bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma
Authors: Zhang XG , Klein B , Bataille R
Published: October 2019

It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.
Downregulation of c-fms gene expression in human monocytes treated with phorbol esters and colony-stimulating factor 1
Authors: Sariban E , Imamura K , Sherman M , Rothwell V , Pantazis P , Kufe D
Published: October 2019

The colony-stimulating factor-1 (CSF-1) regulates survival, growth, and differentiation of monocytes by binding to a single class of high- affinity receptors. The CSF-1 receptor is identical to the product of the c-fms protooncogene. The present studies monitored the effects of TPA and CSF-1 on c-fms gene expression in human monocytes. The results demonstrate that TPA downmodulates the constitutive expression of c-fms mRNA to low but detectable levels. Treatment of human monocytes with TPA was similarly associated with decreases in levels of the 138- and 125-Kd c-fms-encoded proteins. However, the kinetics of c-fms protein downmodulation indicated independent effects of TPA on c-fms expression at the RNA and protein levels. Furthermore, c-fms protein levels subsequently recovered despite persistently low levels of c-fms mRNA. Although previous studies demonstrated that c-fms protein is down- regulated in the presence of CSF-1, the present results indicate that CSF-1 also downregulates levels of c-fms mRNA. Moreover, the results indicate that CSF-1 increases protein kinase C activity in the membrane fraction. Together, these findings suggest that c-fms gene expression is differentially regulated at both the RNA and protein levels after activation of protein kinase C in human monocytes treated with TPA and CSF-1.
Antibodies against platelet glycosphingolipids: detection in serum by quantitative HPTLC-autoradiography and association with autoimmune and alloimmune processes
Authors: Koerner TA , Weinfeld HM , Bullard LS , Williams LC
Published: October 2019

In order to assess the importance of glycosphingolipids (GSL) in the immunology of the platelet, serum antibody binding to immobilized, purified platelet GSLs have been quantitatively measured via high- performance thin-layer chromatography (HPTLC), 125I-radio- immunolabeling, autoradiography, and densitometry. Thirteen neutral GSL bands were detected at Rf.03 through .64 (CHCl3-CH3OH-H2O, 65:25:4) after extraction and chromatography (DEAE-Sephadex and Bio-sil A). Both IgM and IgG serum antibody binding was determined for 50 subjects from four groups: normal blood donors (NBD, n = 18); leukemia patients with nonimmune thrombocytopenia (NIT, n = 10); patients with systemic lupus erythematosus (SLE, n = 10); and patients with chronic autoimmune thrombocytopenia (CATP, n = 12). The ABO typing of these 50 subjects also allowed correlation of serum antibody binding with A blood group antigen expression. These studies reveal: (1) anti-GSL binding at band .06 is associated with blood group A alloantigen expression for both IgG and IgM (P less than .0001) antibodies; (2) binding at bands .17, .27, and/or .46 is associated with general autoimmunity (SLE and CATP) for IgM (P less than .0001); (3) binding at bands .35 and/or .38 is associated with platelet-specific autoimmunity (CATP) for IgG and/or IgM (P less than .005); and (4) binding at bands .03, .20, .23, and/or .43 is frequently observed for sera from all groups. The platelet specificity of bands .35 and .38 was confirmed by comparative studies with human intestinal smooth muscle GSLs. Quantitation of the intensity of CATP-associated anti-GSL binding (86 +/- 88 mm2) is comparable to anti-A alloantigen binding (57 +/- 52 mm2). Two of the GSL bands associated with SLE and CATP appear to be the long-chain fatty-acyl forms of globotriaosyl ceramide (.27) and globotetraosyl ceramide (.17), which are the Pk and P blood group antigens, respectively. These studies indicate that neutral GSLs may be important antigens in both autoimmune and alloimmune processes involving the blood platelet.
Rearrangement and expression of T-cell receptor delta genes in T-cell acute lymphoblastic leukemias
Authors: van Dongen JJ , Wolvers-Tettero IL , Wassenaar F , Borst J , van den Elsen P
Published: October 2019

We have analyzed T-cell receptor delta (TcR-delta) gene rearrangement and transcription in appropriately phenotyped mononuclear cells derived from 12 patients with T-cell acute lymphoblastic leukemia (T-ALL). The T-ALL cells were also analyzed for rearrangement and transcription of the T-cell receptor(TcR)-beta and gamma genes as well as for the presence of TcR-alpha gene transcripts. Four T-ALLs expressed TcR-gamma delta at the cell surface, while three expressed TcR-alpha beta. The other five T-ALLs did not express a TcR-CD3 complex on their cell membrane. The TcR-gamma delta + T-ALL had rearranged both TcR-delta gene alleles and contained mature 2.2 and 1.5 kb TcR-delta transcripts. In one case, immature 1.9 and 1.2 kb TcR-delta transcripts were also found. Furthermore they contained mature TcR-gamma mRNA, mature or immature TcR-beta mRNA, but no TcR-alpha mRNA. The three TcR-alpha beta + T-ALLs contained mature alpha and beta transcripts, but lacked TcR- delta transcripts as a result of deletion of both TcR-delta gene alleles. These data are in line with a mutually exclusive expression of TcR-alpha and -delta genes, which may be important to ensure the presence of only one type of TcR per T cell. One of the five CD3- T- ALLs had germline TcR-beta, gamma, and delta genes. The other four CD3- T-ALLs had rearranged their TcR-beta, gamma, and delta genes and contained immature 1.9 and 1.2 kb TcR-delta gene transcripts. Remarkably, one of these T-ALLs also contained TcR-alpha transcripts in addition to the immature TcR-delta transcripts, which was in line with the deletion of one TcR-delta gene allele and rearrangement of the other allele. This suggests that prevention of dual receptor expression may not only be regulated by the presence of germline TcR-alpha genes in TcR-gamma delta + cells or by deletion of both TcR-delta gene alleles in TcR-alpha beta + cells, but also via other regulation mechanisms. Finally, our data indicated that the combinatorial repertoire of the TcR-delta genes is limited, which has also been described for the TcR-gamma genes.
Autocrine growth mechanisms of the progenitors of blast cells in acute myeloblastic leukemia
Authors: Murohashi I , Tohda S , Suzuki T , Nagata K , Yamashita Y , Nara N
Published: October 2019

