bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes
Authors: Turley EA , Belch AJ , Poppema S , Pilarski LM
Published: October 2019

Migration through extracellular matrix is fundamental to malignant invasion. A receptor for hyaluronan-mediated motility (RHAMM) has previously been shown to play a fundamental role in locomotion of ras- transformed cells as well as functioning in signal transduction. Expression of RHAMM was characterized on B lymphocytes from normal and malignant lymphoid tissues using multiparameter phenotypic immunofluorescence analysis as well as functional analysis of its role in locomotion of malignant hairy cell leukemia B cells. RHAMM is not detectable on most normal B cells located in blood, spleen, or lymph node, but it is detectable on bone marrow and thymic B cells. Among B- cell malignancies, it is expressed on most terminally differentiated B cells from multiple myeloma bone marrows, is present on a subset of non- Hodgkin's lymphomas, and is absent on B chronic lymphocytic leukemia. Activation of peripheral blood B cells by Staphylococcus A cowan (SAC), but not by pokeweed mitogen, induced transient expression of RHAMM at day 3 of culture, suggesting RHAMM may be used by antigen-activated normal B cells. For malignant cells, expression of RHAMM increased on long-term culture of bone marrow plasma cells from multiple myeloma patients, indicating prolonged expression in contrast to the transient expression on SAC-activated normal B cells. Intriguingly, RHAMM was expressed on hairy leukemia cells located in spleen but absent from those in peripheral blood of the same patient. RHAMM, as expressed on splenic hairy cells, was a 58-Kd molecule that binds hyaluronan, is encoded by a 5.2-kb messenger RNA, and participates in locomotion by these cells. Hairy cells locomoted in response to hyaluronan at 4 mu per minute. Monoclonal antibody to RHAMM inhibited this locomotion almost completely as detected using video time-lapse cinemicrography. These observations are consistent with a role for RHAMM in malignant invasion and metastatic growth.
Subtypes of Epstein-Barr virus (EBV) in Hodgkin's disease: association between B-type EBV and immunocompromise [see comments]
Authors: Boyle MJ , Vasak E , Tschuchnigg M , Turner JJ , Sculley T , Penny R , Cooper DA , Tindall B , Sewell WA
Published: October 2019

Epstein-Barr virus (EBV) has been associated with Hodgkin‧s disease (HD) in up to 50% of cases, but the subtype of EBV involved has only recently been studied. In this report, biopsy samples from 30 patients with HD were assessed for EBV sequences using both the polymerase chain reaction (PCR) and in situ hybridization (ISH). EBV sequences were localized to the malignant Reed-Sternberg cells and their mononuclear variants (Hodgkin's cells) in 9 of the 30 cases, with 7 demonstrating A- type and 2 B-type EBV sequences. Both of the patients with B-type EBV- associated HD had features to suggest pre-existing immune compromise: one was infected with human immunodeficiency virus (HIV) and had severe CD4+ T-lymphocyte depletion; the other was a debilitated elderly patient with dementia. A previous study suggested that A-type EBV alone is associated with HD and the finding of predominantly A-type EBV in the present series is in keeping with this report. The presence of B- type EBV in the HD of patients with pre-existing immunodeficiency, taken together with the recent report that B-type EBV occurs in HIV- associated non-Hodgkin‧s lymphoma, suggests that B-type EBV may be an important human pathogen in immunocompromised patients.
P-glycoprotein expression in human plasma cell myeloma: correlation with prior chemotherapy
Authors: Grogan TM , Spier CM , Salmon SE , Matzner M , Rybski J , Weinstein RS , Scheper RJ , Dalton WS
Published: October 2019

Multidrug-resistant (MDR) myeloma patients failing chemotherapy may express P-glycoprotein (PGP), which serves as an efflux pump protecting the neoplastic cells. Unknown is whether PGP expression might relate to prior cytotoxic drug exposure. To address this question, we studied 106 consecutive bone marrow samples from 104 myeloma patients with samples studied either before or after therapy and at the time of relapse. We performed an established immunocytochemical assay of PGP using an MDR-1- specific monoclonal antibody and correlated PGP status with prior chemotherapy dosage. Myeloma patients with no prior therapy had a low incidence of PGP expression (6%, 3/47), whereas those receiving chemotherapy had a significantly higher incidence (43%, 21/49) (P < .0001). A substantially higher incidence of PGP expression (50%, 83%, respectively) occurred when the total vincristine dose exceeded 20 mg and when doxorubicin exceeded 340 mg. In the 11 patients who received both high vincristine and doxorubicin dosages (> 20 mg, > 340 mg total dose) there was 100% incidence of PGP expression in the tumor cells. These data provided the basis for a predictive mathematical model from which dose-related PGP expression normograms were generated. Time with myeloma for PGP-negative patients (mean 33 months) had overlapping confidence limits with PGP-positive patients (mean 42 months), suggesting that disease duration was not a significant variable. PGP expression did not correlate with other clinical factors or immunophenotypic factors. Our findings indicate a strong correlation between PGP expression in myeloma and past chemotherapy in myeloma, in particular, related to prior exposure to the natural product agents vincristine and doxorubicin. Additionally, the proportion of PGP- positive plasma cells was significantly higher in the doxorubicin- treated patients than the nondoxorubicin-treated patients (87.7% v 65.17%; P = .013). Combined high vincristine and doxorubicin total dosage appear highly predictive of PGP expression.
Downregulation of the anti-HLA alloimmune response by variable region- reactive (anti-idiotypic) antibodies in leukemic patients transfused with platelet concentrates
Authors: Atlas E , Freedman J , Blanchette V , Kazatchkine MD , Semple JW
Published: October 2019

