bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular quantification of residual disease in chronic myelogenous leukemia after bone marrow transplantation
Authors: Thompson JD , Brodsky I , Yunis JJ
Published: October 2019

Residual disease remains a major problem in the treatment of human neoplasia. To effectively monitor minimal leukemic activity after bone marrow transplantation (BMT), we used a competitive polymerase chain reaction (PCR) amplification technique to quantify expression of the characteristic bcr-abl fusion message in patients with chronic myelogenous leukemia (CML). Quantitative results were obtained between the 0.001% and 0.1% level in control experiments. This represents a significant advantage over cytogenetic and Southern blotting techniques routinely used to diagnose CML, which may not be sensitive below the 1% level. To illustrate the potential clinical usefulness of the quantitative PCR strategy, we compared results of bcr-abl messenger RNA expression with those obtained using cytogenetic and Southern blotting techniques, in a study of consecutive BM and peripheral blood (PB) samples from two CML patients at high risk for relapse after BMT. One patient received a syngeneic transplant during the chronic phase of the disease and relapse was apparent at the molecular level 4.5 months after BMT, while the patient was in complete clinical remission. The second patient was treated with an allogeneic BMT during the accelerated phase of the disease. A slow, but progressive decrease in bcr-abl expression was observed during the first 12 months after BMT, and expression was undetectable thereafter. Our results indicate that the competitive PCR technique can be used to monitor disease activity in patients at high risk of relapse, while the patients are in complete clinical remission, which should facilitate the early detection of relapse or the identification of progressive disappearance of leukemic activity. The approach used may serve as a model for the study of residual disease in an increasing number of other hematologic malignancies that express cancer-specific RNAs.
Effect of recombinant human macrophage colony-stimulating factor in irradiated murine recipients of T-cell-depleted allogeneic or non- depleted syngeneic bone marrow transplants
Authors: Blazar BR , Aukerman SL , Vallera DA
Published: October 2019

Recombinant macrophage colony-stimulating factor (rM-CSF), which reacts exclusively with cells of monocyte lineage, was evaluated in the murine bone marrow (BM) transplant setting for in vivo effects on recipient survival, hematologic recovery, and engraftment. Two types of fully allogeneic donors were selected based on the expression (BALB/c), or lack of expression (DBA/1), of hybrid hematopoietic histocompatibility (Hh1) antigens. These antigens are established targets for monocyte and/or natural killer (NK) cell-mediated graft rejection. Irradiated C57BL/6 mice were used as recipients for all experiments. Recipients of T-cell-depleted (TCD) BALB/c BM and a 14-day continuous subcutaneous infusion of 16.8 micrograms/d rM-CSF (n = 30) showed a significant decrease in donor cell engraftment as compared with recipients of donor BM administered pumps delivering saline. These mice administered rM-CSF also displayed significantly reduced levels of circulating leukocytes (predominantly lymphocytes) on day 14 posttransplant (compared with saline controls). Neither engraftment effects nor leukocyte effects were observed when C57BL/6 recipients were administered Hh1 nonexpressing TCD DBA/1 BM cells (n = 30), suggesting that the monocyte/macrophage population is important in long-term alloengraftment in certain donor-recipient strain combinations in which donor Hh1 antigens can serve as target antigens for host effector cells, but are not important in strain combinations in which they are not recognized. Circulating tumor necrosis factor alpha (TNF alpha) levels measured at two time periods during rM-CSF infusion were not elevated. Thus, the reduction in alloengraftment is not likely to be directly related to TNF alpha. However, in vivo elimination of NK cells in the BALB/c into C57BL/6 model prevented the impairment of engraftment mediated by rM-CSF. Thus, rM-CSF-mediated inhibition of alloengraftment is contingent on the presence of host NK cells with antidonor reactivity. Survival was unaffected when rM-CSF was administered in either allogeneic BM transplant model, but was significantly reduced when rM-CSF was administered to C57BL/6 recipients of syngeneic BM transplants. These data are the first analyzing the effects of rM-CSF in murine allogeneic BM transplantation and extend our previous studies using the BALB/c into C57BL/6 model in which in vivo infusions of recombinant granulocyte-macrophage CSF, but not recombinant granulocyte-CSF, lead to decreases in alloengraftment. These data show that rM-CSF-induced stimulation of monocytes may increase BM graft rejection in instances in which NK cells are involved in the rejection process. These data may have future clinical implications for the use of rM-CSF in allogeneic BM transplantation.
Mutations in Jewish patients with Gaucher disease
Authors: Beutler E , Gelbart T , Kuhl W , Zimran A , West C
Published: October 2019

DNA from 100 unrelated patients, 97 of whom were Jewish and three half- Jewish, was analyzed for 22 mutations known to cause Gaucher disease. All but seven of the alleles were identified as having previously described mutations. Five of the unidentified mutations proved to be a previously undescribed nucleotide substitution in a splice junction (IVS2+1) that causes skipping of exon 2. Thus, only 2 of 197 alleles remained unidentified. Homozygotes for the most common mutation, that a nucleotide (nt) 1226, manifested, on average, the mildest disease and the latest age of onset. The mutation at nt 84 and the newly described IVS2+1 mutation, which do not produce any enzyme, were associated with earlier onset and more severe disease. Five of the mutations were considered to be “public,” in the sense that they were found in more than one unrelated individual. Screening for these five mutations permitted detection of 97.5% of all Gaucher alleles in this patient population. Because the mutation at nt 1226 is underrepresented in the patient population and because not all homozygotes come to medical attention, screening the Ashkenazi population using DNA analysis should detect approximately 99% of all heterozygotes.
Peripheral blood neutrophils in chronically neutropenic patients respond to granulocyte-macrophage colony-stimulating factor with a specific increase in CR1 expression and CR1 transcription
Authors: Moore FD Jr , Jack RM , Antin JH
Published: October 2019

