bluuebunny/crossref_metadata_2025_split huggingface dataset:
T-lymphocyte killing by T101-ricin A-chain immunotoxin: pH-dependent potentiation with lysosomotropic amines
Authors: Casellas P , Ravel S , Bourrie BJ , Derocq JM , Jansen FK , Laurent G , Gros P 
Published: October 2019

Abstract
To maximize T-lymphocyte killing with anti-pan-T-lymphocyte immunotoxin (IT), prepared by linking ricin A-chain to monoclonal antibody (MoAb) T101 (T101-RTA IT), we have established the nature and the extent of parameters that influence the sensitivity of T lymphocytes to the IT. We showed that peripheral blood T lymphocytes, which are much less susceptible than malignant T cells to the T101-RTA IT, could become highly sensitive to the IT when used in conjunction with NH4Cl. However, enhancement of the IT by NH4Cl only occurred when the pH rose above neutrality. This pH-sensitive process of IT activation by NH4Cl, which led to an all-or-nothing effect within an extremely narrow pH window of 0.7 pH unit width, is due to the fact that NH3 is the effective enhancing component of NH4Cl. We also showed that F(ab')2 or Fab containing IT were much more effective than those produced using the whole IgG counterpart. From these data, we defined a procedure for an optimal and specific elimination of T lymphocytes in vitro by treating them with (Fab)T101-RTA at 10(-8) mol/L at pH 7.8 in the presence of NH4Cl for two hours. This peripheral blood cell processing elicited an abrogation of three logs of functional T-cell response. Under the same conditions, there was no reduction in the number of marrow hematopoietic precursor granulocyte-macrophage colony-forming units (CFU-GM).
Elimination of myeloma cells from bone marrow by using monoclonal antibodies and magnetic immunobeads
Authors: Shimazaki C , Wisniewski D , Scheinberg DA , Atzpodien J , Strife A , Gulati S , Fried J , Wisniewolski R , Wang CY , Clarkson BD 
Published: October 2019

Abstract
The efficacy of immunomagnetic beads to purge human myeloma cells from bone marrow ex vivo was evaluated. The optimal conditions for purging were studied first by using three myeloma cell lines: RPMI-8226, SKO- 007, and SKMM-2. Myeloma cells labeled with the vital fluorescent dye Hoechst 33342 were admixed with normal bone marrow cells, and two monoclonal antibodies reactive with the myeloma cells (PCA-1 and BL-3) were added alone or in combination with the cells. Magnetic beads coated with goat antimouse immunoglobulin G were then added, and the tumor cells to which beads were attached were separated from the mixture with a magnet. The efficacy of tumor cell removal was dependent on the bead-to-tumor ratio; a ratio of more than 500 was optimal in the presence of excess normal marrow cells. The combination of monoclonal antibodies PCA-1 and BL-3 increased the tumor cell removal as compared with either antibody alone. Two cycles of treatment were more effective than one cycle was. Under optimal conditions, 2.3 to 4 logs of tumor cells could be removed from the mixture containing 10% myeloma cells without a significant loss of normal hematopoietic progenitors as measured by CFU-GM, CFU-GEM, and BFU-E. When the efficacy of this procedure was tested on fresh bone marrow from patients with multiple myeloma (MM) by using the combination of PCA-1, BL-3, and J-5, 1.6 to 2.5 logs of tumor cells could be removed by one cycle of treatment, even from marrows containing less than 10% myeloma cells. These observations support the use of monoclonal antibody combinations and immunobeads as a reliable and nontoxic method to eliminate contaminating myeloma cells ex vivo in preparation for autologous bone marrow transplantation in patients with MM.
What radiation dose for DLA-identical canine marrow grafts? [published erratum appears in Blood 1989 Feb;73(2):624]
Authors: Storb R , Raff RF , Appelbaum FR , Schuening FW , Sandmaier BM , Graham TC , Thomas ED 
Published: October 2019

Abstract
In view of reported attempts at marrow grafting after nuclear accidents with a broad range of radiation exposures, the present study explored the total-body irradiation (TBI) conditions needed for engraftment in a canine model by using marrow from DLA-identical littermates. Previous studies have shown that such grafts are consistently successful when recipients are exposed to 920 cGy of TBI delivered at a rate of 7 cGy/min from opposing dual cobalt sources. The present TBI doses were all in the lethal range. Five dogs were administered 450 cGy; seven dogs, 600 cGy; five dogs, 700 cGy; and five dogs, 800 cGy of TBI administered at 7 cGy/min. They received a median of 3.3 x 10(8) marrow cells/kg intravenously after completion of radiation. Results showed transient allogeneic marrow engraftment in all dogs administered the lowest dose of TBI studied (450 cGy). Importantly, transient grafts permitted four of five dogs to live long enough for autologous marrow recovery to occur. At increasing radiation doses, 600, 700, and 800 cGy, the risk of graft failure lessened, with 3 of 7, 2 of 5, and 1 of 5 dogs, respectively, showing graft rejection. Fewer dogs survived with autologous marrow recovery, and more showed sustained allogeneic engraftment (4 of 7, 3 of 5, and 4 of 5 dogs, respectively). We conclude that DLA-identical littermate marrow grafts are beneficial in the setting of otherwise lethal radiation exposures, with most dogs either experiencing sustained allogeneic engraftment or surviving with autologous marrow recovery due to the extended support provided by a transient allogeneic graft.
Correction of canine cyclic hematopoiesis with recombinant human granulocyte colony-stimulating factor
Authors: Lothrop CD Jr , Warren DJ , Souza LM , Jones JB , Moore MA 
Published: October 2019

