bluuebunny/crossref_metadata_2025 huggingface dataset:
Failure of W/Wv mouse-derived cultured mast cells to enter S phase upon contact with NIH/3T3 fibroblasts
Authors: Fujita J , Nakayama H , Onoue H , Ebi Y , Kanakura Y , Kuriu A , Kitamura Y
Published: October 2019

Although W/Wv mutant mice are profoundly deficient in tissue mast cells, these mice do have cells with similar features of mast cells that develop from their bone marrow cells as efficiently as those from congenic +/+ mice in pokeweed mitogen-stimulated spleen cell- conditioned medium (PWM-SCM). With cultured mast cells (CMCs), we analyzed the mechanism of mast-cell deficiency in tissues of W/Wv mice. CMCs were established from bone marrow cells of W/Wv and congenic +/+ mice with PWM-SCM, and then co-cultured with various mouse fibroblast cell lines without PWM-SCM. All the examined mouse embryo-derived fibroblast cell lines maintained CMCs derived from +/+ mice, but not CMCs from W/Wv mice, for greater than 2 weeks. Mast cells in S phase were observed only in CMCs derived from +/+ mice under these conditions. The poor survival of W/Wv CMCs as compared with +/+ CMCs was not owing to a differential death rate but to the inability of W/Wv CMCs to continue active proliferation on fibroblasts without PWM-SCM. By synchronizing CMCs at the G1 phase of the cell cycle, the defect in W/Wv CMCs was further characterized as a failure to transit G1 and enter the S phase upon contact with fibroblasts. This finding indicates the indispensable function of the W gene product(s) for this response.
Four categories of gamma-globin gene triplications: DNA sequence comparison of low G gamma and high G gamma triplications
Authors: Liu JZ , Gilman JG , Cao Q , Bakioglu I , Huisman TH
Published: October 2019

The human fetal gamma chains are produced by closely linked G gamma and A gamma genes, and unequal crossing over between them leads to gamma gene deletions and triplications. Nine gamma gene triplications from seven ethnic groups were analyzed for G gamma and hemoglobin F (Hb F) values of heterozygotes and for the presence of polymorphic XmnI restriction sites 5′ to the gamma genes. Four categories of triplication were found: I had low G gamma and low Hb F values and lacked XmnI sites 5′ to the three gamma genes [---]. II had high G gamma and slightly elevated Hb F values but was also [---]. III was similar to II, except that XmnI was [+--]. IV had very high G gamma and slightly elevated Hb F values, and XmnI was [++-]. One case each of triplications I and IV were cloned into Charon 35. For both, the two 5′gamma gene code for G gamma chain, while the 3′ gamma gene codes for A gamma chain. DNA sequencing showed that the unequal crossover occurred between 472 and 398 base pairs (bp) 5′ to the gamma gene Cap sites (- 472 and -398) for the type IV triplication and between -271 and codon 136 for the type I triplication. In addition, type I had a 4-bp deletion of AGCA from -225 to -222. The high G gamma values of the type IV triplication are explained by its -G gamma-G gamma-A gamma-gene arrangement and the XmnI sites 5′ to the G gamma genes. We hypothesize that the low G gamma value of the type I triplication, which is also -G gamma-G gamma-A gamma-, is due to inactivation of the middle G gamma gene by the AGCA deletion at -225 to -222.
Different distribution of decay-accelerating factor on hematopoietic progenitors from normal individuals and patients with paroxysmal nocturnal hemoglobinuria
Authors: Kanamaru A , Okuda K , Ueda E , Kitani T , Kinoshita T , Nagai K
Published: October 2019

Deficiency of decay-accelerating factor (DAF) occurs in blood cells in paroxysmal nocturnal hemoglobinuria (PNH), characterized by an unusual susceptibility to hemolysis by complement activation. This study examined DAF expression on hematopoietic progenitors from normal individuals and PNH patients using a fluorescence-activated cell sorter (FACS) with monoclonal antibodies to DAF. Nonphagocytic mononuclear marrow cells expressing different density distributions of DAF were sorted into DAF-, DAF+/-, DAF+, and DAF++ fractions. The cells from each fraction were cultured in methylcellulose and assayed for CFU-E, BFU-E, CFU-GM, and CFU-Mix. The percentages of distribution of DAF- negative normal progenitors increased in the order of CFU-E, CFU-GM, BFU-E, and CFU-Mix, whereas those of DAF-positive cells inversely decreased in this order. These results indicate that DAF expression may accompany differentiation from CFU-Mix to CFU-E. On the other hand, most progenitors in PNH patients had little, if any, expression of DAF on their cell surfaces. These findings were supported by another approach using a complement-dependent cytotoxicity method with the anti- DAF monoclonal antibodies. Abnormal expression of DAF was found on the progenitors in the bone marrow as well as on mature cells circulating in the blood in PNH.
Anti-prothrombin antibodies and the lupus anticoagulant
Authors: Fleck RA , Rapaport SI , Rao LV
Published: October 2019

