bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemotactic activity of recombinant human granulocyte colony- stimulating factor
Authors: Wang JM , Chen ZG , Colella S , Bonilla MA , Welte K , Bordignon C , Mantovani A
Published: October 2019

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induced migration across polycarbonate filters of human polymorphonuclear leukocytes (PMN). rhG-CSF was active in inducing PMN migration at concentrations greater than or equal to 10 to 100 U/mL (7 to 70 ng/mL). rhG-CSF did not contain appreciable levels of endotoxin contamination as assessed by Limulus amebocyte assay, and Polymixin B did not affect the chemotactic activity of rhG-CSF. A monoclonal anti-G- CSF antibody blocked the induction of migration by G-CSF, thus establishing that the cytokine was responsible for the activity of the recombinant preparation. Checkerboard analysis was performed by seeding different concentrations of G-CSF above and/or below the filter and revealed that the migratory response to this cytokine was best observed in the presence of a positive concentration gradient between the lower and upper compartments of the chamber, thus indicating an actual chemotactic effect. When different migrating cells were examined, rhG- CSF was inactive on large granular lymphocytes and endothelial cells under conditions in which appropriate reference attractants were active. In contrast, rhG-CSF elicited a chemotactic response in monocytes inhibited by specific antibody. Thus, G-CSF is a chemotactic signal for phagocytes. This cytokine, when produced at inflammatory sites, may contribute to the recruitment of phagocytes from the blood compartment to amplify resistance against certain noxious agents.
In vivo control of differentiation of myeloid leukemic cells by cyclosporine A and recombinant interleukin-1 alpha
Authors: Lotem J , Sachs L
Published: October 2019

There are different types of hematopoietic regulatory proteins that regulate the multiplication and differentiation of normal myeloid cells. These different types include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL- 3), or macrophage and granulocyte inducers, type 1 (MGI-1); another type (called MGI-2) that induces myeloid differentiation of normal myeloid cells without inducing myeloid cell multiplication; and interleukin-1 (IL-1), which can act on myeloid precursor cells. Different clones of myeloid leukemic cells can differ in their ability to be induced to undergo terminal cell differentiation by different hematopoietic regulatory proteins. We have now studied the ability of cyclosporine A and recombinant IL-1 alpha to regulate in vivo differentiation of different clones of myeloid leukemic cells that are either susceptible or resistant to induction of differentiation by IL-1 in vitro. The results show that (a) cyclosporine A, like other immune- suppressing compounds such as cyclophosphamide, inhibited in vivo differentiation of myeloid leukemic cells and differentiation was restored by injecting recombinant GM-CSF; (b) recombinant IL-1 alpha induced in vivo terminal differentiation of IL-1-sensitive but not IL-1- resistant clones of myeloid leukemic cells; (c) IL-1 alpha and GM-CSF synergistically induced differentiation in vivo in a GM-CSF-responsive and IL-1-nonresponsive clone of leukemic cells; and (d) IL-1 alpha induced in vivo the rapid production and release into serum of the differentiation-inducing protein MGI-2 as well as the growth-inducing proteins M-CSF and G-CSF.
X-linked dominant control of F-cells in normal adult life: characterization of the Swiss type as hereditary persistence of fetal hemoglobin regulated dominantly by gene(s) on X chromosome
Authors: Miyoshi K , Kaneto Y , Kawai H , Ohchi H , Niki S , Hasegawa K , Shirakami A , Yamano T
Published: October 2019

Fetal hemoglobin (HbF) levels determined in healthy Japanese adults ranged from 0.3% to 16.0% as F cells and 0.17% to 2.28% as HbF content, which were the same as those obtained in other countries. The frequency distribution of 300 healthy adults with various numbers of F cells consisted statistically of two different groups, low and high F-cell groups. Individuals with greater than or equal to 4.4% of F cells (HbF about 0.7%) were defined as the high F-cell trait, which accounted for 11.3% of males and 20.7% of females. Family studies of 21 probands with this trait and sex-different frequency analyses in the population and probands revealed X-linked dominant inheritance. Two other families of the trait associated with color blindness were described, although no definitive evidence for linkage was obtained between the two. A review of population and family studies reported in the literature indicated that persons with Swiss-type hereditary persistence of fetal hemoglobin (HPFH) are of the same kind as this trait in their incidence and inheritance form, but represent a portion of the trait with higher levels of HbF or F cells. The existence of X chromosome-localized regulatory gene(s) for the developmental switch of human Hb production is discussed.
Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets
Authors: Phillips MD , Moake JL , Nolasco L , Turner N
Published: October 2019

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.
A molecular study of heterozygous protein 4.1 deficiency in hereditary elliptocytosis
Authors: Lambert S , Conboy J , Zail S
Published: October 2019