Approximately 30% to 40% of patients with acute leukemia receiving repeated pooled random-donor platelet transfusions develop anti-HLA alloantibodies. Over time, however, serum anti-HLA concentrations decrease in approximately 50% of these patients, despite continued exposure to platelet and/or red blood cell transfusions. Using an enzyme-linked immunosorbent assay to measure serum Igs, the present study demonstrates that the sera of 67% of 82 transfused patients exhibiting a decrease in anti-HLA contain antibodies (anti-idiotypes) that react with the variable (V) region of anti-HLA antibodies. Anti- HLA binding to platelet membranes could be inhibited by these serum antibodies in 36% of the patients, indicating they had paratope-related reactivity. Protein G sepharose absorption showed that the anti-HLA V region-reactive antibodies were IgG. Of the 43 patients who had a decrease in anti-HLA levels, that were 16 whose anti-HLA decreased to undetectable levels; 7 (44%) developed anti-idiotypic antibodies that could specifically inhibit their own previously anti-HLA-positive serum. In contrast, antibodies with reactivity to the V region of anti- HLA antibodies (anti-idiotypes) were not demonstrable in patients who developed anti-HLA that did not decrease or disappear. The findings suggest that the development of anti-HLA V region-reactive antibodies (anti-idiotypic antibodies) correlates with a decrease in anti-HLA antibody formation in patients multiply transfused with platelet concentrates. The observations indicate that anti-idiotypic antibodies may downregulate alloimmune responses in patients undergoing repeated allostimulation during platelet transfusion therapy.
Unrelated donor marrow transplantation therapy for chronic myelogenous leukemia: initial experience of the National Marrow Donor Program
Authors: McGlave P , Bartsch G , Anasetti C , Ash R , Beatty P , Gajewski J , Kernan NA
Published: October 2019

In the interval from December 1987 to November 1990, 196 consecutive patients with chronic myelogenous leukemia (CML) received unrelated donor marrow transplantation using marrow procured by the National Marrow Donor Program (NMDP) at 21 NMDP-affiliated marrow transplant centers. Baseline donor and recipient data as well as follow-up data were obtained systematically in all cases by the NMDP. The median interval from the initiation of a search for an unrelated donor to bone marrow transplantation was 8.4 months (range, 1.7 to 34.6 months). Median age of the recipients was 33.3 years (4.5 to 54.5 years). Seventy-five recipients were female and 121 were male. At time of transplant, 115 patients were in chronic phase, 51 in accelerated phase, 14 in blast crisis, and 16 in a second or subsequent chronic phase. In 133 cases, donors and recipients were identical at the HLA A, B, and DR loci using standard serologic typing, and in 63 cases, there was nonidentity at one HLA locus. Patients were prepared for transplantation with a combination of high-dose chemotherapy and total body irradiation (N = 169) or with high-dose chemotherapy only (N = 27). Thirty-five patients received marrow depleted ex vivo of T lymphocytes, whereas 161 patients received non-T-depleted marrow. One hundred seventy-four of 196 patients engrafted (absolute neutrophil count > or = 500/mm3 for 3 consecutive days). The median time to engraftment was 22 days (6 to 69 days). Twenty-two patients failed to engraft, and an additional 10 patients experienced late graft failure. The incidence of grades III or IV acute graft-versus-host disease (GVHD) was 0.54 +/- 0.10, and that of extensive chronic GVHD was 0.52 +/- 0.12. A lower incidence of both grades III and IV acute GVHD (P = .0003) and of extensive chronic GVHD (P = .01) were independently associated with use of T-depleted marrow. The actuarial incidence of hematologic relapse at 2 years is 0.11 +/- 0.06. The 2-year actuarial incidence of disease-free survival for patients transplanted in first chronic phase within 1 year of diagnosis is 0.45 +/- 0.21, in chronic phase more than 1 year from diagnosis is 0.36 +/- 0.11, in accelerated phase is 0.27 +/- 0.12, in second or subsequent chronic phase is 0.22 +/- 0.21, and in blast crisis is 0. Fifteen of 55 patients transplanted at 40 to 50 years of age survive.(ABSTRACT TRUNCATED AT 400 WORDS)
Translocation breakpoints are clustered on both chromosome 8 and chromosome 21 in the t(8;21) of acute myeloid leukemia
Authors: Tighe JE , Daga A , Calabi F
Published: October 2019