The investigators have evaluated the frequency and manifestations of anti-prothrombin antibodies in patients with the lupus anticoagulant. Thirty-one of 42 patients with lupus anticoagulants associated with a variety of underlying conditions (74%) had evidence on crossed immunoelectrophoresis of anti-prothrombin antibodies. Twenty-four of 25 patients with an activated partial thromboplastin time exceeding 50 seconds and 14 of 15 patients with a prothrombin time exceeding control by more than two seconds had demonstrable anti-prothrombin antibodies. Three of the 31 patients with anti-prothrombin antibodies had essentially no measurable plasma prothrombin, a presumed result of accelerated clearance of prothrombin/prothrombin antibody complexes. Each of these patients had bled abnormally. The remaining patients with anti-prothrombin antibodies had neither substantial hypoprothrombinemia nor hemorrhagic manifestations, which confirms the non-neutralizing property of anti-prothrombin antibodies associated with the lupus anticoagulant. Since lupus anticoagulant immunoglobulins are known to react with phospholipids, the high prevalence of antibodies binding prothrombin led us to test the hypothesis of antibody polyreactivity. Adsorption of three lupus anticoagulant plasmas with insolubilized prothrombin markedly diminished evidence of both prothrombin/prothrombin antibody complexes and anticoagulant activity. Eluates of the insolubilized prothrombin contained IgG that not only bound prothrombin but possessed lupus anticoagulant activity.
Prednisone and azathioprine compared with prednisone and placebo for treatment of chronic graft-v-host disease: prognostic influence of prolonged thrombocytopenia after allogeneic marrow transplantation
Authors: Sullivan KM , Witherspoon RP , Storb R , Weiden P , Flournoy N , Dahlberg S , Deeg HJ , Sanders JE , Doney KC , Appelbaum FR
Published: October 2019

We conducted a randomized, double-blind comparison of prednisone and placebo (group I) v prednisone and azathioprine (1.5 mg/kg/day) (group II) as early treatment of extensive chronic graft-v-host disease (GVHD). Patients with platelet counts less than 100,000/microL were placed into therapy with prednisone alone (group III). All three groups received identical doses of prednisone (1 mg/kg every other day) and one double-strength trimethoprim-sulfamethoxazole (TMP-SMX) tablet twice daily. Between January 1980 and December 1983, 179 previously untreated patients were enrolled and 164 were evaluable. Patients randomized to group I (n = 63) and group II (n = 63) were well matched for prognostic factors; those placed into group III (n = 38) had more frequent acute GVHD and progressive onset of chronic GVHD. Median duration of therapy was 2 years. Complications included diabetes (5%), aseptic necrosis (5%) and infection. For groups I, II, and III, the respective incidence of infection was disseminated varicella, 11%, 24%, 34%; bacteremia, 6%, 11%, 34%; and interstitial pneumonia, 5%, 14%, 18%. Recurrent malignancy was the most frequent cause of death and did not differ significantly across the groups. Nonrelapse mortality, however, did differ: 21% in group I, 40% in group II, and 58% in group III (I v II, P = .003; I v III, P = .001). Forty patients in group I, 30 in group II, and 10 in group III survive with a minimum follow-up of 3.8 years. Karnofsky performance scores for 68 survivors are 90% to 100%, scores for seven survivors are 70% to 89% and scores for five survivors are less than 70%. Actuarial survival at 5 years after transplant is 61% in group I, 47% in group II, and 26% in group III (I v II, P = .03; I v III, P = .0001). Treatment with prednisone alone results in fewer infections and better survival than prednisone and azathioprine in standard-risk chronic GVHD. Treatment with prednisone alone is less effective in high-risk patients with thrombocytopenia, and other strategies are required.
Granulocyte-macrophage colony-stimulating factor enhances selective effector functions of tissue-derived macrophages
Authors: Coleman DL , Chodakewitz JA , Bartiss AH , Mellors JW
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by a variety of cells at sites of exposure to antigens. GM-CSF has a stimulatory effect on a number of neutrophil functions, but the effect on macrophage function is less clear. We investigated the effect of purified murine recombinant GM-CSF on murine peritoneal macrophage oxidative metabolism, Fc-dependent phagocytosis, anti-Toxoplasma activity, and expression of class II major histocompatibility antigen (Iad). GM-CSF significantly increased phorbol myristate acetate- and zymosan-elicited H2O2 release by resident and thioglycollate-elicited macrophages after 48 hours in vitro. The effect of recombinant GM-CSF was blocked by polyclonal anti-GM-CSF antibody and was not altered by lipopolysaccharide (0.01 to 1.0 microgram/mL). GM-CSF also stimulated Fc-dependent phagocytosis by peritoneal macrophages, although the stimulation of resident macrophages (1.4-fold) was less dramatic than that of thioglycollate-elicited cells (2.1-fold). GM-CSF (at doses up to 100 U/mL) had no effect on macrophage anti-Toxoplasma activity or on expression of Iad. In addition to stimulating macrophage growth, GM-CSF selectively promotes the functional capacity of tissue-derived macrophages.
Acute myeloid leukemia with T-lymphoid features: a distinct biologic and clinical entity
Authors: Cross AH , Goorha RM , Nuss R , Behm FG , Murphy SB , Kalwinsky DK , Raimondi S , Kitchingman GR , Mirro J Jr
Published: October 2019