Autocrine growth mechanisms of leukemic blast progenitors in acute myeloblastic leukemia (AML) were investigated. Colony formation of leukemic blast progenitors was observed in 14 of 14 patients tested when purified blast cell fraction depleted of both T cells and monocytes was plated in methylcellulose without any colony-stimulating factor (CSF). However, there existed a minimal cell density required to initiate blast progenitor growth with marked patient-to-patient variation. To clarify the role of cell density on the spontaneous growth of blast progenitors, we tested whether leukemic cells produced and secreted some stimulatory humoral factor(s). Production of colony- stimulating activity (CSA) by blast cells was observed in 17 of 18 patients tested. Following further depletion of monocytes, the CSA levels decreased markedly in 14 patients, indicating that blast cells with monocytoid differentiation were responsible for CSA production. We also confirmed granulocyte colony-stimulating factor (G-CSF) and/or granulocyte macrophage-colony-stimulating factor (GM-CSF) production by leukemic blasts using specific immunologic assays. When leukemic cells were divided into nonadherent nonphagocytic cell fraction and adherent cell fraction, only nonadherent nonphagocytic cells showed clonogenecity and adherent blast cells lacked the colony-forming capacity. The results indicate that there are at least two blast cell subpopulations in AML: one is proliferating subpopulation with self- renewal capacity and the other is supporting subpopulation with functions such as CSF production. The quite intimate relationship between these two blast cell subpopulations in AML may play an important role on the growth of leukemic blast progenitors in vitro.
Terminal deoxynucleotidyl transferase (TdT)-positive cells in cerebrospinal fluid and development of overt CNS leukemia: a 5-year follow-up study in 113 children with a TdT-positive leukemia or non- Hodgkin's lymphoma
Authors: Hooijkaas H , Hahlen K , Adriaansen HJ , Dekker I , van Zanen GE , van Dongen JJ
Published: October 2019

We investigated whether an indirect nuclear terminal deoxynucleotidyl transferase (TdT) immunofluorescence (IF) assay on single cells present in the cerebrospinal fluid (CSF) is more effective than conventional cytomorphology for early detection or exclusion of (minimal) meningeal leukemic infiltration in patients with a TdT+ malignancy. During a 5- year follow-up study, 1,661 consecutive CSF samples from 113 children with a TdT+ acute lymphoblastic leukemia (ALL) (n = 100), a TdT+ acute nonlymphoblastic leukemia (ANLL) (n = 8), or a TdT+ non-Hodgkin's lymphoma (NHL) (n = 5) were analyzed. In 1,511 (91.9%) of 1,643 evaluable CSF samples, the positive and negative findings of both cytomorphology and the TdT-IF assay were concordant. In 47 (2.9%) samples from 28 patients, the cytomorphology was suspect while the TdT- IF assay was negative; follow-up as long as 58 months revealed no CNS leukemia in any patient. In 85 (5.2%) samples, cytomorphology was negative (n = 70) or suspect (n = 15) but TdT+ cells were detected. RBC contamination seriously hampered evaluation in 31 of these 85 samples. From the remaining 54 TdT+ samples from 29 patients, 40 samples preceded overt CNS leukemia in 20 patients. Two consecutive findings of TdT+ cells in the CSF were always followed by overt CNS leukemia. At initial diagnosis, 11 children had TdT+ cells in their RBC-free CSF. In one of these children, morphology was suspect; a repeated lumbar puncture was positive on both assays. Thus, initial CNS leukemia was diagnosed. In the other ten children, morphology was negative. In six of them, CNS leukemia was diagnosed 2 to 20 months later. In 32 other children examined at initial diagnosis, neither TdT+ cells nor blasts were observed in the CSF. In none of these patients was a CNS leukemia diagnosed after a follow-up of 2.5 to 57 months (median 24 months). In 207 control CSF samples from 58 children with TdT- oncologic, hematologic, or infectious diseases, no TdT+ cells could be detected. The TdT-IF assay is easy to perform and is a more reliable diagnostic tool for detection of CNS leukemia at an early stage than is cytomorphology. At initial diagnosis, the finding of Tdt+ cells in a RBC-free CSF sample with a negative cytomorphology is highly predictive for development of overt CNS leukemia.
Growth and differentiation of a human megakaryoblastic cell line, CMK
Authors: Komatsu N , Suda T , Moroi M , Tokuyama N , Sakata Y , Okada M , Nishida T , Hirai Y , Sato T , Fuse A
Published: October 2019