Abstract
Canine cyclic hematopoiesis (CH) is an autosomal recessive disease of gray collie dogs that is characterized by neutropenic episodes at 14- day intervals. The biochemical basis for CH is not known but may involve a regulatory defect of the response to or production of a hematopoietic growth factor. Administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to two CH and one normal dog caused a marked leukocytosis (greater than 50,000 WBCs) in all three dogs. The leukocytosis was due largely to a greater than tenfold increase in neutrophils. Less pronounced but significant elevations in monocytes occurred during G-CSF treatment. The elevated WBC count was maintained for more than 20 days in all three dogs, and two predicted neutropenic episodes were prevented in both CH dogs during rhG-CSF treatment. A decline in the WBC count occurred simultaneously in all three dogs during the last five treatment days and was presumably associated with the development of neutralizing antibodies to the heterologous rhG-CSF protein. Bone marrow evaluation indicated that the swings in the myeloid/erythroid progenitor cells that are characteristic of CH were eliminated by rhG-CSF treatment in both CH dogs. These results suggest that the regulatory defect in canine CH can be temporarily alleviated by treatment with rhG-CSF and point to the potential treatment of human cyclic neutropenia with this agent.
Pharmacokinetics of human granulocyte-macrophage colony-stimulating factor using a sensitive immunoassay
Authors: Cebon J , Dempsey P , Fox R , Kannourakis G , Bonnem E , Burgess AW , Morstyn G 
Published: October 2019

Abstract
A sensitive and reliable sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The assay is quantitative between 100 pg/mL and 2.5 ng/mL for bacterially synthesized hGM-CSF in human serum and is more sensitive and specific than the semisolid agar bioassay. As part of a phase I study, the pharmacokinetics of intravenous (IV) bolus injection and subcutaneous (SC) administration of hGM-CSF were studied. Following a single IV dose, an initial high blood level of hGM-CSF occurred, followed by a rapid decrease occurring in two apparent phases with a half-life (t1/2)alpha of less than five minutes and a t1/2 beta of 150 minutes. After an SC injection, detectable serum levels occurred within 15 to 30 minutes, and serum levels were sustained for a variable time depending on the dose. At the highest SC dose (10 micrograms/kg), a serum level of greater than 1 ng/mL (65 pmol/L) was maintained for greater than 12 hours after a single injection. This corresponds to the concentration of hGM-CSF supporting near-maximum proliferation in vitro.
Erythroid burst-promoting activity of purified recombinant human GM-CSF and interleukin-3: studies with anti-GM-CSF and anti-IL-3 sera and studies in serum-free cultures
Authors: Sonoda Y , Yang YC , Wong GG , Clark SC , Ogawa M 
Published: October 2019

Abstract
We studied the erythroid burst-promoting activity (BPA) of recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) and Interleukin-3 (IL-3) with two experimental approaches. First we studied the effects of polyclonal antisera prepared against human GM-CSF and gibbon IL-3 on colony formation from 1,000 bone marrow null cells/dish in serum-containing culture. Both GM-CSF and IL-3 independently enhanced erythroid burst formation; however, IL-3 showed more BPA activity than GM-CSF. These data are in agreement with an emerging view that the primary targets of IL-3 are primitive progenitors and that the targets of GM-CSF are intermediate progenitors, including erythroid burst-forming units (BFU-E). The proliferation of one population of BFU- E was independent of GM-CSF or IL-3. To characterize this population of BFU-E further, we developed a serum-free culture assay for the purified progenitors by incorporating insulin-like growth factor-1 (IGF-1) to the serum-free medium. The development of erythroid bursts was supported by IL-3, IGF-1, and erythropoietin (Ep) in a serum-free culture system and to a lesser extent by the combination of GM-CSF, IGF- 1, and Ep. Although the burst-promoting ability of GM-CSF and IL-3 was again demonstrated in this system, unlike serum-containing culture Ep alone did not support burst formation. These results indicate that when fetal calf serum (FCS) is present, the culture system contains BPA that is not GM-CSF or IL-3.
Effect of recombinant hematopoietic growth factors on proliferation of human marrow progenitor cells in serum-deprived liquid culture
Authors: Migliaccio AR , Migliaccio G , Adamson JW 
Published: October 2019