Chronically neutropenic patients from a phase I/II protocol were studied for neutrophil (PMN) abnormalities related to therapeutic use of granulocyte-macrophage colony-stimulating factor (GM-CSF). We analyzed phenotype by flow cytometry to measure indirect immunofluorescent staining and activation of transcription by in situ hybridization. PMN count increased in seven of 17 patients. For the group, PMN expression of complement receptors, CR1 and CR3, increased after GM-CSF administration (P less than .005), while expression of class 1 and FcR III was stable. PMN from both of the patients studied by in situ hybridization demonstrated increased expression of CR1 transcript, which in one case coincided in time and intensity with the course of increased CR1 expression, while in the second case the presence of CR1 mRNA increased but lagged behind the increased CR1 protein expression. Thus, PMN activation was observed after GM-CSF infusion, as indicated by increased complement receptor expression. This effect was due both to translocation of receptors from a preformed intracellular pool to the cell surface, and to transcriptional regulation leading to increased receptor synthesis.
Megakaryocytopoiesis and platelet production are stimulated during late pregnancy and early postpartum in the rat
Authors: Jackson CW , Steward SA , Ashmun RA , McDonald TP
Published: October 2019

Platelet count during uncomplicated pregnancy shows considerable patient variation. To gain a better understanding of thrombocytopoiesis during pregnancy, megakaryocytes and platelets were examined during gestation and the early postpartum period, using as a model the rat. Platelet counts and megakaryocyte concentrations and DNA content distributions of timed-pregnant rats were examined at intervals from day 10 of gestation through parturition on day 22 and days 1 through 7 postpartum. Platelet survival was studied in late gestation and the early postpartum. Platelet volume was measured on gestation day 21. Platelet counts were moderately increased on gestation days 17 and 19 through 22, and on days 2 to 3 postpartum. However, the actual rate of platelet production was much higher than the platelet count suggests because the blood volume increased in late gestation to 1.5 times the nonpregnant level. Mean platelet volume and platelet volume distribution width of day 21 gestation rats were not significantly altered. Platelet survival in pregnant rats was not significantly different from that in nonpregnant females. In contrast, megakaryocyte concentration was significantly increased on gestation days 12, 17, and 19 through 21, and 2 to 3 days postpartum. In addition, in late gestation, megakaryocyte DNA content distributions displayed a marked increase in the proportion of high ploidy cells, which peaked 1 day before parturition. At that time, the proportions of 32N (43%) and 64N cells (3%) were, respectively, three and four times nonpregnant values. In contrast to megakaryocyte concentration, megakaryocyte DNA content distributions had returned to the nonpregnant pattern by day 1 postpartum. The changes in megakaryocyte DNA content distribution were accompanied by changes in megakaryocyte size. These data indicate that thrombopoiesis is substantially increased during late pregnancy, and that this increase is accomplished through an increase in megakaryocyte DNA content and size, as well as megakaryocyte number. The more rapid return of megakaryocyte DNA content than of megakaryocyte concentration to nonpregnant levels postpartum suggests that pregnancy-associated hormonal changes which produce an increase in megakaryocyte DNA content and size differ from those which cause an increase in megakaryocyte number.
CD4+CD7-CD57+ T cells: a new T-lymphocyte subset expanded during human immunodeficiency virus infection
Authors: Legac E , Autran B , Merle-Beral H , Katlama C , Debre P
Published: October 2019

CD7 and CD57 are two cell surface molecules related to the differentiation or functional stages of CD4+ T cells. The CD4+CD7- T cells represent a minor subset of CD4+ cells in normal individuals and are considered to contain the normal counterpart of Sezary T cells; the CD4+CD57+ peripheral blood lymphocytes (PBL) are detectable in long- term renal allograft recipients. We compared the cell surface expression of these CD7 and CD57 markers on CD4+ T lymphocytes in peripheral blood and lymphoid organs from normal individuals and human immunodeficiency virus (HIV)-infected patients. Our results indicate that CD4+CD7- T cells in normal PBL do not express CD57 and were poorly responsive to anti-CD3 monoclonal antibody (MoAb), the activation being restored by addition of anti-CD28 MoAb. This CD4+CD7- cell subset is increased in peripheral blood during HIV infection, and its progressive expansion mirrors both the absolute and relative decrease of CD4+ T cells. The lack of CD7 expression is correlated with CD57 acquisition on CD4+ T cells because CD4+CD7-CD57+ cells represent a major component of the CD4+CD7- subset in HIV-infected patients. Our results suggest that the presence and the expansion of CD4+CD7-CD57+ T lymphocytes, which do not behave as previously defined helper subsets, may participate to the immune dysfunction observed during HIV infection.
Interleukin-5 is an autocrine growth factor for Epstein-Barr virus- transformed B lymphocytes
Authors: Baumann MA , Paul CC
Published: October 2019