Genomic DNA from five kindreds and two individuals with hereditary elliptocytosis [HE(4.1+)] and a partial deficiency of protein 4.1 [HE(4.1+)] was extracted and probed with a cDNA for protein 4.1. When using a fragment of the cDNA that encompassed the coding region of the gene, two restriction fragment length polymorphisms segregating with protein 4.1 deficiency were found in one kindred when using the enzymes BgIII and PvuII but were not seen in the other HE(4.1+) subjects or in 20 random control individuals. DNA digested with three other enzymes (HindIII, EcoRI, TaqI) produced restriction patterns similar to controls. The unique BgIII and PvuII polymorphisms probably reflect a rearrangement of the coding region of the protein 4.1 gene as the underlying cause of the partial protein 4.1 deficiency in this family. A less likely possibility is that these polymorphisms represent coincidental single base changes unrelated to the primary gene defect.
Detection of leukemic clone maturation in vivo by premature chromosome condensation
Authors: Hittelman WN , Agbor P , Petkovic I , Andersson B , Kantarjian H , Walters R , Koller C , Beran M
Published: October 2019

The purpose of this study was to determine the feasibility of using the technique of premature chromosome condensation to detect the in vivo maturation of abnormal elements in patients with chronic myelogenous leukemia (CML), myelodysplastic syndrome, and acute leukemia. Patients were chosen for study if there were a clinical suggestion of in vivo maturation and a leukemic clone exhibiting a distinguishable karyotypic abnormality. Mature peripheral blood granulocytes were enriched by two- step Ficoll-Hypaque gradient sedimentation and fused with mitotic Chinese hamster ovary cells to induce the formation of prematurely condensed chromosomes (PCC). These PCC were then analyzed for chromosome number per cell (in the case of patients with a numerical abnormality) or by G-banding (in the case of specific translocations). Of 13 patients chosen for study, 12 showed karyotypic evidence for maturation of the abnormal elements in vivo. Maturation was observed in a number of clinical situations including before treatment in benign CML and myelodysplasia, after low-dose and high-dose chemotherapy in myelodysplasia and acute myelogenous leukemia (AML), and in remission. These results suggest that the technique of premature chromosome condensation can be a powerful tool in better understanding the biology of disease and mode of response to therapy in vivo in patients with leukemia and preleukemic syndromes, especially during treatment with agents thought to induce maturation of the leukemic elements.
Absence of alternative splicing in bcr-abl mRNA in chronic myeloid leukemia cell lines
Authors: Hermans A , Selleri L , Gow J , Grosveld GC
Published: October 2019

The major consequence of the Philadelphia (Ph) translocation in chronic myeloid leukemia (CML) is the formation of a bcr-abl hybrid oncogene encoding a tumor cell-specific protein P210bcr-abl. In contrast to this, in Ph chromosome-positive acute lymphoblastic leukemia (Ph + ALL), a P190bcr-abl can be observed. This P190bcr-abl has been implicated in acute rather than chronic leukemogenesis. Therefore, it can be hypothesized that the transition from chronic to blast phase in CML is accompanied by an alternative splice in the bcr-abl mRNA, which results in a switch of the production of P210bcr-abl into P190bcr-abl. Initial S1 nuclease protection mapping supported this theory. However, this result appears to be based on an artifact in the S1 analysis. By using the polymerase chain reaction we provide evidence for the absence of alternative splicing in bcr-abl mRNA in two CML blast crisis cell lines.
Differential expression of CD11b/CD18 (Mo1) and myeloperoxidase genes during myeloid differentiation
Authors: Rosmarin AG , Weil SC , Rosner GL , Griffin JD , Arnaout MA , Tenen DG
Published: October 2019

During the course of differentiation of early human myeloid cells toward monocytes and granulocytes, cell surface expression of the cell adhesion molecule, CD11b/CD18 (Mo1) increases dramatically and expression of myeloperoxidase (MPO), a bacteriocidal enzyme, decreases markedly. Using the inducible promyelocytic cell line HL-60 as a model, we studied the mRNA expression of these genes. Differentiation of these cells along both a monocytic and a granulocytic pathway demonstrated that the mRNA levels of the two subunits of CD11b/CD18 increased in a pattern temporally and quantitatively similar to the increase in cell surface expression of this heterodimer. In contrast, the expression of MPO mRNA decreased in a temporal and quantitative pattern similar to the known decrease in MPO protein during differentiation, suggesting that regulation of these myeloid-specific proteins may occur at the level of mRNA expression. These findings have important implications with regard to the nature of the block in differentiation in acute nonlymphocytic leukemia and the regulation of myeloid gene expression.
Vitamin D metabolite-mediated hypercalcemia and hypercalciuria patients with AIDS- and non-AIDS-associated lymphoma
Authors: Adams JS , Fernandez M , Gacad MA , Gill PS , Endres DB , Rasheed S , Singer FR
Published: October 2019