Recently, a human megakaryoblastic cell line, CMK, was established from the peripheral blood of a megakaryoblastic leukemia patient with Down syndrome. Using this cell line, we studied the proliferation and differentiation of megakaryocytic cells in the presence of highly purified human hematopoietic factors and phorbol 12-myristate-13- acetate (PMA). In a methylcellulose culture system, interleukin-3 (IL- 3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitated colony formation by CMK cells in a dose-dependent manner. The maximum stimulating doses of these factors were 10 and 200 U/mL, respectively. These concentrations were comparable to those that stimulate activity in normal hematopoietic cells. In contrast, granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), and erythropoietin (EPO) had no effects on the colony formation of CMK cells. In a liquid culture system, 20% of the CMK cells expressed glycoprotein IIb/IIIa (GPIIb/IIIa) antigen without hematopoietic factors, whereas 40% of the cells expressed GPIIb/IIIa with the addition of IL-3 and GM-CSF. EPO also slightly enhanced expression of GPIIb/IIIa. On the other hand, PMA inhibited growth of CMK cells and induced most of them to express the GPIIb/IIIa antigen. Furthermore, PMA induced CMK cells to produce growth activity toward new inocula of CMK cells. This growth factor (GF) contained colony-stimulating activity (CSA) in normal bone marrow (BM) cells. The activity was believed to be attributable mainly to GM-CSF, since 64% of this activity was neutralized by anti-GM-CSF antibodies and a transcript of GM-CSF was detected in mRNA from PMA-treated CMK cells by Northern blot analysis. These observations suggest that GM-CSF, as well as IL-3, should play an important role in megakaryocytopoiesis.
Establishment and characterization of an Epstein-Barr virus transformed cell line with strong phagocytic activity
Authors: von den Driesch P , Bhardwaj R , Flad HD , Neugebauer DC , Pielken HJ , Urbanitz D , Kolsch E
Published: October 2019

An immunoglobulin M (IgM)-positive cell line, Ms 28, apparently spontaneously transformed by Epstein-Barr virus (EBV) was established from peripheral blood cells of a patient with immature myeloblastic leukemia. It has been characterized according to phenotype, cytochemistry, and membrane antigen pattern. The cell line expresses lymphoid markers like CD 19, CD 22, and CD 30 and synthesizes and secretes IgM. Monocyte markers CD 11c, CD 14, and CD 15 are absent. Neither interleukin-1 (IL-1), nor tumor necrosis factor (TNF-alpha) are produced. But Ms 28 cells show strong phagocytic activity and engulf Latex particles and sheep RBCs (SRBCs) that need not to be opsonized. The phagocytic activity can be inhibited by chloroquine. Both phagocytosis and EBV nuclear-antigen (EBNA) expression can be observed in one and the same cell. Ms 28 cells might be useful to study immunologic activities like antigen processing and presentation.
Feline parvovirus propagates in cat bone marrow cultures and inhibits hematopoietic colony formation in vitro
Authors: Kurtzman GJ , Platanias L , Lustig L , Frickhofen N , Young NS
Published: October 2019

Feline parvovirus (FPV) causes leukopenia in naturally infected cats. We investigated the mechanism of hematopoietic depression by this virus in feline bone marrow cultured in vitro. In suspension cultures we demonstrated FPV propagation and replication using DNA molecular hybridization. Viral RNA and DNA were observed by in situ hybridization in about 10% of marrow cells at day 3. Granulocytes and their precursors were virtually absent from infected cultures after six days. Infected cells showed viral capsid protein predominantly in nuclei by immunofluorescence. In clonal assays, FPV most efficiently inhibited hematopoietic colony formation by myeloid progenitor cells (CFU-GM), but erythroid colony formation (BFU-E and CFU-E-derived) was also depressed in the presence of virus. Inhibition of colony formation could be abrogated by physical inactivation of the virus or preincubation with specific neutralizing antibodies. Recombinant human colony stimulating factors GM-CSF and G-CSF supported feline myelopoiesis in progenitor assays, and FPV completely inhibited factor dependent colony formation.
Interleukin-3 and granulocyte-monocyte colony-stimulating factor receptors on human acute myelocytic leukemia cells and relationship to the proliferative response
Authors: Budel LM , Touw IP , Delwel R , Clark SC , Lowenberg B
Published: October 2019

Interleukin-3 (IL-3) and granulocyte-monocyte-colony-stimulating factor (GM-CSF) stimulate proliferation of human acute myeloid leukemia (AML) in vitro, although patterns of response among clinical cases are diverse. Whether regulatory abnormalities related to growth factor responses in human AML may establish the outgrowth of the neoplasm is unclear. We determined receptor numbers and affinity for IL-3 and GM- CSF on human AML cells using human recombinant IL-3 (rIL-3) and GM-CSF (rGM-CSF). In 13 of 15 cases of primary AML high-affinity (kd 26 to 414 pmol/L) receptors for IL-3 were demonstrable on the cells. The average numbers of IL-3 receptors ranged from 21 to 145 receptors per cell. Normal WBCs showed IL-3 receptors on their surface at similar densities. IL-3 receptor positivity often correlated with GM-CSF receptor positivity of AML; GM-CSF receptors were demonstrated on the cells of 11 of 15 cases, although average numbers of GM-CSF receptors were ten times greater. The in vitro response of the cells to exogenous IL-3 or GM-CSF was examined by measuring thymidine uptake. Because IL-3 and GM-CSF were potent inducers of DNA synthesis in vitro, apparently relatively few receptors are required to permit activation of growth. These experiments did not provide evidence for overexpression or increased receptor sensitivity as an explanation for AML growth. In a minority of cases, however, the cells were unable to respond to IL-3 (four of 15 cases) or GM-CSF (four of 15 cases) despite normal receptor availability on the cell surface.
Production of growth factors by malignant lymphoma cell lines
Authors: Tweeddale M , Jamal N , Nguyen A , Wang XH , Minden MD , Messner HA
Published: October 2019