Because of the recent finding that interleukin-5 (IL-5) is produced by Epstein-Barr virus-transformed B lymphocytes (EBV-B cells), we performed studies to ascertain whether EBV-B cells might use IL-5 by an autocrine mechanism. EBV-B cells known to be IL-5 producers were capable of responding to addition of exogenous IL-5 by dose-related augmented proliferation. The addition of a neutralizing anti-IL-5 antibody reduced these effects and also dose-dependently inhibited proliferation and reduced viability of unsupplemented EBV-B cells, having a maximum effect at about 120 hours. In contrast, no stimulatory effect of IL-5 was noted on Burkitt's lymphoma cell lines, nor were these lines growth-inhibited by anti-IL-5 antibody. With biotinylated IL-5, (b-IL-5) second labeling with streptavidin-FITC, and flow cytometric analysis, binding of IL-5 to EBV-B cells cultured in fresh medium was demonstrated and could be competed for by excess unlabeled IL-5, suggesting the presence of IL-5-specific binding sites. Binding of IL-5 was reduced on cells cultured for longer periods before study but could be restored by extensively washing cells before labeling them with b-IL-5, suggesting that surface binding sites had become occupied by endogenously produced IL-5. These findings support a role for IL-5 in autocrine support of EBV-B cell growth.
An alanine-to-threonine substitution in protein 4.2 cDNA is associated with a Japanese form of hereditary hemolytic anemia (protein 4.2NIPPON)
Authors: Bouhassira EE , Schwartz RS , Yawata Y , Ata K , Kanzaki A , Qiu JJ , Nagel RL , Rybicki AC
Published: October 2019

Erythrocyte (RBC) protein 4.2 (P4.2)-deficiency observed in Japanese individuals results in a hemolytic anemia associated with abnormally shaped (spherocytic, ovalocytic, and elliptocytic), osmotically fragile RBCs, the clinical presentation of which resembles hereditary spherocytosis (HS). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, P4.2-deficient individuals contain less than 1% of the normal membrane content of P4.2 and immunologic analysis shows that the P4.2 present exists as an equimolar doublet of 74-Kd and 72-Kd bands, in contrast to normal RBC membranes where a discrete 74-Kd band is not observed. RBC membranes from both of the biologic parents of a P4.2- deficient individual contained both the 74-Kd and the 72-Kd bands, demonstrating their heterozygosity for the P4.2 defect. The molecular basis of Japanese P4.2-deficiency was investigated by reverse transcription of total reticulocyte RNA, followed by polymerase chain reaction (PCR) amplification, subcloning, and sequencing. The complete cDNA sequence of a P4.2-deficient patient showed a single point mutation that changes codon 142 from GCT (alanine) to ACT (threonine) (Protein 4.2NIPPON). The mutation also eliminated an HgaI restriction site, therefore allowing rapid screening for the presence of the mutation. Screening of PCR-amplified genomic DNA showed that the mutation was present in the homozygous state in four (eight chromosomes) unrelated Japanese P4.2-deficient individuals and absent in 35 (70 chromosomes) P4.2-normal controls (including 15 Japanese [30 chromosomes]). The presence of the mutation was confirmed by allele- specific hybridization. The mutation occurred in an alternatively spliced exon that is present in two of four P4.2 mRNA splicing isoforms. These results demonstrate that Japanese P4.2-deficiency is closely associated with the P4.2 gene and does not arise secondarily to a defect in another membrane protein, and further suggest that the P4.2- deficiency is related to the pathogenesis of the hemolytic anemia in this variant form of recessively inherited spherocytosis.
Decreased concentrations of tumor necrosis factor-alpha in supernatants of monocytes from homozygotes for hereditary hemochromatosis
Authors: Gordeuk VR , Ballou S , Lozanski G , Brittenham GM
Published: October 2019

To determine whether release of tumor necrosis factor-alpha (TNF- alpha), a cytokine that affects iron homeostasis, may be selectively altered in hereditary hemochromatosis, we measured concentrations of TNF-alpha and interleukin-1 beta (IL-1 beta) in supernatants of cultured peripheral blood monocytes from 11 homozygotes for hereditary hemochromatosis, 11 healthy individuals, and five patients with iron- loading anemia. The gene for hereditary hemochromatosis is tightly linked to the HLA locus on chromosome 6, but its exact site and product are not known. The gene for TNF-alpha also is located within the HLA region. Monocytes were incubated from 4 to 36 hours in medium alone or with added lipopolysaccharide. Mean concentrations of immunoreactive TNF-alpha in supernatants were significantly lower for subjects with hereditary hemochromatosis as compared to healthy controls (P less than .037) and patients with iron-loading anemia (P less than .005); differences between homozygotes for hemochromatosis and healthy controls were up to 4.5-fold at 4 hours (P = .008), 1.9-fold at 12 hours (P = .036), and 7.0-fold at 36 hours (P = .001). Importantly, concentrations of IL-1 beta in supernatants were not significantly different among the three groups. We conclude that release of TNF-alpha by monocytes may be selectively impaired in hereditary hemochromatosis. Deficient activity of TNF-alpha may contribute to the disordered iron metabolism of this disease.
Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment [see comments]
Authors: Wagner JE , Broxmeyer HE , Byrd RL , Zehnbauer B , Schmeckpeper B , Shah N , Griffin C , Emanuel PD , Zuckerman KS , Cooper S
Published: October 2019