Fifteen patients with lymphoma and hypercalcemia (greater than or equal to 11.0 mg/dL) were identified by screening the serum chemistry profile obtained from patients upon admission to the Los Angeles County/USC Medical Center. Seven of the 15 (47%) possessed a frankly elevated serum concentration of 1,25-dihydroxyvitamin D [1,25-(OH)2-D]. An additional patient with severe hypercalcemia (16.2 mg/dL) had a serum 1,25-(OH)2-D concentration in the midnormal range, not a suppressed value. To examine the potential existence of hypercalciuria in absence of overt hypercalcemia, prospective screening of 23 normocalcemic patients with lymphoma was undertaken. Four of the 23 patients (17%) had increased fractional urinary calcium excretion rates (0.35 +/- 0.3 mg calcium/100 mL glomerular filtrate [GF], mean +/- SE; normal, less than 0.16 mg/100 mL GF); two of the hypercalciuric patients had a frankly elevated serum 1,25-(OH)2-D concentration. Of the 19 hypercalcemic/hypercalciuric lymphoma patients identified, none had an elevated serum immunoreactive parathyroid hormone concentration. Fourteen of the 19 hypercalcemic/hypercalciuric patients (74%) suffered from B-cell neoplasms, three had Hodgkin's lymphoma, and two had adult T-cell leukemia/lymphoma. All hypercalcemic/hypercalciuric patients had widespread disease (stage III or IV). Six patients, four with hypercalcemia and two with hypercalciuria, had acquired immunodeficiency syndrome (AIDS). These data suggest that the deregulated synthesis of a 1,25-(OH)2-D-like metabolite is a common cause of hypercalcemia and hypercalciuria in patients with lymphoma including patients with AIDS-associated tumors.
Granulocyte-macrophage colony-stimulating factor (CSF) and multilineage CSF recruit human monocytes to express granulocyte CSF
Authors: Oster W , Lindemann A , Mertelsmann R , Herrmann F
Published: October 2019

We assessed the capacity of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and multilineage (Multi)-CSF to induce release of granulocyte-CSF (G-CSF) by highly purified peripheral blood monocyte (Mo) preparations. Our results reveal that GM-CSF and Multi-CSF, either alone or in a synergistic concert, activate Mo to transcribe G-CSF messenger (m) RNA and release biologically active G- CSF protein into their culture supernatants. G-CSF had no regulatory effect on Mo expression of cytoplasmic G-CSF mRNA levels and G-CSF protein secretion by itself. These differential actions of CSFs provide further insight into self-regulatory mechanisms within the growth factor hierarchy system.
Demonstration of a blood-bone marrow barrier to macrophage colony- stimulating factor
Authors: Shadduck RK , Waheed A , Wing EJ
Published: October 2019

Several previous studies suggested that murine macrophage colony- stimulating factor (CSF-1) might have impaired access to hematopoietic cells in the marrow. The apparent lack of hematopoietic responses to exogenous CSF and the finding of available or unoccupied CSF receptors despite saturating CSF levels in the serum led to studies of a potential blood-bone marrow barrier for this factor. Groups of mice were injected with pure unlabeled CSF-1 by either intravenous (IV) or intraperitoneal (IP) routes. Marrow and spleen cells were obtained at intervals after injection, held at 0 degree C, and assessed for changes in binding of 125I-CSF. Saturation of all available CSF receptors is achieved in vitro with 100 to 150 U CSF/mL. Despite achieving serum levels of 5,000 to 7,000 U/mL after IV injection of 25,000 units of CSF, less than 50% of the marrow receptors and less than 85% of the splenic receptors were saturated or downregulated. The decline in receptor availability was transient, with return of receptor sites in two to four hours. Increasing the IV dose to 125,000 units increased serum CSF values to approximately 20,000 U/mL and led to a virtual disappearance of available receptors for two to three hours. When administered IP, only approximately 40% of marrow and 80% of splenic receptors were affected for two hours. It was necessary to increase the dose of CSF to 250,000 units IP to saturate or downregulate receptors for three to four hours after injection. These observations indicate a marked blood-bone marrow barrier and lesser blood-spleen barrier for the transfer of serum CSF to responsive hematopoietic cells in vivo.
Rearrangement of the T-cell receptor delta genes in human T-cell leukemias
Authors: Foroni L , Laffan M , Boehm T , Rabbitts TH , Catovsky D , Luzzatto L
Published: October 2019

Two distinct types of T-cell receptors (TCR), designated alpha beta and gamma delta, have been identified on the surface of T cells. In the adult, T cells bearing the gamma delta TCR are a minority and they have the phenotype CD3+, CD4-, CD8-/+. By using appropriate probes, rearrangements of the TCR alpha, beta, and gamma genes have been extensively investigated in a variety of lymphoproliferative disorders. Because the TCR delta gene has been cloned only recently, no comparable information exists with respect to this in human leukemias. We report the analysis of the TCR delta gene configuration in 21 T-cell acute and chronic leukemias, 40 B-cell leukemias, 4 acute myeloid leukemias of difficult classification, and 12 normal controls. The TCR delta genes were structurally modified in all T-cell disorders and in germ-line configuration in all controls and all but one case of non-T-cell leukemias tested. In one case of T-chronic lymphocytic leukemia (CD3+, CD4-, CD8+) we found rearrangement and expression of TCR gamma and delta (but not alpha and beta), suggesting that leukemic transformation took place in a cell bearing a TCR gamma delta rather than a TCR alpha beta. In two cases of pre-T-acute lymphoblastic leukemia, only delta was rearranged out of the three TCR genes tested. This finding is in keeping with the suggestion that the TCR delta gene might be the first to rearrange in T cell ontogeny, and that its mode of rearrangement may play a role in the subsequent choice of the cell between production of a TCR alpha beta or gamma delta. Thus, TCR delta chain gene analysis can provide novel information of the clonal nature of T-cell disorders, particularly if the analysis of the beta and gamma genes has not been helpful.
Alternating v repeated postremission treatment in adult acute myelogenous leukemia: a randomized phase III study (AML6) of the EORTC Leukemia Cooperative Group
Authors: Zittoun R , Jehn U , Fiere D , Haanen C , Lowenberg B , Willemze R , Abels J , Bury J , Peetermans M , Hayat M
Published: October 2019