We studied the clinical and biologic features of 10 cases of acute leukemia that met standard French-American-British (FAB) criteria for acute myeloid leukemia (AML) but in which the blast cells also expressed the T-cell-associated CD2 surface antigen. All cases had greater than 3% myeloperoxidase and Sudan black B-positive leukemic blasts, and blasts from seven cases contained Auer rods. Reactivity of the cells with a panel of monoclonal antibodies (MAbs) indicated that leukemic cells in all cases expressed myeloid-associated (CD11b, CD13) surface antigens, further supporting the diagnosis of AML. However, blasts from every patient coexpressed the T-cell-associated surface CD2 and CD7 as well as cytoplasmic CD3 antigens. Blasts from five patients expressed surface CD25, whereas blasts from only one expressed surface CD3. Five patients had rearranged T-cell receptor beta-chain genes, whereas only three had rearranged T-cell receptor gamma-chain genes. This pattern of lineage-related gene expression appears to define a distinct subtype of AML with T-lymphoid features (CD2+ AML) and could reflect either aberrant gene expression in leukemic blasts or transformation of a pluripotent stem cell having a flexible pattern of gene expression. Clinically, these 10 patients presented at an older age with a higher leukocyte count and a higher frequency of lymphadenopathy than did children whose blast cells were characteristic of myeloid leukemia. Patients with CD2+ AML also had poorer responses to remission induction therapy (50% v 80% entered complete remission, P = .05). However, each of the five children who failed induction chemotherapy on AML protocols had a striking response to drug combinations usually reserved for lymphoid leukemia. We conclude that this leukemia with mixed lymphoid and myeloid characteristics is a distinct biologic and clinical entity.
DNA ligation in relation to DNA strand breaks in phytohemagglutinin- stimulated blast cells from acute lymphoblastic and nonlymphoblastic leukemia
Authors: Feon SA , Valerius RM , Genetet NM , Bernard-Griffiths I , Le Prise PY , Le Gall EJ , David JC
Published: October 2019

DNA ligase activity was determined in the WBCs from 306 cases of acute lymphoblastic leukemia (ALL) and acute nonlymphocytic leukemia (ANLL). In T-ALL cells this activity was either low or absent. DNA analysis by nucleoid, alkaline elution, and alkaline sucrose centrifugation after cells were embedded in agarose inserts has shown more DNA breaks in T- ALL than in ANLL blasts. Phytohemagglutinin stimulation of T-ALL blasts resulted in the apparent joining of the DNA breaks. Apparent identical results can be obtained by the incubation of DNA with exogenous DNA ligase. The authors suggest that this enzyme is a crucially regulated step of replication and subsequent proliferation in this type of leukemia.
Frequent c-myc oncogene activation and infrequent presence of Epstein- Barr virus genome in AIDS-associated lymphoma
Authors: Subar M , Neri A , Inghirami G , Knowles DM , Dalla-Favera R
Published: October 2019