The t(8;21)(q22;q22) is consistently associated with acute myeloid leukemia (AML) M2. Recent data have suggested that breakpoints on chromosome 21 are clustered within a single intron of a novel gene, AML1, just downstream of a region of homology to the runt gene of D melanogaster. In this report, we confirm rearrangement at the same location in at least 12 of 18 patients with t(8;21). Furthermore, we have isolated recombinant clones spanning the breakpoint regions on both the der(8) and the der(21) from one patient. By using a chromosome 8 probe derived from these clones, we show that t(8;21) breakpoints are also clustered on chromosome 8.
Relationship of deoxycytidine kinase and cytoplasmic 5'-nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine
Authors: Kawasaki H , Carrera CJ , Piro LD , Saven A , Kipps TJ , Carson DA
Published: October 2019

The agent 2-chlorodeoxyadenosine (2-CdA) has chemotherapeutic activity in hairy cell leukemia (HCL) and in refractory chronic lymphocytic leukemia (CLL). The cytotoxic activity of 2-CdA requires the intracellular accumulation of 2-CdA nucleotides. Deoxycytidine kinase (dCK) and cytoplasmic 5′-nucleotidase (5′-NT) are the principal enzymes that phosphorylate 2-CdA and dephosphorylate 2-CdA 5′-monophosphate, respectively. The net accumulation of 2-CdA nucleotides may therefore depend on both dCK and 5′-NT. The purpose of the present experiments was to determine if there is a relationship between pretreatment levels of dCK and 5′-NT in HCL and in CLL cells, and the clinical outcome of 2- CdA treatment. As measured by a direct immunoassay for dCK in 25 CLL patients, and by a 5′-NT activity assay in 23 patients, mean dCK levels were significantly higher in 2-CdA responders than in nonresponders (P < .01), whereas mean 5′-NT levels were significantly lower in 2-CdA responders than in nonresponders (P < .05). Mean dCK levels were higher in six HCL 2-CdA responders than in one nonresponder, whereas mean 5′- NT levels were lower in the 2-CdA responders than in the nonresponder. These results suggest that both dCK and 5′-NT are determinants of 2-CdA responsiveness.
Growth factor requirement for survival in cell-cycle dormancy of primitive murine lymphohematopoietic progenitors
Authors: Katayama N , Clark SC , Ogawa M
Published: October 2019

We used enriched marrow cells from mice administered three doses of 150 mg/kg 5-fluorouracil (5-FU) 1, 3 and 7 days before they were killed to study the effects of different growth factors on the survival of primitive, cell-cycle dormant progenitors in culture. This cell population yielded substantially fewer colonies in response to single growth factors than corresponding preparations from day 2 post-5-FU bone marrow samples, and the majority of progenitors were multipotential in nature. These observations were consistent with the prediction that multiple cycles of 5-FU treatment would further enrich for primitive cells. With this cell population, we found that among all the factors tested, interleukin-3 (IL-3) and steel factor (SF) as single factors are the most effective in supporting survival of dormant primitive progenitors. Interleukin-6 (IL-6), granulocyte colony- stimulating factor (G-CSF), interleukin-11 (IL-11), interleukin-4 (IL- 4), interleukin-1 alpha (IL-1 alpha), and tumor necrosis factor-alpha (TNF-alpha) also supported survival of a few progenitors, but much less effectively than either IL-3 or SF. The hematopoietic progenitors that survived for 1 week in liquid culture supplemented with either IL-3 or SF retained the capability to develop pre-B-cell colonies in secondary culture. Our results demonstrate that survival of dormant murine lymphohematopoietic cells in culture is dependent on the presence of specific growth factors, and that this growth factor requirement can be satisfied well by SF or IL-3.
A role for manganese superoxide dismutase in radioprotection of hematopoietic stem cells by interleukin-1
Authors: Eastgate J , Moreb J , Nick HS , Suzuki K , Taniguchi N , Zucali JR
Published: October 2019