The value of a postremission treatment in acute myelogenous leukemia (AML), with alternating combinations of non-cross-resistant drugs, has been prospectively assessed. Of 515 evaluable patients, 347 (67.4%) entered into complete remission (CR), following induction treatment with daunorubicin (DNR), vincristine (VCR), and cytosine arabinoside (ara-C). After one consolidation course, 248 patients were randomized for six courses of intensive maintenance: either repeated treatment with DNR-VCR-ara-C, or alternating treatment where amsacrine (AMSA) was combined with high dose ara-C on cycle 1,3, and 5 and with 5- azacytidine on cycle 2, 4, and 6. Ninety-nine patients were not randomized: 57 were introduced in a bone marrow transplantation (BMT) program, and 42 went off study, mainly for treatment toxicity or refusal. The main prognostic factors for achievement of CR were performance status, cytogenetics, and age, and for the disease-free survival (DFS): age and number of courses to CR. The rate of second remission was fairly high (64%) for patients relapsing off therapy. The DFS appeared identical (median, 53 weeks), in the two randomized arms, the alternating treatment not showing superiority to the repeated one, in spite of an increased toxicity. The median overall survival for patients achieving a CR was 90 weeks. The reason for the failure of alternating maintenance treatment to improve the DFS is probably related to an insufficient dose intensity: five patients who relapsed during maintenance arm B achieved a second CR with a more intensive combination of high-dose ara-C and AMSA. In addition, 60 patients underwent a BMT (43 allogeneic and 17 autologous). The DFS of patients treated with allogeneic BMT tended to be superior to the one obtained with the chemotherapy program. However the overall survival, as well as the event-free survival, seemed equivalent, including patients who relapsed before the planned BMT. Comparisons between allogeneic BMT, autologous BMT, and intensive consolidation during first CR deserve further prospective studies in AML.
Inhibition against CFU-C and CFU-E colony formation by soluble factor(s) derived from hairy cells
Authors: Taniguchi N , Kuratsune H , Kanamaru A , Tokumine Y , Tagawa S , Machii T , Kitani T
Published: October 2019

The inhibitory effect by hairy cell conditioned medium (HCCM) on the growth of granulocyte and erythrocyte colony forming cells was studied in vitro. The percent inhibition of CFU-C formation by HCCM from four hairy cell leukemia (HCL) patients ranged from 36% to 76%, while no inhibition was observed with conditioned medium (CM) obtained from three B-cell chronic lymphocytic leukemia (B-CLL) patients nor from two normal controls. HCCM inhibited specially the growth of rG-CSF responding stem cells. The hairy cell-derived colony inhibitory factor from HCCM was nondialyzable, fairly stable to heat treatment, and trypsin sensitive. Its maximal inhibitory activity against granulopoiesis was observed in the fractions of 5,000 to 6,000 daltons. Moreover HCCM inhibited CFU-E colony formation but not BFU-E. These results indicate that hairy cells produce a factor that inhibits granulopoiesis and erythropoiesis in vitro. This factor may play a role in neutropenia and anemia observed in HCL.
Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin
Authors: Bolyard MG , Lord ST
Published: October 2019

The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.
Immune reconstitution following bone marrow transplantation: comparison of recipients of T-cell depleted marrow with recipients of conventional marrow grafts
Authors: Keever CA , Small TN , Flomenberg N , Heller G , Pekle K , Black P , Pecora A , Gillio A , Kernan NA , O'Reilly RJ
Published: October 2019

The reconstitution of hematopoietic cells and in vitro assays of immunologic function have been followed in leukemic patients after conventional bone marrow transplantation (BMT) (N = 34) and T-cell depleted BMT (N = 52) from human leukocyte antigen (HLA)-identical sibling donors. No effects of the T-cell depletion could be seen on the recovery of myeloid or lymphoid cells as measured by the day to engraftment or by the absolute number of cells through day 100. Normal numbers of lytically active natural killer cells returned the earliest and were rapidly followed in both groups of patients by the appearance of circulating B cells and normalization of the responses to B-cell mitogens. However, the recovery of normal T-cell proliferative responses were more delayed in recipients of T-cell depleted grafts. Significant quantitative differences were seen only during the first 3 months after transplantation. Neither the number of CD3+ T cells nor the ratio of CD4:CD8 positive cells differed markedly between the two transplant groups. Mitogen-induced immunoglobulin production by peripheral blood lymphocytes (PBL) from patients following T-cell depleted BMT was quantitatively less than that of conventional marrow recipients through the first year, with low normal IgM production reached by 4 to 6 months in both groups. IgG production reached low normal 7 to 9 months after conventional BMT but did not remain at this level until 1 year following either type of transplant. Assessment of the incidence of infections from the day the absolute neutrophil count reached 500 until day 180 after transplant revealed no significant differences between the two groups; indeed, the overall nonleukemic mortality was higher in the recipients of conventional bone marrow. Thus, in our series, the removal of mature cells from the marrow graft did not affect the rate or degree of recovery of myeloid and lymphoid cells but did affect the regeneration of in vitro T-cell dependent functions. We noted early quantitative differences and a delay in the normalization of the T-cell functions measured rather than prolonged absolute deficiencies. The in vitro deficiencies did not result in significant clinically apparent differences between the two groups.
Endogenously generated activated killer cells circulate after autologous and allogeneic marrow transplantation but not after chemotherapy
Authors: Reittie JE , Gottlieb D , Heslop HE , Leger O , Drexler HG , Hazlehurst G , Hoffbrand AV , Prentice HG , Brenner MK
Published: October 2019