Fourteen Epstein-Barr virus (EBV)-negative cell lines were raised from bone marrow (BM), peripheral blood (PB), or lymph node samples of patients with intermediate- or high-grade malignant lymphoma. The cell lines were propagated in liquid suspension culture. They contain clonogenic progenitors capable of forming lymphoma colonies in semi- solid culture medium. Cells of these lines were used to examine the growth factor requirements of their clonogenic progenitors and to assess their ability to produce their own growth factors. Two of the cell lines (OCI-Ly9 and OCI-Ly13.1) required addition of exogenous factors for colony growth. These factors were routinely provided by media conditioned by phytohemagglutinin-stimulated leukocytes (PHA- LCM). Three lines formed some and nine lines gave rise to optimal numbers of colonies without addition of growth factors. Eight of these factor-independent lines were able to function as feeder cells and promoted colony formation by both factor-dependent lines. Cell lines that displayed feeder cell function released activities into supernatants able to replace their cellular source. Some of these endogenously produced growth-promoting activities could be replaced by known hematopoietic growth factors. Both factor-dependent cell lines were cultured with recombinant IL-1 alpha, IL-2, IL-3, IL-6, and GM colony-stimulating factor (CSF) and semipurified B-cell growth factor (BCGF) interleukin-4 (IL-4). A heterogeneous response pattern was observed. Both lines formed colonies with IL-4. The colonies were comparable in frequency and size with colonies observed with (PHA-LCM). OCI-Ly9 responded to IL-6 but showed no growth with IL-2. In contrast, the TAC-positive line OCI-Ly13.1 gave rise to colonies with IL-2 while remaining unresponsive to IL-6. A moderate number of colonies was observed when cells of this line were cultured with GM-CSF. Colony formation of both lines was uninfluenced by IL1 alpha or IL-3.
The in vivo effects of recombinant human interleukin-3: demonstration of basophil differentiation factor, histamine-producing activity, and priming of GM-CSF-responsive progenitors in nonhuman primates
Authors: Mayer P , Valent P , Schmidt G , Liehl E , Bettelheim P
Published: October 2019

Recently human interleukin-3 (IL-3) produced by molecular cloning was characterized as a growth factor for basophils and eosinophils in human bone marrow cultures. Since we found a similar activity of the human factor on simian bone marrow cells, we investigated the in vivo effects of recombinant human (rh) IL-3 in healthy rhesus monkeys (n = 10). rh IL-3 was administered subcutaneously (SC) to monkeys at different doses (11, 33, and 100 micrograms/kg/d) for 14 days followed by subsequent rh GM-CSF administration (5.5 micrograms/kg/d SC) for another two weeks. During the second week of rh IL-3 administration monkeys responded with a twofold to threefold increase of WBCs caused by a dose-dependent elevation of basophils (up to 40% of WBCs) and eosinophils. rh IL-3 also induced a dose-dependent increase of histamine (up to 700-fold above normal values) in monkey blood cells. Administration of rh GM-CSF to rh IL-3 pretreated monkeys resulted in a twofold enhanced increase in WBCs (due mainly to eosinophils and neutrophils) compared with animals treated with rh GM-CSF alone. Simultaneous administration of both cytokines (100 micrograms/kg rh IL-3 + 5.5 micrograms/kg rh GM-CSF SC) to two separate monkeys for 14 days induced a WBC elevation similar to that observed in monkeys treated with rh GM-CSF alone. In conclusion, our results indicate that rh IL-3 is a differentiation factor for blood basophils and primes the hematopoietic system for subsequent rh GM-CSF actions.
Quantitative tracing of mRNAs for T- and B-lymphocyte receptor genes in individual cells by in situ hybridization with fluorochrome-labeled gene probes. I. Expression in malignancies carrying B-lineage associated antigens
Authors: Mar P , Pachmann K , Reinecke K , Emmerich B , Thiel E
Published: October 2019

Acute and chronic lymphatic leukemias were investigated on the single- cell level for the activity of genes coding for the IgM heavy chain and the alpha and beta chains of the T-cell antigen receptor (TCR). We used a new method for preparing highly fluorochrome-labeled gene probes for in situ hybridization, which allowed rapid and quantitative detection of mRNA at the individual cell level. Leukemic cell populations classified as belonging to the B lineage according to their surface antigenic patterns revealed increasing expression of mRNA for the IgM heavy chain (mu mRNA) in a maturation-dependent fashion, which was not correlated to rearrangement of the immunoglobulin mu chain gene--only 66% of the leukemias with rearranged mu gene also transcribed it. TCR mRNA was detected in B-antigen positive leukemic cells. High levels of both TCR and mu mRNA expression in all cells of some of these leukemias allowed the conclusion that these cells simultaneously transcribed the genes for T and B cell antigen receptors. TCR mRNA was also found in what are considered relatively mature B leukemias, lineage cross-over on the mRNA level being observed at a frequency of 23% (five of 22 cases), comparable with that of “inappropriate” receptor gene rearrangement. The quantitation of mRNA with fluorochrome-labeled gene probes in situ may allow determining the degree of gene activation in individual antigenically defined cells and may thus contribute a new tool for characterization of normal and malignant cells.
Platelet-melanoma cell interaction is mediated by the glycoprotein IIb- IIIa complex
Authors: Boukerche H , Berthier-Vergnes O , Tabone E , Dore JF , Leung LL , McGregor JL
Published: October 2019