Pretreatment with interleukin-1 (IL-1) has been shown to protect mice from the myelotoxicity associated with irradiation via a mechanism potentially mediated through the induction of the antioxidant enzyme manganese superoxide dismutase (MnSOD). In this study, we have compared the ability of IL-1 to induce MnSOD mRNA in murine bone marrow cells and human cell lines with its ability to protect these cells against the damaging effects of ionizing radiation. Bone marrow cells obtained from mice 6 hours after a single injection of IL-1 demonstrate a dose- dependent increase in the expression of MnSOD RNA. In this same study, IL-1 was also shown to be radioprotective when given to mice 20 hours before lethal irradiation. Similarly, in vitro treatment with IL-1 of bone marrow cells isolated from 5-fluorouracil-treated mice results in elevated levels of MnSOD RNA. Pretreatment with IL-1 also protected bone marrow long-term culture-initiating cells capable of reconstituting irradiated stromal cultures from an irradiation insult. Furthermore, IL-1-treated human bone marrow cells display both elevated MnSOD RNA and protein levels when compared with media controls. The human A375 melanoma, A549 adenocarcinoma, and factor-dependent TF-1 leukemic cell lines demonstrate low basal MnSOD RNA levels that increase following treatment with IL-1. For the A375 cells, this correlates with increased MnSOD protein expression and radioprotection by IL-1 using a colony assay. In contrast, the chronic myelogenous leukemic cell line, K562, displays a high basal MnSOD RNA level, and this RNA expression is not further increased by IL-1 treatment. In addition, these cells are comparatively radioresistant and are not further protected by IL-1 treatment. Finally, the Mo-7 cell line displays a low basal level of MnSOD RNA that correlates with a high sensitivity to irradiation and IL-1 pretreatment has no effect on MnSOD RNA levels. Our results indicate that increased radioprotection by IL-1 correlates with the induction of the antioxidant enzyme MnSOD and this induction may be an important factor in IL-1 radioprotection.
Soluble stem cell factor in human serum
Authors: Langley KE , Bennett LG , Wypych J , Yancik SA , Liu XD , Westcott KR , Chang DG , Smith KA , Zsebo KM
Published: October 2019

Stem cell factor (SCF) is a recently described factor active in the early stages of hematopoiesis. It can exist in membrane-bound form and in proteolytically released soluble form. The levels and nature of SCF in human serum are described. As determined by an enzyme-linked immunosorbent assay performed for 257 samples, SCF level in serum averaged 3.3 +/- 1.1 ng/mL. The serum SCF was partially purified by immunoaffinity chromatography and analyzed by glycosidase treatments in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the SCF has N- linked and O-linked carbohydrate and corresponds to the soluble form, at or about 165 amino acids in length. The findings suggest functional importance for soluble SCF in humans.
Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro
Authors: Srour EF , Brandt JE , Briddell RA , Grigsby S , Leemhuis T , Hoffman R
Published: October 2019

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.
Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa binding proteins on the HepG2 cell surface
Authors: Kazama Y , Komiyama Y , Kisiel W
Published: October 2019

Previous studies indicated that human factor Xa bound to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule. Factor Xa binding to this cell line was not measurably affected by pretreatment of the cells with anti- factor V IgG and to a large extent (approximately 70%) was calcium- independent, suggesting the presence of cell-surface binding proteins specific for factor Xa other than factor V/Va. In the present study, we have further characterized the interaction of factor Xa with the HepG2 cell and performed chemical cross-linking and immunoprecipitation studies to determine the identity of the HepG2 surface protein(s) interacting with factor Xa. Initial studies demonstrated that HepG2- bound 125I-factor Xa was not significantly displaced by unlabeled factor Xa blocked at the active site with dansyl-L-glutamyl-glycyl-L- arginine (DEGR)-chloromethyl ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no 125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an intact factor Xa active site was a prerequisite for binding. 125I-factor Xa binding to HepG2 cells was inhibited approximately 70% by pretreatment of the cells with anti- tissue factor pathway inhibitor (TFPI) IgG in the presence or absence of calcium ions, but was without effect on the expression of prothrombinase activity. Immunoprecipitation of 125I-factor Xa chemically cross-linked to its cell-surface binding protein with anti- factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a complex with an apparent molecular weight of 96,000. An identical molecular weight complex was observed following immunoprecipitation of this radiolabeled complex with anti- TFPI IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1) as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These results suggest that approximately 1% to 2% of the factor Xa interacts with HepG2 cell-surface factor V/Va to form a productive prothrombinase complex, while the remaining factor Xa forms a non-productive complex with either TFPI or PN-1.
Increased thrombin generation in a child with a combined factor IX and protein C deficiency
Authors: Negrier C , Berruyer M , Durin A , Philippe N , Dechavanne M
Published: October 2019

We report a quantitative protein C deficiency combined with a factor IX deficiency in a one-year-old boy. The inheritance of the two deficiency states was independent, the factor IX defect coming from the mother and the protein C defect from the father. Both factor IX activity and antigen were below 1%, and protein C activity as well as antigen were close to 27% of normal values. This association raises a real therapeutic and prognostic question. Protein C deficiency is indeed associated with a significant thrombotic risk and some factor IX concentrates seem to carry a potential thrombogenicity, particularly following infusion of repeated doses. We evaluated in this patient the potential activation of the coagulation system by measuring the levels of prothrombin fragment F1 + 2 at the basal state and after a single administration of 20 U/kg of a high purity factor IX concentrate. We found an unexpected basal activation of the hemostatic system before infusion (F1 + 2 = 1.6 nmol/L), which further increased during 8 hours. Despite the clinical predominant expression of the hemophilic trait, our results seem to assess the biologic prevalence of the protein C deficiency. This emphasizes the need for a careful follow-up after infusions of repeated doses of factor IX, as used during a surgical procedure. Furthermore, this raises the question of the prognosis because the risk of thrombotic manifestations associated with a protein C deficiency increases with age. Finally, these results highlight a part of the in vivo activation process of prothrombin in case of failure of the intrinsic pathway of coagulation. The protein C defect seems to be responsible for an upregulation of the prothrombin activation through the extrinsic pathway.
Phosphorylation of factor Va and factor VIIIa by activated platelets
Authors: Kalafatis M , Rand MD , Jenny RJ , Ehrlich YH , Mann KG
Published: October 2019