After marrow transplantation, major histocompatibility complex (MHC)- unrestricted natural killer (NK) lymphocytes are among the first cells to appear in the circulation. After T-cell-depleted bone marrow transplantation (TD-BMT), these cells have an activated pattern of target cell killing; they also secrete lymphokines including gamma- interferon (gamma-IFN), interleukin-2 (IL-2), and tumor necrosis factor (TNF) and may have a significant role as a primary defense against viral reactivation and in the elimination of residual host malignancy. We studied 43 patients with hematologic malignancy, treated by allogeneic TD-BMT, autologous nondepleted BMT, or chemotherapy alone to investigate (a) the mechanisms underlying the generation of these activated killer cells, (b) the range of conditions under which they are produced, and (c) their surface phenotype. We showed that gamma-IFN- secreting activated killer cells with the capacity to kill MHC- nonidentical NK-resistant targets are generated 4 to 6 weeks after either allogeneic TD-BMT or autologous BMT but do not appear after treatment with chemotherapy. Production therefore is not owing to T- cell depletion per se or to host donor alloreactivity, nor is it caused by stimulation by alloantigens contained in blood product support since no significant difference exists between allograft and chemotherapy patients in the number of units of blood platelet support given in the posttreatment period. Because most patients had no evidence of stimulation from virus reactivation/infection, the phenomenon of activation therefore appears to represent posttransplant immune disregulation following repopulation of the host immune system with lymphoid subsets derived exclusively from blood and marrow. Activated killing is predominantly mediated by the CD16+ CD3- subset, but substantial activity remains in the CD16- CD3+ cell fraction. Monoclonal antibodies (MoAbs) that block interaction with class-I MHC molecules at the level of target cell (W6/32 anti-HLA class I) or effector cell (CD8) do not inhibit killing by CD16- CD3+ cells. Activated killer cells may contribute to the lower risk of relapse after marrow transplantation as compared with intensive chemotherapy.
Deleterious effects of irradiation and bone marrow transplantation therapy in the genetically anemic an/an mouse
Authors: Eppig JT , Barker JE
Published: October 2019

The efficacy and outcome of bone marrow transplantation therapy following lethal irradiation were examined in syngeneic mice that had a hereditary macrocytic anemia (an/an) or were genotypically normal (+/+). Successful RBC and WBC replacement, based on blood cell parameters and donor genetic markers, were observed in all combinations of transplant therapy. Nevertheless, the an/an mice died prematurely several months after treatment, whether they received +/+ or an/an marrow cells. In contrast, the +/+ recipients of either +/+ or an/an marrow cells survived for at least 1 year after transplantation. Premature death of the an/an mice was associated with lymphopenia, anemia, kidney lesions, and severe pathogen-free pneumonitis. On the basis of our results, we hypothesize that the premature deaths of an/an mice are caused by a kind of chronic irradiation damage to which an/an mice are especially susceptible.
Calcium-dependent proteolysis of actin during storage of platelet concentrates
Authors: Snyder EL , Horne WC , Napychank P , Heinemann FS , Dunn B
Published: October 2019

In an ongoing study of the changes that occur in platelet concentrates during storage, we investigated two 28–26-Kd proteins designated SP–1 and SP–2, respectively, which increase markedly during blood-bank storage of platelet concentrates at room temperature. Formation of SP–1 and SP–2 was inhibited by storage at 4 degrees C as well as by treatment of the concentrates with leupeptin, N–ethylmaleimide, and EDTA; DFP and PPACK had no effect. The calcium ionophore A23187 markedly stimulated production of SP–1 and SP–2. After partial purification, the two proteins were found to be associated with platelet cytoskeletal protein. Two-dimensional peptide mapping and amino acid sequencing identified SP-1 and SP-2 as fragments of actin formed by cleavage on the N-terminal side of residues Thr–106 and Ala- 114, respectively. Generation of SP–1 and SP–2 during storage of platelet concentrate is likely attributable to calcium-dependent neutral protease degradation of actin and may have implications for development of the platelet storage lesion.
Platelet membrane glycoprotein IIb heavy chain forms a complex with glycoprotein IIIa that binds Arg-Gly-Asp peptides
Authors: Lam SC , Plow EF , Ginsberg MH
Published: October 2019