The possibility that umbilical cord and placental blood from an HLA- identical sibling might produce stable donor-derived lymphohematopoietic engraftment was tested in a patient with juvenile chronic myelogenous leukemia (JCML). After conditioning with high-dose busulfan and cyclophosphamide, cryopreserved umbilical cord blood, containing 0.5 x 10(8) nucleated cells/kg and 2.7 x 10(4) colony forming units-granulocyte, macrophage (CFU-GM)/kg, was infused. A leukocyte count greater than 1,000/microL, absolute neutrophil count (ANC) greater than 500/microL, and platelet count greater than 20,000/microL (untransfused) were observed on days 39, 39, and 47 after transplantation, respectively. Donor cell engraftment was documented in the peripheral blood and bone marrow by cytogenetic analysis, restriction fragment length polymorphism (RFLP), and polymerase chain reaction (PCR) as early as day 21. Furthermore, the donor origin of each lymphohematopoietic lineage (ie, CD5+ T cells, CD19/20+ B cells, CFU-GM, and burst-forming unit-erythrocyte [BFU-E]) was confirmed. On day 200, assays of the peripheral blood and bone marrow showed an abnormal proliferation of CFU-GM at low seeding densities in the absence of exogenous growth factors, as well as a hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), both pathophysiologic characteristics of JCML. Recurrent disease was confirmed histologically on day 225. Together, these results demonstrate that umbilical cord blood contains sufficient numbers of hematopoietic stem cells necessary for the engraftment of leukemia patients treated with myeloablative therapy and that the detection of “spontaneous” CFU-GM and hypersensitivity to GM-CSF after treatment is a marker of residual or recurrent disease in patients with JCML.
Proof of differentiative mode of action of all-trans retinoic acid in acute promyelocytic leukemia using X-linked clonal analysis
Authors: Elliott S , Taylor K , White S , Rodwell R , Marlton P , Meagher D , Wiley J , Taylor D , Wright S , Timms P
Published: October 2019

Using X-linked clonal analysis, mechanism of action of all-trans retinoic acid (ATRA) was sought in a 16-year-old female with relapsed clonally evolved acute promyelocytic leukemia (APL), who achieved complete remission. On ATRA, metamorphosis of peripheral blood leukemic promyelocytes to mature neutrophils was observed, despite the persistence of t(15;17) in 100% of bone marrow metaphases. DNA was extracted from fractionated serial blood specimens, collected at diagnosis, in first complete remission (CR), relapse, and during ATRA treatment. Using a phosphoglycerokinase (PGK) probe, the patient was heterozygous for both Bgl I and Bst XI PGK polymorphisms. Methylation analysis showed monoclonal leukemic promyelocytes with a polyclonal first CR achieved by standard chemotherapy. Subsequent examination, in relapse, of granulocytes appearing during ATRA treatment showed these to be monoclonal, proving these were derived from the neoplastic clone. The X-linked clonal analysis methodology has provided in vivo evidence of cellular differentiation as the mechanism of action of ATRA. Parallel studies of cytogenetic and clonal analysis showed a regression of the t(15;17) cytogenetic abnormality and return of a polyclonal PGK methylation pattern in 5 weeks, indicating a repopulation of marrow by normal stem cells. As standard cytogenetic techniques are inappropriate for nondividing cells, X-linked clonal analysis provides a marker system to allow insight into mechanism of drug action in malignant hematologic disease.
Acute- and chronic-phase chronic myelogenous leukemia colony-forming units are highly sensitive to the growth inhibitory effects of c-myb antisense oligodeoxynucleotides
Authors: Ratajczak MZ , Hijiya N , Catani L , DeRiel K , Luger SM , McGlave P , Gewirtz AM
Published: October 2019

We have previously demonstrated that malignant hematopoietic colony- forming units (CFUs) may be purged from normal CFU by exposure to c-myb antisense oligodeoxynucleotides (oligomers). This novel strategy appeared particularly promising for patients with chronic myelogenous leukemia (CML) in blast crisis, since in some cases complete elimination of bcr-abl-expressing cells was accomplished. We have examined 11 additional patients, including seven in chronic phase, in order to extend these initial observations. We sought in particular to determine if elimination of bcr-abl-expressing clones was a usual event. Exposure of CML cells to c-myb antisense oligomers resulted in inhibition of CFU-granulocyte, macrophage (CFU-GM)-derived colony formation in eight of 11 (73%) cases evaluated. Inhibition was antisense sequence-specific, dose-dependent, ranged between 58% and 93%, and was statistically significant (P less than or equal to .03) in seven of the eight cases. In two cases, CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM)-derived colony formation was also examined and found to be inhibited by the c-myb antisense oligomers in a sequence-specific manner. To determine whether CML CFU had been reduced or eliminated after exposure to the antisense oligomers, we examined cells in the residual colonies for bcr-abl mRNA expression using a reverse transcription-polymerase chain reaction detection technique (RT-PCR). Eight cases were evaluated and in each case where antisense myb inhibited growth, bcr-abl expression as detected by RT- PCR was either greatly decreased or nondetectable. No residual leukemic CFU were demonstrable on replating of treated cells. These results suggest that c-myb antisense oligomers substantially inhibit the growth and survival of CML CFU in both chronic and blast phase of disease. They may therefore prove useful for both ex vivo and in vivo treatment of CML.
Multilineage phenotypes of interleukin-3-dependent progenitor cells
Authors: Ford AM , Healy LE , Bennett CA , Navarro E , Spooncer E , Greaves MF
Published: October 2019