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II- like kinase may downregulate the activity of the two cofactors.
Degradation of von Willebrand factor in patients with acquired clinical conditions in which there is heightened proteolysis
Authors: Federici AB , Berkowitz SD , Lattuada A , Mannucci PM
Published: October 2019

The behavior of plasma von Willebrand factor (vWF) in patients with acute leukemia (n = 5), decompensated cirrhosis (n = 10), and acute pancreatitis (n = 5) was investigated to evaluate whether the systemic proteolytic states associated with these diseases had affected the structure and function of the molecule. vWF antigen and, to a lesser degree, ristocetin cofactor activity in patient plasma were high. Multimeric analysis of plasma vWF revealed loss of high molecular weight multimers. The subunit composition and proteolytic pattern of vWF immunopurified from patient plasmas and reduced were studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by transblotting and probing with monoclonal antibodies that distinguish cleavages caused by plasmin from those caused by other proteases. There was marked reduction of the relative concentration of the native vWF subunit of 225 Kd in all patient groups, indicating heightened cleavage of the protein. The concentrations of 189- and 140- Kd vWF fragments, normally present in plasma, were increased in cirrhosis and pancreatitis but not in leukemia. Novel fragments, ranging in size from less than 225 to approximately 120 Kd were present in leukemia and cirrhosis, including plasmin-generated fragments of 176 and 145 Kd. These data indicate that in clinical conditions in which there is heightened proteolysis vWF is degraded in vivo by plasmin and other proteases. Degraded vWF may be less effective than native vWF in supporting primary hemostasis, thereby being a cofactor in the multifactorial bleeding diathesis accompanying systemic proteolytic states.
Immunoglobulin heavy chain gene VH-D junctional diversity at diagnosis in patients with acute lymphoblastic leukemia
Authors: Kitchingman GR
Published: October 2019

Acute lymphoblastic leukemia (ALL) represents the clonal outgrowth of transformed hematopoietic progenitor cells. We have found that blast cells in some cases of B-precursor cell ALL contain Ig heavy chain gene rearrangements with considerable diversity at the junctions of the variable (VH), diversity (D), and joining (JH) regions. This diversity consists of heterogeneous nucleotide sequences at the VH-D and, less frequently, the D-JH junctions. In two cases, different VH segments were attached to the same D-JH rearrangement. In all cases studied there was a much higher than expected frequency of nucleotide sequence changes in the VH segment. At least three mechanisms may produce these changes in different cases: (1) continuing rearrangement of the heavy chain gene, in some cases by VH addition to a preexisting D-JH; (2) VH replacement; and (3) an open-and-shut mechanism. These findings suggest that an active VDJ recombinase system is present at the time of transformation in a high percentage of ALLs. An active recombinase in the rapidly growing leukemic cell population could lead to genomic instability.
Calcium influx in induced differentiation of murine erythroleukemia cells
Authors: Gillo B , Ma YS , Marks AR
Published: October 2019

Murine erythroleukemia cells (MELC) have served as a model for examining the regulation of erythroid differentiation. However, the role of Ca2+ in the signal transduction pathways regulating differentiation remains unclear. To begin to address this uncertainty we have characterized the regulation of cytoplasmic Ca2+ and the possible role of calcium channels during induced differentiation in MELC. MELC can be induced to terminal differentiation using the polar/apolar compound hexamethylene bisacetamide (HMBA). We found that HMBA stimulated Ca2+ influx within 3 to 6 minutes and that Ca2+ entry was required but not sufficient for MELC growth and differentiation. Nifedipine (1 to 10 mumol/L), a calcium channel antagonist, blocked HMBA-induced Ca2+ influx and inhibited differentiation by approximately 60%. Depolarization of the MELC membrane did not induce Ca2+ influx and whole-cell patch-clamp recordings failed to detect a voltage-activated Ca2+ current, suggesting that MELC do not express detectable levels of a functional voltage-dependent calcium channel (VDCC). However, a cDNA probe encoding a portion of the alpha 1 subunit of the cardiac VDCC detected an approximately 8-kb mRNA on Northern blots of total MELC RNA. Taken together, these data show that Ca2+ influx is an early event associated with HMBA-induced differentiation in MELC, blockade of this calcium influx inhibits induced differentiation, and a voltage- insensitive dihydropyridine-sensitive calcium channel may be involved in Ca2+ influx in MELC.
Acute graft-versus-host disease prophylaxis with methotrexate and cyclosporine after busulfan and cyclophosphamide in patients with hematologic malignancies
Authors: von Bueltzingsloewen A , Belanger R , Perreault C , Bonny Y , Roy DC , Lalonde Y , Boileau J , Kassis J , Lavallee R , Lacombe M
Published: October 2019