Platelet membrane GPIIb is comprised of a disulfide-linked heavy chain (GPIIb(H)) and light chain (GPIIb(L)). We have examined the role of the two chains of GPIIb in the maintenance of the GPIIb-IIIa heterodimer and Arg-Gly-Asp (RGD) peptide-binding function. Lysates of surface radioiodinated platelets were treated with 1% 2-mercaptoethanol for 18 hours at 4 degrees C. Reduction of the interchain disulfide in GPIIb was followed by immunoprecipitation with antipeptide antibodies specific for GPIIb(H) or GPIIb(L). In addition to the GPIIb-IIIa complex, a polypeptide of 120 Kd was precipitated by anti-GPIIb(H) and a polypeptide of 23 Kd was precipitated by anti-GPIIb(L) from reduced platelet lysates. To determine whether GPIIb(H) or GPIIb(L) remained complexed with GPIIIa, reduced platelet lysates were immunoprecipitated with AP3, a monoclonal anti-GPIIIa antibody, resulting in the coimmunoprecipitation of GPIIb(H) but not GPIIb(L). Conversely, the monoclonal anti-GPIIb(H) antibody PMI-1 immunoprecipitated GPIIIa with GPIIb(H). Thus GPIIb(H) maintains its association with GPIIIa. Furthermore, the GPIIb(H)-IIIa complex retains its reactivity with AP2, a monoclonal antibody (MoAb) specific for the nondissociated GPIIb-IIIa complex. Affinity chromatography of reduced platelet lysates on immobilized KYGRGDS resulted in binding and specific elution of the GPIIb(H)-IIIa complex. These findings indicate that GPIIb(H) contains sufficient information for maintenance of a complex with GPIIIa and support of the binding of the heterodimer to RGD peptides.
Radiation sensitivity of resting and activated nonspecific cytotoxic cells of T lineage and NK lineage
Authors: Zarcone D , Tilden AB , Lane VG , Grossi CE
Published: October 2019

Natural killer (NK) cell-mediated killing of tumor cells is a radiation- sensitive function that in most subjects is completely abrogated by treatment of the effector cells with 3,000 cGy. The radiation sensitivity of LAK (lymphokine-activated killer) cells and their precursors, the bulk of which are NK cells, is undetermined. In this study, functional cytotoxicity assays and electron microscopy were used to determine the effect of radiation on the cytotoxic function of NK cells, LAK cells (generated by three-day culture of peripheral blood lymphocytes with IL-2), and LAK cell precursors (lymphocytes irradiated prior to culture with IL-2). For comparison, we analyzed the radiation sensitivity of lectin-dependent cell-mediated cytotoxicity (LDCC), which is primarily a function of CD3+ CD8+ granular lymphocytes. We also analyzed the radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells (AK activity). Following 3,000 cGy irradiation, NK cells retained their ability to bind to tumor cell targets but, as shown by both morphologic and functional analyses, they did not undergo activation after conjugate formation, and were unable to release the content of their granules. In order to evaluate LDCC, lymphocytes were depleted of CD16+ cells and tested in a cytotoxicity assay in the presence of Con A. The radiation sensitivity curve was comparable to that of NK cell-mediated cytotoxicity. IL-2-treated lymphocytes (LAK cells) were relatively radioresistant as compared with untreated NK cells, and their cytotoxic function was not abrogated until treatment with greater than 10,000 cGy. Cells receiving such radiation doses displayed cytoplasmic blebbing and damage of their cytoskeletal structures, with disruption of centrioles and microtubules, and disarray of the intermediate filaments. As was shown with NK cells, irradiated LAK cells formed conjugates with tumor targets but failed to degranulate. The radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells was identical to that of LAK effector cells. Doses up to 2,000 cGy did not prevent generation of LAK cells from blood lymphocytes, but 3,000 cGy did so. Blast transformation similar to that observed in IL-2- stimulated controls occurred when lymphocytes irradiated with 3,000 cGy were cultured with IL-2. These transformed cells were not cytotoxic and displayed a normal cytoskeletal apparatus but did not bear electron- dense granules.(ABSTRACT TRUNCATED AT 400 WORDS)
Profiles of expression of activated cell antigens on peripheral blood and lymph node cells from different clinical stages of adult T-cell leukemia
Authors: Shirono K , Hattori T , Hata H , Nishimura H , Takatsuki K
Published: October 2019

The expression of activated cell antigens (Ags) on adult T cell leukemia (ATL) cells at different clinical stages (acute, chronic, and smoldering), and ATL cells from enlarged lymph nodes were studied using monoclonal antibodies (MoAbs). The expressions of CD25 (Tac), CD28, T9 (transferrin receptor), and Ki-67 (proliferating cell nuclear antigen) Ags were high, and that of CD7 Ag was low in both acute and chronic ATL cells compared with that in normal T cells. The expressions of T9 and Ki-67 Ags were higher in acute ATL cells than in chronic ATL cells. The expression of CD38 Ag was higher in acute ATL cells but not in chronic ATL cells compared with normal T cells. On the contrary, the expression of HLA-DR Ag on the cell surface and the transcript of the HLA-DR Ag gene were high in chronic ATL cells, but not in acute ATL cells. A high percentage of lymph node ATL cells expressed T9, CD38, and Ki-67 Ags, although the peripheral blood ATL cells of the same patients showed significantly lower levels of expression of the same Ags. Interestingly, CD7 and HLA-DR Ags were detected on the majority of lymph node ATL cells. Moderately high percentage of smoldering ATL cells expressed CD25, CD28, and HLA-DR Ags, though a few numbers of leukemic cells were present in the samples. These findings confirm that the modes of activation of ATL cells in different clinical stages differ, and suggest that the differences might be reflected in the clinical features. The low expression of HLA-DR Ag in acute ATL cells, despite high expression of the Ag in chronic ATL cells, suggests that successive events that suppress HLA-DR Ag expression are necessary for the transformation of chronic ATL cells into acute ATL cells. Moreover, the high expressions of T9, CD38, Ki-67, CD7, and HLA-DR Ags on lymph node ATL cells indicate that ATL cells preferentially proliferate in lymph nodes.
Interleukin-3 is significantly more effective than other colony- stimulating factors in long-term maintenance of human bone marrow- derived colony-forming cells in vitro
Authors: Kobayashi M , Van Leeuwen BH , Elsbury S , Martinson ME , Young IG , Hapel AJ
Published: October 2019