Abstract
We investigated the effects of recombinant interleukin-3 (IL-3), granulocyte-macrophage and granulocyte colony-stimulating factors (GM- CSF and G-CSF), and erythropoietin (Ep) on the number of human hematopoietic progenitors after two to ten days of incubation in liquid cultures deprived of fetal bovine serum (FBS). The source of progenitor cells was normal human marrow depleted of T lymphocytes and/or adherent cells. When adherent cell-depleted marrow was cultured without growth factors, the number of progenitor cells was relatively constant for periods up to eight days. In contrast, a progressive decline in the number of progenitor cells was detected in cultures of nonadherent, T- cell-depleted marrow cells. In both cases, the addition of IL-3 increased by two- to fourfold over input the number of erythroid burst- forming cells (BFU-E) per culture. The number of BFU-E peaked either at day 4 or 8. G-CSF had no effect on the number of progenitor cells per culture. GM-CSF and Ep had no effect in cultures of nonadherent marrow cells but maintained the number of BFU-E in cultures of nonadherent, T- cell-depleted marrow cells. The addition of a neutralizing anti-GM-CSF monoclonal antibody, but not anti-IL-3 neutralizing antiserum, decreased the number of BFU-E in cultures of nonadherent marrow cells. None of the growth factors investigated enhanced the number of GM progenitors to the same degree as the number of BFU-E. However, in cultures of nonadherent, T-cell-depleted marrow cells, IL-3 and GM-CSF maintained the number of GM progenitors up to eight days. These results indicate that IL-3 alone is capable of increasing the number of BFU-E and of maintaining the number of GM progenitors in liquid culture, whereas GM-CSF and Ep are capable of maintaining, but not increasing, BFU-E in this system.
Identification of monoclonal antibodies that inhibit the function of protein C inhibitor. Evidence for heparin-independent inhibition of activated protein C in plasma
Authors: Meijers JC , Vlooswijk RA , Kanters DH , Hessing M , Bouma BN 
Published: October 2019

Abstract
Monoclonal antibodies specific for protein C inhibitor (PCI) partially blocked the inactivation of activated protein C (APC) in plasma, whereas in a purified system, the PCI activity could be completely blocked. The inactivation of APC in normal and in PCI-depleted plasma was similar in the absence of heparin. The addition of heparin did not change the rate of inactivation of APC in PCI-depleted plasma, whereas in normal plasma a rapid phase of inhibition of APC was followed by a slower phase of inhibition. The slower phase was identical to the rate of inhibition of APC in the absence of heparin. After incubation of normal plasma with a monoclonal antibody specific for PCI that blocked its activity, there was no difference in heparin-dependent or heparin- independent inhibition of APC. These results indicate that in the absence of heparin PCI is unable to inactivate APC in a plasma environment.
Flow as a regulator of the activation of factor X by tissue factor
Authors: Gemmell CH , Turitto VT , Nemerson Y 
Published: October 2019

Abstract
A novel enzyme reactor for phospholipid-dependent reactions was used to study the effects of flow on tissue factor-initiated coagulation. Microcapillaries were coated with a phospholipid bilayer containing tissue factor, a transmembrane protein that is an essential cofactor for a plasma procoagulant enzyme, factor VII. We show that, in contrast to static, closed systems, the steady-state catalytic activity is independent of enzyme concentration and the time to steady state becomes a function of the enzyme concentration.
Antithrombin-III-Hamilton: a gene with a point mutation (guanine to adenine) in codon 382 causing impaired serine protease reactivity
Authors: Devraj-Kizuk R , Chui DH , Prochownik EV , Carter CJ , Ofosu FA , Blajchman MA 
Published: October 2019