The combination of two powerful immunosuppressive agents, methotrexate (MTX) and cyclosporine (CSP), has resulted in a significant decrease in the morbidity and mortality after allogeneic bone marrow transplantation (BMT). However, the additive toxicities from ablative preparative regimens may lead to suboptimal use of this combined immunoprophylaxis. We evaluated the efficacy and feasibility of administering MTX/CSP with busulfan (4 mg/kg/d for 4 days) and cyclophosphamide (50 mg/kg/d for 4 days) (BuCy4) in 101 consecutive patients with hematologic malignancies categorized into high- and low- risk groups receiving HLA-matched marrow grafts. Postgrafting immunosuppression consisted of MTX short course (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11) and intravenous CSP (1.5 mg/kg every 12 hours). Eighty-three patients (82.1%) received 100% of MTX calculated dose and 87 (86.1%) achieved a CSP therapeutic level (250 to 600 ng/mL) within a median of 16 days. Seventy-three patients (72.2%) received optimal immunosuppressive therapy comprising a full MTX course and achieving CSP therapeutic concentrations. The Kaplan-Meier estimated incidence of grade II to IV acute graft-versus-host disease (GVHD) was 9.2% for all patients and 5.5% in patients receiving optimal GVHD prophylaxis. Eighty-nine patients (88.2%) survived > or = 100 days posttransplant and 43 (48.3%) developed chronic GVHD, the majority of which were de novo (31 of 43). The estimated incidence of relapse was 28.9% for all patients and 14.8% in the low-risk group, with a median follow-up of 24.5 months. High-risk features and the absence of chronic GVHD were significantly associated with relapse (P = .002 and .036, respectively) in multivariate analyses. Projected disease-free survival at 2 years was 52.3% for all patients and 65.2% in low-risk patients. Disease-free survival was significantly improved in optimally treated patients (P = .03) due to a lower incidence of early deaths from acute GVHD and infectious episodes. In conclusion, optimal delivery of MTX/CSP in association with BuCy4 resulted in a near complete abrogation of acute GVHD in HLA-matched transplants and a significantly improved disease-free survival.
Double-negative (CD4- CD8-) T cells from adult T-cell leukemia patients also have poor expression of the T-cell receptor alpha beta/CD3 complex
Authors: Suzushima H , Asou N , Nishimura S , Nishikawa K , Wang JX , Okubo T , Naito M , Hattori T , Takatsuki K
Published: October 2019

We present four patients with adult T-cell leukemia (ATL) derived from a novel T-cell subset (CD4-, CD8- [double-negative, DN], T-cell receptor [TCR] alpha beta+). In the ATL cells of these patients, neither gene nor surface expression of CD4 and CD8 antigens was detected. Clinical and laboratory data showed no difference between DN- ATL and CD4+ATL patients. In contrast to typical CD4+ATL cells, DN-ATL cells were shown to express the protein and messenger RNA (mRNA) for S100 beta in immunocytochemical assay and the reverse-transcription polymerase chain reaction assay. The mean fluorescence intensity of the TCR/CD3 complex was extremely low in all four DN-ATL patients as well as in typical CD4+ ATL. All four patients had TCR beta and gamma chain gene rearrangements, with deletion of TCR delta chain gene and mRNA expression for TCR alpha, beta, and CD3 delta but not for TCR gamma and delta chain genes. Thus, CD4- CD8- TCR alpha beta T cells are also a target for human T-cell lymphotrophic virus type I-induced leukemogenesis. In addition, expression of the TCR alpha beta/CD3 complex on the DN-ATL cells was further diminished by the addition of anti-CD3 or anti-TCR alpha beta monoclonal antibody. These results suggest that the decreased expression of the TCR alpha beta/CD3 complex by ATL cells plays a key role in the development of ATL, irrespective of CD4 expression.
Long persistent bcr-abl positive transcript detected by polymerase chain reaction after marrow transplant for chronic myelogenous leukemia without clinical relapse: a study of 64 patients
Authors: Miyamura K , Tahara T , Tanimoto M , Morishita Y , Kawashima K , Morishima Y , Saito H , Tsuzuki S , Takeyama K , Kodera Y
Published: October 2019