Human bone marrow cells cultured for 21 days in the presence of recombinant human interleukin-3 (IL-3) produced up to 28 times more colony-forming cells (CFC) than could be obtained from cultures stimulated with granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF). IL-3-cultured cells retained a multipotent response to IL-3 in colony assays but were restricted to formation of granulocyte colonies in G-CSF and granulocyte or macrophage colonies in GM-CSF. Culture of bone marrow cells in IL-3 also led to accumulation of large numbers of eosinophils and basophils. These data contrast with the effects of G-CSF, GM-CSF, and IL-3 in seven-day cultures. Here both GM-CSF and IL-3 amplified total CFC that had similar multipotential colony-forming capability in either factor. G-CSF, on the other hand, depleted IL-3-responsive colony-forming cells dramatically, apparently by causing these cells to mature into granulocytes. The data suggest that a large proportion of IL-3- responsive cells in human bone marrow express receptors for G-CSF and can respond to this factor, the majority becoming neutrophils. Furthermore, the CFC maintained for 21 days in IL-3 may be a functionally distinct population from that produced after seven days culture of bone marrow cells in either IL-3 or GM-CSF.
Evidence for a pretranslational defect in hereditary and acquired myeloperoxidase deficiency
Authors: Tobler A , Selsted ME , Miller CW , Johnson KR , Novotny MJ , Rovera G , Koeffler HP
Published: October 2019

Myeloperoxidase (MPO) is a heme containing enzyme involved in the oxygen-dependent microbicidal activity of human polymorphonuclear leukocytes (PMN). Complete hereditary and acquired MPO deficiencies are defined as lack of peroxidase activity in PMN. Using this criterion, we studied a patient with complete hereditary MPO deficiency, and a MPO deficient variant cell line of HL-60 (HL-60-A7), which we used as a model for acquired MPO deficiency. Western blot analysis showed complete absence of mature and precursor protein of MPO both in PMN from the patient and in HL-60-A7 cells. PMN from both parents had one half of normal levels of these proteins. To study further the molecular basis of this defect, we isolated an intron specific probe for MPO and used it and a cDNA probe. Both normal human bone marrow cells and the promyelocytic HL-60 leukemia cells contained MPO mRNA species of 2.8, 3.3, approximately 4, and greater than 8 kilobase (kb). The transcripts of greater than 8 and approximately 4 kb contained sequences hybridizing to a probe specific for intron 7 of the MPO gene. Bone marrow cells of the MPO deficient patient contained two species of heterogeneous nuclear (hn) RNA of greater than 8 and approximately 4 kb, but only trace amounts of the normal sized 3.3 kb MPO mRNA and undetectable 2.8 kb MPO mRNA. HL-60-A7 cells contained both greater than 8 and approximately 4 kb hnRNA, but only small amounts of normal sized 2.8 kb MPO mRNA and undetectable levels of the 3.3 kb mRNA. Southern blot analyses revealed no gross alteration of the MPO gene in both cases. Our results suggest that a pretranslational defect is one mechanism leading to MPO deficiency.
Microangiopathy in patients on cyclosporine prophylaxis who developed acute graft-versus-host disease after HLA-identical bone marrow transplantation
Authors: Holler E , Kolb HJ , Hiller E , Mraz W , Lehmacher W , Gleixner B , Seeber C , Jehn U , Gerhartz HH , Brehm G
Published: October 2019

Severe microangiopathy has been reported as a rare complication of cyclosporine A (CsA) prophylaxis in allogeneic bone marrow transplantation (BMT). We found morphological and biochemical changes indicative of generalized endothelial damage in 49 of 66 allogeneic marrow graft recipients receiving cyclosporine, but none in 11 patients treated with methotrexate for prophylaxis of graft-v-host disease (GVHD). Changes occurred after engraftment of bone marrow and consisted of intravascular hemolysis with red cell fragmentation and de novo thrombocytopenia. They were preceded by a decrease in activated partial thromboplastin time and fibrinogen indicating activation of coagulation. Endothelial damage as the central lesion of microangiopathy was confirmed by a simultaneous increase of factor VIII related antigen. Severe microangiopathy was observed in ten patients and was fatal in seven. Risk factor analysis revealed a highly significant association of microangiopathy with severity of acute GVHD (aGVHD) (P less than .001) and use of CsA prophylaxis (P less than .001). Our data suggest endothelial damage as a result of cellular activation and subsequent release of cytokines in the course of a aGVHD, which is not inhibited by CsA prophylaxis.
Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts
Authors: Lapidot T , Terenzi A , Singer TS , Salomon O , Reisner Y
Published: October 2019