Interleukin-3 (IL-3)-dependent murine FDCP-mix cells have multilineage differentiation capacity; they are nonleukemic, have a normal karyotype, and are nonimmortalized. These cells coexpress on their cell surface the “early” B-lineage marker B220/CD45R and the myeloid marker Mac-1/iC3b receptor (CR3), transcribe germline T-cell receptor gamma genes, and express the macrophage lineage growth factor receptor gene c- fms as a predominant 8.4-kb transcript. They do not detectably express at the stable mRNA or protein level other lymphoid precursor cell genes including CD2, TdT, lambda 5, and BP1. Induction of granulocyte/macrophage differentiation in these cells closes down expression of the lymphoid genes and activates stable expression of genes specific to the myeloid lineage, including myeloperoxidase. Expression of the c-fms gene at the mRNA level is upregulated and the dominant stable transcript is now in the 4.1-kb form typical of the macrophage lineage. These data provide a plausible explanation for the coexpression of lymphoid and myeloid lineage markers on human leukemic cells of stem cell or progenitor cell origin and have implications for the programming of lineage potential in normal multipotential hematopoiteic progenitor cells.
Factor IXa-factor VIIIa-cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo
Authors: Bauer KA , Mannucci PM , Gringeri A , Tradati F , Barzegar S , Kass BL , ten Cate H , Kestin AS , Brettler DB , Rosenberg RD
Published: October 2019

We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.
Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets
Authors: Kroner PA , Kluessendorf ML , Scott JP , Montgomery RR
Published: October 2019

von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574---- Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non- ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.
Two types of murine CD34 mRNA generated by alternative splicing
Authors: Suda J , Sudo T , Ito M , Ohno N , Yamaguchi Y , Suda T
Published: October 2019

To characterize and clarify the function of CD34 antigen experimentally, we isolated two types of CD34 mRNA from a cDNA library of murine stromal cell line, PA-6 stimulated with lipopolysaccharide (LPS) and 12-o-tetra-decanoylphorbol 13-acetate (TPA) using a human CD34 probe. In addition to the clone (open reading frame [ORF]:1149bp) reported by Brown et al, a novel clone (ORF:978 bp) was obtained. The difference between the two clones was in the cytoplasmic portion of CD34; the former has 73 amino acids, while the latter has 16. We investigated the genomic sequence of cytoplasmic portion and found conserved nucleotide sequences at the exon-intron junction (GT ... AG). Thus, it was concluded that alternative splicing gave two types of CD34 mRNA. A novel clone contains the longer cDNA, including a insert of 156 bp, but results in a shorter predicted coding sequence because of the introduction of an inframe stop codon. Northern blot analysis using a murine cDNA probe (HindIII fragment, 900 bp) showed that CD34 was highly expressed in the brain and testis, and moderately in the thymus, spleen, and bone marrow, but not in adult liver. However, day 12 to 14 fetal liver cells showed significant expression of CD34. Quantitative reverse transcription polymerase chain reaction showed that spleen, thymus, bone marrow, and testis RNA gave two bands of almost equal intensity, but in the brain a novel clone was expressed three times more than the other clone. Furthermore, Northern blot analysis using a probe (156 bp) specific for the spliced intracellular region confirmed the significant mRNA expression of a novel clone. Although the biologic significance of alternative splicing remains to be elucidated, it is suggested that a different carboxyterminal tail causes a change in signal transduction.
Isolation and molecular characterization of the human CD34 gene
Authors: He XY , Antao VP , Basila D , Marx JC , Davis BR
Published: October 2019

The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5′ termini of mRNAs by 5′-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5′ and 3′ untranslated regions, amino acid coding sequences, and 5′ flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.
The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb- IIIa on nonactivated platelets is accessible to high-affinity macromolecules
Authors: Tomiyama Y , Tsubakio T , Piotrowicz RS , Kurata Y , Loftus JC , Kunicki TJ
Published: October 2019