A human malignant melanoma cell line (M3Dau) was observed by electron microscopy to interact directly with human platelets and induced platelet aggregation. Fab fragments of a monoclonal antibody MoAb (LYP18), directed against the platelet glycoprotein (GP) IIb-IIIa complex, inhibited platelet-melanoma interactions and platelet-platelet aggregation. M3Dau melanoma cells bind LYP 18 and synthesize IIb-IIIa- like GPs. When the melanoma cells were preincubated with LYP 18, tumor- platelet interaction did not occur, suggesting that the interaction may be mediated by the IIb-IIIa-like GPs present on the melanoma cell surface. Glanzmann's thrombasthenic platelets, lacking GPIIb and IIIa, did not interact with melanoma cells, indicating that the platelet GPIIb-IIIa complex is also necessary for the platelet-melanoma cell interaction. This work demonstrates the importance of the IIb-IIIa-like GPs, present on M3Dau melanoma cells, in mediating tumor-platelet interactions.
Montreal platelet syndrome: a defect in calcium-activated neutral proteinase (calpain)
Authors: Okita JR , Frojmovic MM , Kristopeit S , Wong T , Kunicki TJ
Published: October 2019

Platelets from patients with Montreal platelet syndrome (MPS) consistently display a defect in the mechanisms that regulate platelet size during shape change and undergo spontaneous aggregation and stir- induced microaggregate formation. We now provide data that the surface glycoprotein composition of MPS platelets is indistinguishable from that of normal platelets. However, a defect in calcium-activated neutral proteinase (calpain) was detected in MPS platelets. The specific activity of calpain in the cytosolic fraction of platelets from four MPS patients was found to be only 30% of that in platelets from normal control donors (n = 18, P less than .001). Additionally, platelets from MPS patients (n = 3) contained only 50% (P less than .001) of the calpain I catalytic subunit antigen found in platelets from normal control donors (n = 9). Platelets from the asymptomatic father/grandfather of the MPS patients had normal amounts of both total calpain proteolytic activity and calpain I catalytic subunit antigen. This represents the first report of a defect in calpain in human cells. The abnormally low calpain activity in MPS platelets may account for the platelet defects characteristic of this disorder.
Plasminogen binding to rat hepatocytes in primary culture and to thin slices of rat liver
Authors: Gonias SL , Braud LL , Geary WA , VandenBerg SR
Published: October 2019

Human 125I-plasminogen bound readily to rat hepatocytes in primary culture at 4 degrees C and at 37 degrees C. Binding was inhibited by lysine and reversed by lysine, epsilon-aminocaproic acid, or nonradiolabeled plasminogen. The Kd for binding of 125I-plasminogen to hepatocytes was 0.59 +/- 0.16 mumol/L, as determined from the saturation isotherm by nonlinear regression (r2 = 0.99) and the Scatchard transformation by linear regression (r2 = 0.93). The number of sites per cell was 14.1 +/- 1.1 x 10(6). Fibrinogen synthesis and secretion by hepatocytes was insufficient to account for the major fraction of plasminogen binding, as determined by enzyme-linked immunosorbent assay (ELISA). Polyacrylamide gel electrophoresis and trichloroacetic acid precipitation studies demonstrated that plasminogen is neither activated nor degraded when bound to hepatocytes at 37 degrees C. Thin slices of whole rat liver (500 microns), isolated and prepared totally at 4 degrees C, bound 125I-plasminogen. Binding was inhibited by lysine. 125I-albumin binding to liver slices was minimal and not inhibited by lysine. Activation of plasminogen by tissue plasminogen activator (t-PA) was enhanced by hepatocytes in primary culture. When lysine was included in the media, the enhanced rate of activation was no longer observed. After activation with t-PA, much of the plasmin remained associated with hepatocyte surfaces and was partially protected from inhibition by alpha 2-antiplasmin. These studies suggest that hepatocyte plasminogen binding sites may provide important surface anticoagulant activity.
A codon 338 nonsense mutation in the factor IX gene in unrelated hemophilia B patients: factor IX338 New York
Authors: Driscoll MC , Bouhassira E , Aledort LM
Published: October 2019

Hemophilia B is an X-linked recessive bleeding disorder resulting from a deficiency of the coagulation factor IX (FIX) protein activity, a vitamin K-dependent serine protease active in both the intrinsic and extrinsic coagulation systems. DNA analyses of the factor IX gene in two unrelated patients with severe hemophilia B, with a IX coagulant activity less than 1% and undetectable FIX antigen, detected the loss of the second TaqI site in exon h (VIII) in both individuals. Polymerase chain reaction (PCR) amplification of 576 base pairs of exon h (VIII) with cloning and dideoxy sequencing of cloned DNA from one hemophiliac revealed a single C----T transition in codon 338 that changes an arginine residue codon CGA to a nonsense codon TGA. Allele- specific oligonucleotide probe hybridization with a mutant (C----T) and a wild-type allele confirmed the same mutation in amplified genomic DNA of the second hemophilia patient. The C----T transition represents another example of mutation at a CpG dinucleotide. DNA polymorphism analysis of the FIX gene in both individuals revealed each to be on a separate FIX haplotype; therefore, predicting each to be a separate mutation event.
T-cell activation and subset patterns are altered in B-CLL and correlate with the stage of the disease
Authors: Totterman TH , Carlsson M , Simonsson B , Bengtsson M , Nilsson K
Published: October 2019