A major problem in using murine models for studies of bone marrow allograft rejection in leukemia patients is the narrow margin in which graft rejection can be analyzed. In mice irradiated with greater than 9 Gy total body irradiation (TBI) rejection is minimal, whereas after administration of 8 Gy TBI, which spares a significant number of clonable T cells, a substantial frequency of host stem cells can also be detected. In current murine models, unlike in humans, bone marrow allograft rejection is generally associated with full autologous hematopoietic reconstitution. In the present study, we investigated the effect of the myeloablative drug dimethyl myleran (DMM) on chimerism status following transplantation of T cell-depleted allogenic bone marrow (using C57BL/6 donors and C3H/HeJ recipients, conditioned with 8 Gy TBI). Donor type chimerism 1 to 2 months post-transplant of 1 to 3 x 10(6) bone marrow cells was markedly enhanced by using DMM one day after TBI and prior to transplantation. Conditioning with cyclophosphamide instead of DMM, in combination with 8 Gy TBI, did not enhance engraftment of donor type cells. Artificial reconstitution of T cells, after conditioning with TBI plus DMM, by adding mature thymocytes, or presensitization with irradiated donor type spleen cells 1 week before TBI and DMM, led to strong graft rejection and consequently to severe anemia. The anti-donor responses in these models were proportional to the number of added T cells and to the number of cells used for presensitization, and they could be neutralized by increasing the bone marrow inoculum. These results demonstrate the potential of DMM to facilitate engraftment in unsensitized mice in which the host stem cells may compete with donor type cells; the use of DMM to create models in which mechanisms of immune rejection can be studied without interference due to stem cell competition; and that bone marrow allograft rejection may be overcome by increasing the bone marrow inoculum in these stringent models.
Antibody-radionuclide conjugates as part of a myeloablative preparative regimen for marrow transplantation
Authors: Appelbaum FR , Brown P , Sandmaier B , Badger C , Schuening F , Graham T , Storb R
Published: October 2019

The behaviors of an anti-Ia antibody (7.2) and an antibody directed at a lymphocyte adhesion molecule (S.5) radiolabeled with 131I were studied in normal dogs. Antibody 7.2 localized to spleen and, to a lesser extent, to marrow and lymph nodes. Antibody S.5 rapidly localized to marrow and spleen, achieving tissue/blood ratios greater than 6:1 within three hours of injection that were maintained for at least 48 hours. Prior treatment with cyclophosphamide (CY) markedly altered the distribution of S.5 but had much less effect on the distribution of 7.2 and almost no effect on the distribution of a control antibody. When animals were treated with increasing doses of 131I labeled to S.5, lethal myelosuppression occurred when a dose of 6 mCi/kg was reached. At this dose, the otherwise lethal effects of 131I could be reversed with autologous marrow transplant support.
Human plasma extrinsic pathway inhibitor activity: I. Standardization of assay and evaluation of physiologic variables
Authors: Warr TA , Warn-Cramer BJ , Rao LV , Rapaport SI
Published: October 2019

An assay was standardized to measure extrinsic pathway inhibitor (EPI) activity in human plasma. Variables that could potentially influence its measurement were systematically examined. The coefficient of variation of the assay was 6.3% for the same sample assayed on different days. The linear regression line for a plot of observed v expected values of mixtures of plasmas with different EPI levels was Y = 1.01X - 2.7%. Single samples from 21 healthy adults under 60 years of age varied between 74% and 159% of a pooled reference plasma. The plasma level of a given individual (eight subjects) did not vary on repeat sampling over weeks to months. EPI activity was significantly lower in plasma from umbilical cord blood (64.3% +/- 12.7%, n = 16) than in plasma from adults. Mean EPI activity in adults greater than or equal to 60 years of age was slightly but significantly higher (112% +/- 16.8%, n = 23) than in adults less than 60 years of age (97.2% +/- 19.0%, n = 21). EPI levels in the third trimester of pregnancy were slightly higher than in nonpregnant women. Plasma EPI levels fell slightly after surgical procedures that caused fibrinogen levels to rise, which suggests that EPI is not an acute phase reactant. Administration of 1-desamino-8-d-arginine vasopressin (DDAVP) did not alter plasma EPI levels. In two patients subjected to plasmapheresis and volume replacement with albumin and isotonic saline, plasma EPI levels returned to one-half of the levels before pheresis within about one day.
Molecular cloning of a cDNA encoding canine factor IX
Authors: Evans JP , Watzke HH , Ware JL , Stafford DW , High KA
Published: October 2019

Factor IX (F.IX) is a vitamin K-dependent plasma protein, a deficiency of which results in hemophilia B. A canine model of hemophilia B exists; attempts to use this model for gene transfer experiments or characterization of the hemophilic defect require elucidation of normal canine F.IX structure. We report the isolation and characterization of the coding region for canine F.IX cDNA. Canine F.IX possesses 86% identity at the amino-acid level with human F.IX. The leader peptide, Gla domain, EGF domains, and the carboxy-terminal portion of the heavy chains show extensive sequence conservation between the canine and human. All Glu residues undergoing gamma-carboxylation in humans are conserved in canines. The complete coding sequence for canine F.IX has been determined, and the derived translation product has been analyzed. A similar approach should allow identification of the causative mutation in canine hemophilia B. Furthermore, this clone may prove a valuable resource in gene transfer experiments for this disease.