We have characterized a murine IgG monoclonal antibody, OP-G2, specific for platelet glycoprotein (GP) IIb-IIIa (alpha IIb beta 3). OP-G2 Fab fragments inhibit fibrinogen-mediated platelet aggregation and competitively inhibit adenosine diphosphate-induced binding of 125I- fibrinogen to washed platelets. OP-G2 binding to GPIIb-IIIa is specifically inhibited by RGD-containing peptides but not the fibrinogen gamma-chain carboxy-terminal peptide, and OP-G2 Fab fragments, like RGD-containing peptides, alter the conformation of GPIIb-IIIa resulting in the expression of a ligand-induced binding site (LIBS) recognized by PMI-1. OP-G2 fails to bind to the recombinant Cam variant of GPIIb-IIIa (alpha III beta 3Cam) wherein an Asp119 to Tyr119 substitution in GPIIIa abrogates the ability to recognize RGD. These data indicate that OP-G2 recognizes an epitope at or in very close proximity to the RGD recognition site of GPIIb-IIIa and that, in every aspect tested, OP-G2 behaves like a macromolecular RGD ligand. Interestingly, two-color flow cytometry shows that OP-G2 IgG can bind to nonactivated platelets. Quantitative binding assays indicate that nonactivated platelets bind approximately 50,000 125I-OP-G2 molecules/platelet. Furthermore, the affinity of OP-G2 for platelets activated with thrombin is roughly fivefold higher (nonactivated, kd = 24.8 nmol/L; activated, kd = 4.9 nmol/L). These results suggest that the RGD recognition site of GPIIb-IIIa is available to macromolecules that contain RGD even on nonactivated platelets, provided that the affinity of the ligand is adequate.
Molecular cloning and expression of rabbit antithrombin III
Authors: Sheffield WP , Brothers AB , Wells MJ , Hatton MW , Clarke BJ , Blajchman MA
Published: October 2019

A cDNA containing the complete open-reading frame encoding rabbit antithrombin III (AT-III) was isolated from a rabbit liver cDNA expression library, using a specific antibody as a probe. Sequence analysis showed 84% identity between the deduced amino acid sequences of the rabbit and human proteins. A previously described cell-free expression system was used to verify the identity of the clone. The full-length cDNA was inserted into an expression vector, and messenger RNA (mRNA) transcripts generated. In vitro translation of these transcripts, in the presence of [35S]methionine, in an mRNA-dependent rabbit reticulocyte lysate system resulted in the synthesis of a 51-Kd polypeptide, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This nonglycosylated protein was capable of forming SDS-stable complexes with human alpha-thrombin. Complex formation was significantly enhanced following the deletion of nucleotides encoding the signal peptide, and the resultant generation of a 47-Kd nonglycosylated mature protein product. When the template DNA giving rise to this product was internally truncated, two rabbit AT- III deletion mutants were generated that lacked the ability to interact with thrombin, but retained the ability to bind heparin. Cell-free expression plasmids encoding the human and rabbit AT-III mature molecules were manipulated to produce two interspecies fusion proteins. For the first, human codons were used to replace rabbit codons from residue 369–433, while in the second human codons replaced rabbit codons from residue 217–433. Both fusion proteins exhibited less efficient thrombin-complexing ability than the original cell-free- derived mature rabbit AT-III. Thus, portions of AT-III molecules from the two species, despite their high degree of homology, are not interchangeable. Knowledge of the structure of rabbit AT-III, combined with the availability of the rabbit cDNA, will permit defined experimentation aimed at understanding antithrombin III structure relative to its function in vivo.
Direct detection of activated protein C in blood from human subjects
Authors: Gruber A , Griffin JH
Published: October 2019

The antithrombotic enzyme, activated protein C (APC) was measured in blood using an enzyme capture assay (ECA). The ECA involved (1) collection of blood into anticoagulant containing a reversible inhibitor of the enzyme, (2) specific affinity capture of the enzyme by an immobilized antibody that does not inhibit the enzyme, (3) removal of the reversible inhibitor by washing, and (4) direct assay of the captured enzyme's amidolytic activity. The ECA for APC used benzamidine for inhibition, anti-PC light-chain murine monoclonal antibody for capture, and the oligopeptide substrate S-2366 for enzyme assay. The sensitivity of this assay was 5 pmol/L (0.3 ng/mL) APC. The APC activity in normal pooled plasma corresponded to the amidolytic activity of 38 pmol/L (2.26 +/- 0.2 ng/mL) purified human plasma- derived APC in the ECA. APC levels in 41 normal donors ranged from 64% to 143%, averaging 104.9% +/- 19.6% (SD). Thus, APC is a measurable and normal component of circulating human blood, and this ECA may be useful for identifying APC deficiency. Moreover, similar ECAs for other enzymes in the circulation may be useful.
Bone marrow-derived stromal cells prevent apoptotic cell death in B- lineage acute lymphoblastic leukemia
Authors: Manabe A , Coustan-Smith E , Behm FG , Raimondi SC , Campana D
Published: October 2019