Abstract
Antithrombin-III-Hamilton is a structural mutant of antithrombin III with defective serine protease reactivity, demonstrable in three members of a French Canadian family. The propositus, a 54-year-old man with a history of recurrent thromboembolic events, and his two asymptomatic grown children are heterozygous for the mutant antithrombin III gene. In all three individuals, the immunoreactive antithrombin III level is normal, while the antithrombin and antifactor Xa activity is approximately 50% of the control value. Two dimensional immunoelectrophoresis of antithrombin-III-Hamilton in the presence of heparin is normal. Purified antithrombin-III-Hamilton did not form thrombin-antithrombin III complex when incubated with thrombin for up to 30 minutes. The normal and mutant antithrombin III alleles of the propositus could be distinguished by linkage to Pstl restriction fragment length polymorphisms (RFLP). Genomic DNA from the propositus was cloned into EMBL 3 phage vectors and two clones containing nearly complete copies of the antithrombin-III-Hamilton allele were identified. Exon 6 of both clones was subcloned into M13 phage vector and sequenced, revealing a G----A point mutation in the first base of codon 382. Codon 382 codes for alanine in the normal allele and for threonine in the antithrombin-III-Hamilton allele. Alanine-382, 12 residues from the reactive center, is a highly conserved amino acid in the family of serine protease inhibitors known as the serpins. We postulate that, as a result of the substitution of threonine for alanine in antithrombin-III-Hamilton, either the tertiary structure or the hydrophobicity of the thrombin-binding region is altered, causing aberrant conformation of the Arg-393-Ser-394 bond at the reactive center impairing the interaction between antithrombin-III-Hamilton and the activated serine proteases.
Plasminogen interactions with platelets in plasma
Authors: Adelman B , Rizk A , Hanners E 
Published: October 2019

Abstract
In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.
A novel monoclonal antibody based diagnostic test for alpha-thalassemia- 1 carriers due to the (-SEA/) deletion
Authors: Luo HY , Clarke BJ , Gauldie J , Patterson M , Liao SK , Chui DH 
Published: October 2019

Abstract
The presence of minute amounts of embryonic zeta-globin chains in adult hemolysates is a marker for carriers of alpha-thalassemia-1 resulting from (--SEA/) deletion. Recently, we developed a murine monoclonal antihuman embryonic zeta-globin chain antibody, 8E8. By using this antibody, we have now established a slot-blot immunobinding assay for the rapid detection of zeta-globin chains in adult hemolysates. zeta- globin chains were found to be present in 30 blood samples obtained from individuals who were carriers of alpha-thalassemia-1. In another 30 blood samples from individuals who were not carriers of the (--SEA/) deletion, zeta-globin chains were not detected. This simple diagnostic test can be used in appropriate populations to identify those couples at risk of conceiving fetuses afflicted with the Hb Bart's hydrops fetalis syndrome due to homozygous alpha-thalassemia.
Calcium transport and ultrastructure of red cells in beta-thalassemia intermedia
Authors: Bookchin RM , Ortiz OE , Shalev O , Tsurel S , Rachmilewitz EA , Hockaday A , Lew VL 
Published: October 2019

Abstract
Reported findings of elevated total calcium (Ca) contents in erythrocytes (RBCs) from patients with beta-thalassemia intermedia (beta-TI) prompted the question of whether the state and transport of Ca in these RBCs are similar to those in sickle cell anemia (SS) RBCs where the increased Ca is compartmentalized in endocytic inside-out vesicles and extracted by exposure of the cells to the Ca ionophore A23187 and a Ca chelator (ethylene glycol tetraacetic acid) and the levels of cytoplasmic free ionized Ca [( Ca2+]i) are normal. We confirmed a high total Ca content of 51 +/- 13 mumol/L RBCs in splenectomized (SPX) beta-TI and 24 +/- 1 mumol/L RBCs in non-SPX beta- TI. Unlike SS RBCs, however, most of the increased Ca was in the lighter, presumably younger beta-TI RBCs, and about half the Ca was not ionophore mobilizable but apparently firmly bound, possibly to remnants of organelles in nucleated and other young RBCs. In the denser RBCs from non-SPX beta-TI, total and extractable Ca amounts were normal. beta-TI RBCs loaded with the Ca chelator Benz 2 showed an initial influx of 45Ca in the normal range, which indicated normal Ca permeability, and near-steady-state levels of [Ca2+]i that were normal (22 +/- 7 nmol/L RBCs in non-SPX beta-TI) or minimally increased (40 +/- 19 nmol/L RBCs in SPX beta-TI). Serial-section electron microscopy of beta-TI ghosts from the denser cell fractions showed more fully enclosed vesicles in non-SPX ghosts than were seen in normal ghosts and many large vesicles and structured, electron-dense material in SPX ghosts. A delayed extrusion of ionophore-preloaded 45Ca only by the SPX beta-TI RBCs together with normal [Ca2+]i suggested compartmentalization of the loaded Ca in these RBCs, perhaps in endocytic inside-out vesicles, and normal Ca pumps. Since beta-TI RBCs show essentially normal levels of [Ca2+]i and normal Ca influx, their high total Ca content should not be associated with any of the deleterious effects observed in vitro with increased levels of [Ca2+]i.
In vitro inhibition of normal human hematopoiesis by marrow CD3+, CD8+, HLA-DR+, HNK1+ lymphocytes
Authors: Vinci G , Vernant JP , Nakazawa M , Zohair M , Katz A , Henri A , Rochant H , Breton- Gorius J , Vainchenker W 
Published: October 2019

Abstract
We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E- C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+- sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co- culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T- cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.