Two-color FACS analysis was used to study activated and “functional” T and natural killer (NK) cell subsets of circulating lymphocytes in 23 patients with B-type chronic lymphocytic leukemia (B-CLL) and in 30 healthy subjects. As compared with controls, B-CLL patients had increased absolute numbers of phenotypically activated, HLA-DR+ CD4+ and CD8+ cells and T suppressor/effector (CD11b+CD8+) cells. When patients in Rai stages II through IV (n = 11) were compared with cases in Rai stages O through I (n = 12), the former group of patients had higher numbers of activated CD4+ and CD8+ T cells and decreased levels of suppressor/effector T cells. The absolute numbers of T suppressor/inducer (CD45R+CD4+) cells were elevated in patients with stage O through I disease but within normal range in stage II through IV leukemia. We further showed that the absolute numbers of NK-like (CD16+) cells and their activated counterparts (DR+CD16+) are elevated in B-CLL patients as compared with healthy subjects. The comparison of relative T and NK subsets in the blood of patients and controls showed that the proportions of CD4+, CD8+, and CD16+ cells expressing the activation marker HLA-DR were increased in B-CLL. Furthermore, the percentage of T-suppressor/inducer (CD45R+) cells within the CD4+ population was decreased in the patients. The proportion of T- suppressor/effector (CD11b+) cells within the CD8+ subset was reduced in subjects with stage II-IV disease only. When stimulated in vitro with the T-cell-dependent inducer TPA, B-CLL cells from patients in Rai stages II through IV secreted larger amounts of IgM as compared with cells from stage O through I patients. A positive correlation was observed between the degree of phenotypic activation of CD4+ T-helper cells and their functional capacity to augment IgM secretion by autologous B-CLL cells. Our findings indicate a tumor cell-directed regulatory role of T cells (and possibly NK cells as well) in B-CLL. Furthermore, monitoring of phenotypically activated and functional T- cell subsets may be helpful in the prediction of disease progression and timing of therapy in B-CLL.
Human neutrophil-mediated lysis of ovarian cancer cells
Authors: Lichtenstein A , Seelig M , Berek J , Zighelboim J
Published: October 2019

Because of recent questions concerning the sensitivity of human tumor cells to neutrophil-induced oxidative injury, we studied six freshly obtained human ovarian cancer (OC) specimens. Stimulation of neutrophils (PMNs) by phorbol myristate acetate (PMA) did not result in OC cytolysis during the first nine hours of incubation. However, three of six specimens were significantly lysed by stimulated PMNs when assay length was increased to 18 hours. Cytotoxicity was mediated by PMN production of reactive oxidative intermediates (ROIs). Presentation of ROIs to OC targets as preformed or enzymatically generated molecules in cell-free systems duplicated the enhanced lysis at 18 hours (as compared with six hours). Since addition of catalase at three or six hours did not inhibit enhanced lysis at 18 hours (achieved by PMNs or in cell-free systems), it appears that an initial ROI-mediated lethal event occurs early, but longer incubations are required for the event to become manifested as cell death. These data suggest that shorter assays may underestimate the potential of PMNs as effector cells against human tumor cells.
Demonstration of monoclonal EBV genomes in Hodgkin's disease and Ki-1- positive anaplastic large cell lymphoma by combined Southern blot and in situ hybridization [see comments]
Authors: Anagnostopoulos I , Herbst H , Niedobitek G , Stein H
Published: October 2019

Forty-two cases of Hodgkin's disease (HD) and 22 cases of Ki-1-positive anaplastic large cell (Ki-1 + ALC) lymphoma were examined by Southern blotting for the presence of Epstein-Barr virus (EBV) DNA. Seven cases of HD and one case of Ki-1 + ALC lymphoma scored positive with a probe specific for the internal repetitive region of the EBV genome. Subsequently, these viral genomes could be demonstrated to be monoclonal in origin using an EBV terminal region DNA probe. In situ hybridization revealed that in two HD cases, the EBV infected cells had the distinct morphology of Hodgkin and Reed-Sternberg cells, thus suggesting a direct pathoetiological relationship between EBV and some cases of HD.
Molecular analysis of beta zero-thalassemia intermedia in Sardinia
Authors: Galanello R , Dessi E , Melis MA , Addis M , Sanna MA , Rosatelli C , Argiolu F , Giagu N , Turco MP , Cacace E
Published: October 2019

In this study we have carried out alpha- and beta-globin gene analysis and defined the beta-globin gene polymorphisms in a group of patients with thalassemia intermedia of Sardinian descent. A group of patients (109) with thalassemia major of the same origin served as control. Characterization of the beta-thalassemia mutation showed either a frameshift mutation at codon 6 or a codon 39 nonsense mutation. We found that homozygotes for the frameshift mutation at codon 6 or compound heterozygotes for this mutation and for the codon 39 nonsense mutation develop thalassemia intermedia more frequently than thalassemia major. The frameshift mutation at codon 6 was associated with haplotype IX that contains the C-T change at position -158 5′ to the G gamma globin gene implicated in high gamma chain production and thus the mild phenotype. In patients' homozygotes for codon 39 nonsense mutation, those with thalassemia intermedia more frequently had the two- gene deletion form of alpha-thalassemia, or functional loss of the alpha 2 gene as compared with those with thalassemia major. In a few siblings with thalassemia major and intermedia, the thalassemia intermedia syndrome correlated with the presence of the -alpha/-alpha genotype. No cause for the mild phenotype was detected in the majority of patients who had not inherited either haplotype IX or alpha- thalassemia.
Model of reticuloendothelial iron metabolism in humans: abnormal behavior in idiopathic hemochromatosis and in inflammation
Authors: Fillet G , Beguin Y , Baldelli L
Published: October 2019