We report here the results of polymerase chain reaction (PCR) for bcr- abl transcript and clinical details derived from 64 chronic myelogenous leukemia (CML) patients after allogeneic bone marrow transplantation (BMT). A total of 139 samples (2 to 220 weeks after BMT) were analyzed and bcr-abl transcript was detected in 99 samples from 52 patients. Patients were defined as bcr-abl early negative (EN) if they had > or = 1 negative PCR result < or = 1 year post-BMT (n = 13), and bcr-abl late positive (LP) if they had > or = 1 positive PCR result > or = 1 year post-BMT (n = 21). Among LP patients, only two patients had hematologic/cytogenetic (clinical) relapse. Another 19 LP patients remained in clinical remission 7 to 130 weeks after positive analysis for bcr-abl transcript, including 5 patients who had persistent bcr-abl transcript detectable even 2 years after BMT. To estimate the relationship between clinical data and residual bcr-abl transcript, EN patients are compared with LP patients. However, no clinical data studied were significantly associated with the persistent PCR positivity. If only patients in chronic phase are compared, the t-test showed significant correlation between leukocyte count just before BMT and sustained bcr-abl transcript (P < .05). These results suggest that PCR positivity is frequently observed in CML patients who sustain clinical remission after BMT, without being predictive of imminent clinical relapse. Tumor burden at the time of BMT may play an important role in the latency of bcr-abl positivity after BMT.
Interleukin-3 expression by activated T cells involves an inducible, T- cell-specific factor and an octamer binding protein
Authors: Davies K , TePas EC , Nathan DG , Mathey-Prevot B
Published: October 2019

Interleukin-3 (IL-3) is exclusively expressed by activated T and natural killer cells, a function that is tightly controlled both in a lineage-specific and in a stimulation-dependent manner. We have investigated the protein binding characteristics and functional importance of the ACT-1-activating region of the IL-3 promoter. This region binds an inducible, T-cell-specific factor over its 5′ end, a site that is necessary for the expression of IL-3 in the absence of other upstream elements. Over its 3′ end, it binds a factor that is ubiquitously and constitutively expressed. This factor is Oct-1 or an immunologically related octamer-binding protein, and it plays a role in coordinating the activity of several regulatory elements. These characteristics make the ACT-1 site analogous to the activating ARRE-1 site in the IL-2 promoter. Furthermore, and despite a lack of sequence homology, the promoters of IL-3 and IL-2 share an organizational pattern of regulatory elements that is likely to be important for the T- cell-specific expression of these genes.
Erythropoietin-specific cell cycle progression in erythroid subclones of the interleukin-3-dependent cell line 32D [published erratum appears in Blood 1993 Jun 1;81(11):3168]
Authors: Shimada Y , Migliaccio G , Ralph H , Migliaccio AR , Shaw H$[corrected to Ralph H]
Published: October 2019

Recently, a variety of growth factor-dependent subclones of the murine interleukin-3 (IL-3)-dependent cell line 32D have been isolated. These subclones include those dependent for growth on erythropoietin (Epo) (32D Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) (32D GM), or granulocyte colony-stimulating factor (G-CSF) (32D G). 32D Epo1.1 is a revertant of 32D Epo and is capable of growing in IL-3. These cell lines express the differentiation program appropriate to the specific growth factor and depend on the growth factors not only for proliferation but also for survival. To determine how the signal for proliferation is triggered by various growth factors, we examined the DNA histograms and the expression of cell cycle-specific genes in the different cell lines. The cell cycle-specific genes analyzed were myc (early G1), myb (late G1), and the structural genes for the calcium- binding protein 2A9 (middle G1) and histone H3 (G1-S boundary). The DNA histogram analysis of cells in the logarithmic phase of growth showed that approximately 40% of 32D, 32D GM, 32D G, and 32D Epo1.1 (growing in IL-3) were cells with a 2N DNA content (and therefore in G0/G1), and another 40% have a DNA content intermediate between 2N and 4N (in S phase). In contrast, 32D Epo and 32D Epo1.1 (growing in Epo) had fewer cells in the G0/G1 phase of the cell cycle compared with the number of cells that were in the S phase (19% to 31% v 69% to 78%, respectively). Because all the cell lines have comparable doubling times (15 to 18 hours), the cell distribution among the phases of the cell cycle is proportional to the length of the phase. Therefore, cells growing in IL- 3 (32D and 32D Epo1.1), GM-CSF (32D GM), or G-CSF (32D G) progress along the cycle in a manner typical of previously reported nontransformed cell lines. In contrast, cells growing in Epo (32D Epo or 32D Epo1.1) spend relatively less time in G0/G1 and correspondingly more time in S. These data were confirmed by the analysis of the tritiated thymidine (3H-TdR) suicide rate and of the expression of cell cycle-specific genes. The 32D and 32D Epo1.1 cells growing in IL-3 had a suicide rate of congruent to 50%, whereas the suicide rate of 32D Epo and 32D Epo1.1 growing in Epo was higher than 75%.(ABSTRACT TRUNCATED AT 400 WORDS)
T- and B-lymphocyte differentiation potentials of spleen colony-forming cells
Authors: Lepault F , Ezine S , Gagnerault MC
Published: October 2019