Establishing requirements for the survival of human immature B cells in vitro has proved elusive. In this article, we report prolonged survival of B-lineage leukemic cells on ‘feeder layers’ of bone marrow (BM)- derived stromal cells in a serum-free environment. In 15 of 18 cases of B-lineage acute lymphoblastic leukemia (ALL), there was a greater than 50% decrease in the number of viable cells after 72 hours of culture in medium alone. Cell loss was preceded by molecular and cellular changes characteristic of programmed cell death, or apoptosis, and was not suppressed by adding interleukin-7 to the tissue culture medium. By contrast, the use of allogeneic BM stromal cells as feeder layers prevented apoptosis in 10 of 12 cases of ALL, leading to extended survival of the blast cells. This method was not successful when the allogeneic marrow cells were replaced with established cell lines. In six of eight cases in which the numbers of intact CD19+ lymphoblasts were counted by flow cytometry after 7 days of culture, the proportion of such cells recovered in the presence of BM stromal cells was 68.8% to 124.7% (median, 95.3%) of that originally seeded, as opposed to the 0.3% to 15.9% fraction (median, 0.7%) obtained in the absence of stromal cells. Survival requirements of the B-lineage lymphoblasts appeared to be heterogeneous, as cells from 3 of the 18 cases studied showed no signs of apoptosis in serum-free tissue culture medium that lacked BM stromal cells. The only cells not giving rise to viable cultures were from two hyperdiploid (greater than 50 chromosomes) cases with identical karyotypes. The serum-free assay described here can be used to compare the survival requirements of normal and leukemic B-cell progenitors as well as to identify the molecules involved in the interaction between BM stroma and immature B cells.
False-positive flow cytometric platelet glycoprotein IIb/IIIa expression in myeloid leukemias secondary to platelet adherence to blasts
Authors: Betz SA , Foucar K , Head DR , Chen IM , Willman CL
Published: October 2019

Because neither morphologic nor routine cytochemical features are pathognomonic, the diagnosis of acute megakaryoblastic leukemia (AML- M7) is difficult, requiring either specialized ultrastructural or immunologic techniques. Because the ultrastructural techniques are cumbersome, immunologic assays for expression of several platelet- specific antigens, such as CD41a (platelet glycoprotein IIb/IIIa), have become the primary method used to detect megakaryoblastic differentiation. In our flow cytometric analysis of the immunophenotypes of over 1,000 cases of AML from patients registered to Southwest Oncology Group (SWOG) Treatment Protocols, we found that 38% of cases demonstrated CD41a reactivity. Because this frequency of CD41a expression by flow cytometry greatly exceeded the number of morphologically defined cases of AML-M7, we postulated that the reaction may be caused by platelets adherent to leukemic blasts. To investigate this hypothesis, we performed a side-by-side comparison of flow cytometric and cytospin immunofluorescence studies on 37 cases of adult de novo AML that demonstrated a wide range of CD41a expression by flow cytometric analyses. We found that the expression of CD41a detected by flow cytometric techniques was secondary to adherent platelets or platelet fragments in 85% of cases. Many of these cases also expressed the lineage-specific carbohydrate, LNF III (CD15), which may mediate platelet adhesion to mature monocytes and neutrophils. Only 15% of the CD41a flow cytometrically positive cases demonstrated true diffuse membrane and cytoplasmic positivity on cytospin slides indicative of megakaryoblastic differentiation. Cytospin immunofluorescence for CD41a should be performed on all cases of suspected AML-M7. If only flow cytometric techniques are used, adherent platelets may result in the erroneous diagnosis of this AML subtype.
Elliptocytogenic alpha I/36 spectrin Sfax lacks nine amino acids in helix 3 of repeat 4. Evidence for the activation of a cryptic 5'-splice site in exon 8 of spectrin alpha-gene
Authors: Baklouti F , Maréchal J , Wilmotte R , Alloisio N , Morlé L , Ducluzeau MT , Denoroy L , Mrad A , Ben Aribia MH , Kastally R , Delaunay J
Published: October 2019

Elliptocytogenic alpha I/36 spectrin Sfax is a new variant found in a Tunisian family. The alpha I/36 allele yielded a clinically manifest picture only when occurring in trans to a recently identified, low expression level polymorphism referred to as the alpha V/41 allele. Spectrin dimers were slightly increased in 4 degrees C extracts. On peptide maps, the alpha I domain split into two abnormal fragments of 36 and 33 Kd. The mutated alpha-chain represented 20% and 44% of total alpha-chain in alpha/alpha I/36 and alpha V/41/alpha I/36 heterozygotes, respectively. Peptide sequencing showed that the 36-Kd fragment started at Ala 357 and displayed a deletion extending from amino acids 363 to 371. The corresponding 27-nucleotide deletion was found in alpha-spectrin mRNA. However, exon 8 of spectrin alpha-gene failed to disclose this deletion. Instead, an A to G substitution appeared in position 3 of codon 362, leading to the occurrence of the critical GU dinucleotide within a cryptic 5′-splice site surrounding codon 362. This event would account for the splicing out of codons 363 to 371. The reading frame was preserved and even amino acid 362 (AGG, Arg) remained unaltered. As in most spectrin alpha-chain elliptocytogenic variants, the change involved a helix 3. This is the first elliptocytogenic mutation recorded in repeat alpha 4.
Synergism of interleukin-2 and cyclosporine A in induction of a graft- versus-tumor effect without graft-versus-host disease after syngeneic bone marrow transplantation
Authors: Charak BS , Agah R , Mazumder A
Published: October 2019