Iron transport in the reticuloendothelial (RE) system plays a central role in iron metabolism, but its regulation has not been characterized physiologically in vivo in humans. In particular, why serum iron is elevated and RE cells are much less iron-loaded than parenchymal cells in idiopathic hemochromatosis is not known. The processing of erythrocyte iron by the RE system was studied after intravenous (IV) injection of 59Fe heat-damaged RBCs (HDRBCs) and 55Fe transferrin in normal subjects and in patients with iron deficiency, idiopathic hemochromatosis, inflammation, marrow aplasia, or hyperplastic erythropoiesis. Early release of 59Fe by the RE system was calculated from the plasma iron turnover and the 59Fe plasma reappearance curve. Late release was calculated from the ratio of 59Fe/55Fe RBC utilization in 2 weeks. The partitioning of iron between the early (release from heme catabolism) and late (release from RE stores) phases depended on the size of RE iron stores, as illustrated by the inverse relationship observed between early release and plasma ferritin (P less than .001). There was a strong correlation between early release and the rate of change of serum iron levels during the first three hours in normal subjects (r = .85, P less than .001). Inflammation produced a blockade of the early release phase, whereas in idiopathic hemochromatosis early release was considerably increased as compared with subjects with similar iron stores. Based on these results, we describe a model of RE iron metabolism in humans. We conclude that the RE system appears to determine the diurnal fluctuations in serum iron levels through variations in the immediate output of heme iron. In idiopathic hemochromatosis, a defect of the RE cell in withholding iron freed from hemoglobin could be responsible for the high serum iron levels and low RE iron stores.
Abnormalities in the mechanical properties of red blood cells caused by Plasmodium falciparum
Authors: Nash GB , O'Brien E , Gordon-Smith EC , Dormandy JA
Published: October 2019

Although changes in the mechanical properties of infected red cells may contribute to the pathophysiology of malaria, such changes have not previously been described in detail. In this study, the physical properties of individual cells from both clinical and cultured samples infected with Plasmodium falciparum were tested using micropipette aspiration techniques. Cells containing ring forms took about 50% longer to enter 3 microns pipettes compared with nonparasitised cells, and there was a similar increase in the critical pressure required to induce cell entry. These abnormalities were similar in clinical and cultured samples. More mature cultured parasites (ie, trophozoites and schizonts containing pigment) caused much greater loss of deformability, with entry time and pressure increased four to sixfold. The decrease in deformability of the ring forms was attributable to a deficit in cell surface area/volume ratio (based on micropipette measurement of the surface area and volume of individual cells) and slight stiffening of the cell membrane (shear elastic modulus increased 13%, as measured by pipette aspiration of small membrane tongues). Measurement of the rate of cell shape recovery indicated that the membrane of parasitised cells was not more viscous. The main factor in the drastic loss of deformability of the trophozoites and schizonts was the presence of the large very resistant parasite itself. Otherwise, the cell surface area/volume deficit was slightly less and membrane rigidification slightly greater compared with ring forms. The above abnormalities should cause the trophozoites and schizonts to have great difficulty in traversing splenic or marrow sinuses and could contribute to microvascular occlusion and sequestration. On the other hand, the ring forms may be expected to circulate relatively unhindered.
Loss of hematopoietic stem cell self-renewal after bone marrow transplantation
Authors: Mauch P , Hellman S
Published: October 2019

The quality of long-term hematopoietic engraftment after bone marrow transplantation (BMT) has not been well characterized. Clinical autologous BMT involves removal of less than 5% of the total content of the recipient marrow followed by ablation of the remaining marrow and reinfusion. To study long-term consequences of transplanting limited numbers of BM stem cells further, we evaluated the hematopoietic reserve in recipient animals after transplantation of varying quantities of BM. Recipient animals demonstrated a donor BM cell dose- dependent decrease in stem cell content and self-renewal capacity that was not reflected in peripheral blood (PB) counts or BM cellularity. This decrease was observed after initial BM recovery and did not change with time after transplantation, demonstrating a permanent loss in BM self-renewal capacity. In addition, animals alive at 3 months, a time selected to allow BM recovery, also demonstrated a donor BM cell dose- dependent decrease in survival at 1 year. These results emphasize the importance of optimizing stem cell number in BMT.
Inhibition of vascular endothelial cell prostacyclin synthesis by plasmin
Authors: Schafer AI , Rodriguez R , Loscalzo J , Gimbrone MA Jr
Published: October 2019