Cells that generate splenic colonies within 8 days (day-8 colony- forming units-spleen [CFU-s]) are generally thought to differentiate only into erythroid/myeloid cells. The T and B lymphocyte differentiation potentials of day-8 CFU-s were evaluated and compared with those of day-12 and 5-fluorouracil (5-FU) CFU-s. This was achieved by analyzing, after intravenous and intrathymic injection, the lymphocyte progeny of cells contained within individual splenic colonies collected at day 8 and day 12 post-bone marrow cell transfer into irradiated congenic recipients. A large majority of day-8 spleen colonies generated T cells when transferred intrathymically. After intravenous (IV) injection of day-8 colonies, donor-type thymocytes emerged in 33% of the animals reconstituted with only 1 day-8 colony, but in 83% of those inoculated with a pool of 5 colonies. All post-5-FU and 75% of day-12 colonies gave rise to thymocytes after IV injection. B cells were generated by a high proportion of day-8 colonies, and by all day-12 and post 5-FU colonies. These results demonstrate that progenitors of T and B lymphocytes are generated within spleen colonies produced by at least some day-8 CFU-s and virtually all day-12 CFU-s. Whether these progenitors are CFU-s themselves or committed precursors remains an open question.
In vivo effects of recombinant interleukin-11 on myelopoiesis in mice
Authors: Hangoc G , Yin T , Cooper S , Schendel P , Yang YC , Broxmeyer HE
Published: October 2019

Purified recombinant human interleukin-11 (rhuIL-11) was assessed for its in vivo effects on the proliferation and differentiation of hematopoietic progenitors as well as its capacity to accelerate the recovery of a drug-suppressed hematopoietic system. Dosage and time sequence studies demonstrated that administration of IL-11 to normal mice resulted in increases in absolute numbers of femoral marrow and splenic myeloid (granulocyte-macrophage colony-forming unit [CFU-GM], burst-forming unit-erythroid [BFU-E], CFU-granulocyte, erythroid, macrophage, megakaryocyte) progenitor cells and in stimulation of these progenitors to a higher cell cycling rate. This was associated with increased numbers of circulating neutrophils. Administration of IL-11 to mice pretreated with cyclophosphamide decreased the time required to regain normal levels of neutrophil and platelet counts in peripheral blood. In addition, IL-11 accelerated reconstitution to normal range of myeloid progenitors from bone marrow and spleen of myelosuppressed mice. These data suggest that IL-11 may play an important role in the regulation of hematopoiesis, and the application of this novel cytokine may have clinical therapeutic benefits.
The place of high-dose BEAM therapy and autologous bone marrow transplantation in poor-risk Hodgkin's disease. A single-center eight- year study of 155 patients
Authors: Chopra R , McMillan AK , Linch DC , Yuklea S , Taghipour G , Pearce R , Patterson KG , Goldstone AH
Published: October 2019

Although high-dose chemotherapy and autologous bone marrow transplantation (ABMT) are increasingly being used for the treatment of relapsed and resistant Hodgkin's disease, there have been few large, single-center studies reported with adequate follow-up to allow full evaluation of this therapeutic modality. We present 155 poor-risk Hodgkin's disease patients who received high-dose BEAM (BCNU, etoposide, cytosine arabinoside, and melphalan) chemotherapy and ABMT who have been studied over a period of 8 years. All patients had either not attained a remission on mechlorethamine, vincristine, procarbazine, prednisone-type therapy and had poor prognostic features at presentation, not attained a complete remission or relapsed within 1 year of an initial alternating regimen, or not attained remission with two or more lines of treatment. At the time of ABMT the relapse status of the patients was as follows: 46 patients were primarily refractory to induction therapy, 7 were good partial responders, 52 were in first relapse, 37 in second relapse, and 13 in third relapse. Seventy-eight patients had chemoresistant disease, 33 had chemosensitive disease at the time of ABMT, and 44 were untested for chemosensitivity at latest relapse. The procedure related mortality in the first 90 days post-ABMT of 10% overall. At 3 months 43 patients (28%) were assessed as complete responders, 72 patients had a partial response (46%), and 24 patients (16%) had no response or progression of disease. However, by 6 months, 53 (24%) patients were assessed as complete responders and 51 (33%) patients had nonprogressive disease. Forty-five patients had received radiotherapy post-ABMT to residual masses (41 patients) or to previous sites of bulk disease (4 patients). The actuarialoverall and progression-free survival at 5 years was 55% and 50%, respectively. On multivariate analysis patients with bulk (masses >10 cm), heavily pretreated patients (those receiving three or more lines of treatment) and females had a significantly poorer prognosis. Relapse status was also significant for progression-free survival in that patients in second (60%) and third relapse (70%) had a better prognosis than those in first relapse (44%) or with primary refractory disease (33%). Response to prior chemotherapy did not predict for progression-free survival. These results enable comparisons to be made between high-dose chemotherapy with ABMT and conventional dose salvage therapy. Furthermore, although the results as a whole are highly encouraging, certain groups carry an unfavorable prognosis.