demonstrate enhanced cytotoxic activity without proliferation in response to high (nanomolar) concentrations of IL-2. In the present study, we characterized IL-10R expression on human NK cells and the functional consequences of IL-10 binding directly to highly purified subsets of CD56bright and CD56dim NK cells. Binding studies using 125I-IL-10 indicated that resting human NK cells constitutively express the IL-10 receptor protein at a surface density of approximately 90 receptor sites per cell, with a kd of approximately 1 nmol/L. Alone, IL-10 did not induce proliferation of CD56bright or CD56dim NK cell subsets. However, at low concentrations (0.5 to 5 ng/mL), IL-10 significantly augmented IL-2-induced proliferation of the CD56bright NK cell subset mediated via the high-affinity IL-2R. In the absence of IL-2, IL-10 was able to induce significant NK cytotoxic activity against NK-resistant tumor cell targets in both subsets of NK cells in a dose-dependent fashion. Furthermore, the combination of IL-10 and IL-2 had an additive effect on NK cytotoxic activity, whereas that of IL-10 and IL-12 did not. Production of interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor by IL-2-activated NK cells was also significantly enhanced by IL-10. Neither resting nor activated human NK cells appear to produce human IL-10 protein. In summary, NK cells constitutively express the IL-10R protein in low density, and the functional consequences of IL-10 binding directly to human NK cell subsets appear to be stimulatory and dose-dependent. In contrast to its direct effects on human T cells and monocytes/macrophages, IL-10 potentiates cytokine production by human NK cells.
Cytokine activation during attacks of the hyperimmunoglobulinemia D and periodic fever syndrome
Authors: Drenth JP , van Deuren M , van der Ven-Jongekrijg J , Schalkwijk CG , van der Meer JW
Published: October 2019

The hyperimmunoglobulinemia D and periodic fever (hyper-IgD) syndrome is typified by recurrent febrile attacks with abdominal distress, joint involvement (arthralgias/arthritis), headache, skin lesions, and an elevated serum IgD level (< 100 U/mL). This familial disorder has been diagnosed in 59 patients, mainly from Europe. The pathogenesis of this febrile disorder is unknown, but attacks are joined by an acute-phase response. Because this response is considered to be mediated by cytokines, we measured the acute-phase proteins C-reactive protein (CRP) and soluble type-II phospholipase A2 (PLA2) together with circulating concentrations and ex vivo production of the proinflammatory cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, and tumor necrosis factor alpha (TNF alpha) and the inhibitory compounds IL-1 receptor antagonist (IL-1ra), IL-10, and the soluble TNF receptors p55 (sTNFr p55) and p75 (sTNFr p75) in 22 patients with the hyper-IgD syndrome during attacks and remission. Serum CRP and PLA2 concentrations were elevated during attacks (mean, 213 mg/L and 1,452 ng/mL, respectively) and decreased between attacks. Plasma concentrations of IL-1 alpha, IL-1 beta, or IL-10 were not increased during attacks. TNF alpha concentrations were slightly, but significantly, higher with attacks (104 v 117 pg/mL). Circulating IL-6 values increased with attacks (19.7 v 147.9 pg/mL) and correlated with CRP and PLA2 values during the febrile attacks. The values of the antiinflammatory compounds IL-1ra, sTNFr p55, and sTNFr p75 were significantly higher with attacks than between attacks, and there was a significant positive correlation between each. The ex-vivo production of TNF alpha, IL-1 beta, and IL-1ra was significantly higher with attacks, suggesting that the monocytes/macrophages were already primed in vivo to produce increased amounts of these cytokines. These findings point to an activation of the cytokine network, and this suggests that these inflammatory mediators may contribute to the symptoms of the hyper-IgD syndrome.
The PML gene encodes a phosphoprotein associated with the nuclear matrix
Authors: Chang KS , Fan YH , Andreeff M , Liu J , Mu ZM
Published: October 2019

The t(15;17)(q22;q12) translocation is the cytogenetic hallmark of acute promyelocytic leukemia (APL). The PML and retinoic acid receptor-alpha (RAR alpha) transcription factor genes are involved at translocation breakpoint. To elucidate the biologic function of PML, antipeptide antibody against PML protein was raised in rabbits. This antibody was able to detect a 90-kD PML protein and a 110-kD PML-RAR alpha fusion protein by Western blotting and a nuclear speckled pattern in all cell lines by immunofluorescent staining. In K562 and NIH/3T3 cells transfected with a PML expression plasmid, we found PML to be associated with the nuclear matrix. Our results also showed that PML is a phosphorprotein. A weak signal was detected in a Western blot containing the immunoprecipitated PML protein using the phosphotyrosine-specific monoclonal antibody. Therefore, at least one of the sites was phosphorylated by a tyrosine kinase. From our analysis of the phosphoamino acids of the PML protein by complete hydrolysis and thin-layer chromatography, we concluded that both tyrosine and serine residues of PML are phosphorylated. To investigate whether expression of the PML protein is cell-cycle related, HeLa cells synchronized at various phases of the cell cycle were analyzed by immunofluorescence staining and confocal microscopy for PML expression. We found that PML was expressed at a lower level in S, G2, and M phases and at a significantly higher level in G1 phase. Our study showed that PML has many similar properties compared with the tumor suppressor, eg, Rb. These findings further support the important role of PML in APL pathogenesis.