Interleukin-2 (IL-2) therapy generates killer cells with major histocompatibility complex (MHC)-unrestricted cytotoxicity against most tumors but not normal tissues. Cyclosporine A (CsA) has been reported to break tolerance to self and to induce killer cells with specificity against class II MHC (Ia) antigens both on the host and the tumor cells, resulting in a mild graft-versus-host disease (GVHD) in an autologous bone marrow transplantation (BMT) setting in the rat. We used these two agents in a syngeneic BMT model in a strain of mice that does not develop GVHD with CsA. Therapy with either agent alone was ineffective, whereas a combination of CsA plus IL-2 after BMT induced a potent graft-versus-tumor (GVT) effect against a melanoma and an acute myeloid leukemia. The antitumor effect could be adoptively transferred by infusing spleen cells harvested from mice treated with CsA plus IL-2 into secondary recipients that received chemoradiotherapy. The cytotoxicity of these cells was not influenced by treatment of tumor cells with gamma-interferon or Ia antibody. The cytotoxic effect was mediated by Thy 1+ and asialo GM 1+ cells. There was no GVHD either in the primary recipients of CsA and IL-2 or in those receiving the adoptively transferred spleen cells. Our findings show that combination therapy with CsA and IL-2 after syngeneic BMT induces a potent GVT effect in a non-MHC-restricted manner, and point to the existence of differences between the mechanisms of GVT and GVHD.
Expression of selected human HOX-2 genes in B/T acute lymphoid leukemia and interleukin-2/interleukin-1 beta-stimulated natural killer lymphocytes
Authors: Petrini M , Quaranta MT , Testa U , Samoggia P , Tritarelli E , Care A , Cianetti L , Valtieri M , Barletta C , Peschle C
Published: October 2019

Although the key role of human homeobox (HOX) genes in development is well established, their function in adult cells is still under scrutiny. We have analyzed, in normal adult blood cell subpopulations, acute lymphoid leukemia (ALL) cells lines, and primary blasts, the RNA expression of all HOX-2 cluster genes (5′-2.5, 2.4, 2.3, 2.2, 2.1, 2.6, 2.7, 2.8, 2.9, 3′) and nine genes in the HOX-1, -3, and -4 cluster by Northern blotting, RNAse protection, and/or reverse transcriptase polymerase chain reaction (RT-PCR). The analyzed HOX-1, -3, and -4 genes were never expressed in all tested cell populations. Natural killer (NK) cells activated in interleukin-2 (IL-2)/IL-1 beta-treated cultures exhibit a gradually increasing, abundant expression of three HOX-2 genes (2.2, 2.6, 2.8), while three other genes (2.3, 2.1, 2.7) are expressed at a lower level at late culture times. However, no HOX-2 gene is expressed in quiescent lymphocytes (NK, B and T [T-cell receptor (TCR) alpha/beta, gamma/delta lymphocytes, thymocytes] cells), granulocytes, and monocytes. In B- and T-ALL cell lines, HOX-2 genes are expressed according to different patterns: (1) widespread transcription (seven of nine genes, including 2.3 and 2.6) in the Peer line bearing the TCR gamma/delta; (2) expression of 2.5, 2.2, and 2.6 in the SEZ 627 line, which derives from an HTLV-1+ T-helper leukemia; (3) transcription of 2.3 and 2.6 in both the T-ALL CEM line and four B- ALL lines (interestingly, CALLA- B-ALL lines are constantly 2.3/2.6 RNA+); (4) no HOX-2 gene expression was detected in one T- and two B- ALL lines. Primary blasts from five T- and five pre-B-ALL showed selective expression of one or more HOX-2 genes, namely 2.5, 2.2, 2.6, and 2.7. Our data are compatible with the hypothesis that selected HOX- 2 genes play a role in the IL-2/IL-1 beta-induced activation and/or proliferation of normal NK lymphocytes and possibly in the oncogenetic process of some T- and B-ALL.
Effects of gamma-interferon on human neutrophils: protection from deterioration on storage
Authors: Klebanoff SJ , Olszowski S , Van Voorhis WC , Ledbetter JA , Waltersdorph AM , Schlechte KG
Published: October 2019

Human polymorphonuclear leukocytes (PMN) preincubated overnight with 100 U/mL gamma-interferon (IFN-gamma) had an increased metabolic response, as measured by iodination and/or superoxide production, to stimulation by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionyl-leucyl- phenylalanine (FMLP), opsonized zymosan, and lipopolysaccharide (LPS), as compared with cells comparably preincubated in the absence of IFN- gamma. The decline in the staphylocidal activity of the stored PMN was also prevented in part by IFN-gamma, as was the depressed adherence of PMN stimulated with phorbol myristate acetate (PMA), FMLP, TNF, GM-CSF, and LPS. This protective effect of IFN-gamma on PMN function was associated with the prolonged surface expression of the complement receptor three (CR3) alpha-chain (CD11b), CR3 beta-chain (CD18), FcRII (CD32), and FcRIII (CD16), and the appearance of surface FcRI (CD64). The polymerase chain reaction (PCR) was used to amplify neutrophil RNA- derived cDNA recognized by synthetic oliogonucleotides designed from published nucleotide sequences for specific proteins. Using this procedure, mRNA for gp91-phox, p67-phox, p47-phox, CD64, two forms of CD32, CD16, CD11b, CD18, and actin were found to be depressed after overnight storage of neutrophils, and this decrease in steady-state mRNA levels was in part or totally prevented by IFN-gamma. CD64 and gp91-phox mRNA were generally increased by IFN-gamma to a level greater than that of freshly isolated neutrophils. Northern analysis of CD64 and p47 phox mRNAs confirmed the findings with the PCR method. These findings suggest that storage of PMN in a functionally active state is favored by the presence of IFN-gamma.