Vascular endothelial cells (EC) play an active role in the synthesis and assembly of components of the fibrinolytic system and the generation of the major fibrinolytic protease plasmin. However, the reciprocal effects of plasmin on EC function have not been previously examined. We have studied the actions of plasmin on the production of prostacyclin (PGI2) by cultured human umbilical vein (HUVEC) and bovine aortic (BAEC) endothelial cells. Plasmin causes little or no direct stimulation of PGI2 formation by EC. Preincubation of EC with plasmin, however, produces a time- and concentration-dependent inhibition of ionophore A23187-, thrombin-, and histamine-induced PGI2 synthesis; a smaller inhibitory effect on arachidonate- and PGH2-induced PGI2 synthesis is found. Incubation of HUVEC or BAEC with a physiologic concentration of plasminogen (180 micrograms/mL) and recombinant tissue plasminogen activator (tPA) generates tPA dose-dependent plasmin activity that exceeds that generated in the absence of EC. In the presence of plasminogen, tPA also causes a tPA dose-dependent inhibition of thrombin- and ionophore A23187-stimulated PGI2 production. PGI2 inhibitory plasmin activity is generated within the concentration range of tPA achieved in plasma during pharmacologic therapy with tPA. These findings suggest that vascular endothelial cells not only regulate activation of the fibrinolytic system but may also be targets of plasmin action on PGI2 synthesis in the modulation of hemostasis and thrombosis.
Comparison of fractionated to single-dose total body irradiation in conditioning canine littermates for DLA-identical marrow grafts
Authors: Storb R , Raff RF , Appelbaum FR , Graham TC , Schuening FG , Sale G , Pepe M
Published: October 2019

We explored the ability of fractionated total body irradiation (TBI) given at a rate of 7 cGy/min from opposing dual 60Co sources at otherwise lethal doses of 450, 600, 700, 800, and 920 cGy to condition dogs for marrow grafts from DLA-identical littermates. Results were compared with those of a previously reported study using single-dose TBI administered under otherwise identical conditions. Fractionated TBI was less immunosuppressive than single-dose TBI, as evidenced by a significantly higher rate of graft rejection (P = .001). Specifically, sustained allogeneic engraftment was observed in only two of 18 (11%) dogs that received 600 to 800 cGy fractionated TBI as compared with 11 of 17 (65%) dogs that received comparable doses of single-dose TBI. Only at 450 cGy (none of the ten dogs studied had sustained engraftment) and at 920 cGy (four of five dogs that received fractionated and 20 of 21 dogs that received single-dose TBI engrafted) were we unable to find differences between the two modes of radiation. Most dogs that rejected their graft survived with autologous recovery (13 of 22 that received fractionated and eight of 12 that received single-dose TBI; P = .49), presumably the result of extended support provided by the transient allogeneic grafts. We conclude that at equivalent doses fractionated TBI is significantly less effective than single-dose TBI to condition DLA-identical littermate dogs for marrow transplantation. These findings have implications for the design of conditioning programs in clinical transplantation, especially when T- cell-depleted marrow grafts are used.
Autologous transplantation of bone marrow purged in vitro with anti-CD7- (WT1-) ricin A immunotoxin in T-cell lymphoblastic leukemia and lymphoma
Authors: Preijers FW , De Witte T , Wessels JM , De Gast GC , Van Leeuwen E , Capel PJ , Haanen C
Published: October 2019

Seven patients with high-risk acute T-cell lymphoblastic leukemia (T- ALL) and six with T cell lymphoma (T-LL) were treated with autologous bone marrow transplantation (ABMT) after in vitro purging of their bone marrow with WT1 (CD7)-ricin A-chain immunotoxin. CD7 expression on the tumor cells showed large variations between the individual patients and was highly related to the specific cytotoxicity of WT1-ricin A. Incubation of bone marrow with up to 10(-8)mol/L WT1-ricin A in the presence of 6 mmol/L NH4Cl did not compromise the growth potential of the hematopoietic progenitors CFU-GM, CFU-GEMM, and BFU-E. Hematologic engraftment (greater than 10(9) leukocytes/L) occurred within a normal time period (median, 17 days). Seven patients are alive and in complete remission (CR) at 48+, 44+, 40+, 26+, 11+, 7+, and 6+ months after ABMT. Four patients relapsed within 6 months after ABMT. Two of them had the lowest CD7 expression on their tumor cells, the other two were transplanted in CR2 and CR3. Two patients died from transplantation related infections. The immunologic reconstitution was delayed, although the numbers of T cells reached normal levels within 1 month. The number of CD7+ cells remained low up to 1 year after transplantation. The T4/T8-ratio was decreased for at least 6 months. The T-cell response to mitogens recovered to normal levels after 1 year. This study shows that ABMT with WT1-ricin A purged bone marrow in high-risk T-cell malignancies results in a complete hematopoietic and a delayed immunologic reconstitution. The actuarial relapse free survival is 61% at 3 years.
Interleukin-4 stimulates human monocytes to produce tissue-type plasminogen activator
Authors: Hart PH , Burgess DR , Vitti GF , Hamilton JA
Published: October 2019

Tissue-type plasminogen activator (t-PA) is involved in the lysis of blood clots (fibrinolysis) and is used clinically for this purpose. Endothelial cells are one source of the t-PA present in blood. We report here that interleukin-4 (IL-4) (0.1 to 0.25 U/mL; 1 to 3 x 10(- 11) mol/L), but not interferon-gamma (IFN-gamma), elevates t-PA messenger (m)RNA expression and secretion of t-PA activity by human monocytes, with the maximum response at 2.5 U/mL. Supernatant t-PA activity was detected within three hours of exposure to IL-4 and maximum activity within six hours. Thus, IL-4 may control fibrin deposition at sites of inflammation during cell-mediated immune responses, as well as having a therapeutic role in thrombolysis.