Sixteen cases of histologic intermediate-grade and high-grade AIDS- associated non-Hodgkin's lymphoma (NHL) were studied for the presence and patterns of c-myc gene and bcl-2 locus rearrangements. The presence of Epstein-Barr virus (EBV) sequences and proteins and HTLV-I sequences were also investigated. c-myc gene rearrangements analogous to those observed in sporadic Burkitt lymphomas were detected in 12 of 16 cases. Six of 16 cases had detectable EBV sequences and proteins. None of the cases displayed bcl-2 rearrangements or contained HTLV-I sequences. These data suggest a frequent role for c-myc activation in the pathogenesis of AIDS-associated NHL, independent of histologic type. Conversely, EBV does not appear to be directly involved in lymphomagenesis in the majority of AIDS-associated NHLs.
Immunophenotypic diagnosis of clinical and preclinical chronic lymphatic leukemia by using monoclonal antibodies against the cCLLa, a CLL-associated antigen
Authors: Faguet GB , Agee JF
Published: October 2019

The clinical usefulness of monoclonal antibodies (MoAbs) against the cCLLa, an antigen restricted to B-chronic lymphatic leukemia (CLL) and its variants, was ascertained in 65 patients with overt CLL and 25 individuals with unexplained mild lymphocytosis. Healthy volunteers (n = 25) and patients with malignant and nonmalignant disorders (n = 58) served as controls. The following observations were made in CLL. (a) Anti-cCLLa MoAbs identified neoplastic CLL cells as judged by the high correlation (r = .985) between monoclonal surface immunoglobulins (Slgs) and cCLLa expression in all patients, and dual-label flow cytometry studies showing cCLLa expression by monoclonal Slg-bearing B- CLL cells but not by normal B lymphocytes. (b) The size of the circulating cCLLa-positive clone paralleled the degree of lymphocytosis (r = .987) and was associated with reciprocal (r = .893) relative T lymphopenia. Ten patients with borderline lymphocytosis exhibited a subset of monoclonal Slg/cCLLa-positive cells ranging from 16% to 45% of the total. These patients were indistinguishable from those with CLL in terms of age, clone lineage, and reciprocal relative T lymphopenia. Two patients have progressed to overt CLL within 19 months, but eight have not (observation time, 18 to 82 months). These data suggest that anti-cCLLa MoAbs are sensitive probes useful to identify and monitor cCLLa clones during their clinical and preclinical phases.
Interleukin-3 (IL-3) stimulates the clonal growth of pulmonary alveolar macrophage of the mouse: role of IL-3 in the regulation of macrophage production outside the bone marrow
Authors: Chen BD , Mueller M , Olencki T
Published: October 2019

Interleukin-3 (IL-3) is one of the hematopoietic growth factors that regulates the growth and differentiation of pluripotent stem cells, thereby leading to the production of all the major blood cell types. The role of IL-3 in the regulation of pulmonary alveolar macrophage (PAM) production was investigated. IL-3 stimulated the proliferation and clonal growth of murine PAM with a dose-response curve similar to that of bone marrow granulocyte-macrophage colony-forming cells. The IL- 3-induced colony formation by cells outside the bone marrow appeared to be unique to PAM; IL-3 failed to cause colony formation by both peritoneal exudate macrophages (PEM) and blood monocytes. Unlike bone marrow stem cells, PAM are unipotential and in vitro gave rise to only mononuclear phagocytes under the influence of IL-3. Nevertheless, cells derived from PAM cultures in media containing IL-3 displayed a high degree of heterogeneity in terms of their Fc receptor-mediated phagocytic activity. At low concentrations, IL-3 induced a synergistic response with colony-stimulating factor 1 (CSF-1), which resulted in an enhanced proliferative capacity of PAM. A synergistic effect was also observed by short-term exposure of PAM to IL-3 followed by incubating with CSF-1 alone. This study shows that IL-3 exhibited a macrophage growth factor activity unique to PAM.
Synthesis and characterization of an antihuman T-lymphocyte saporin immunotoxin (OKT1-SAP) with in vivo stability into nonhuman primates
Authors: Siena S , Lappi DA , Bregni M , Formosa A , Villa S , Soria M , Bonadonna G , Gianni AM
Published: October 2019

The authors conjugated, by a disulphide bond, the antihuman T- lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis. The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen- induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC50 0.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomolgus monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2 alpha = 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2 beta = 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double- antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1-SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans.
Variable Philadelphia breakpoints and potential lineage restriction of bcr rearrangement in acute lymphoblastic leukemia
Authors: Secker-Walker LM , Cooke HM , Browett PJ , Shippey CA , Norton JD , Coustan-Smith E , Hoffbrand AV
Published: October 2019

Philadelphia (Ph1) chromosome breakpoints in acute lymphoblastic leukemia (ALL) are of two kinds: those within the breakpoint cluster region (bcr+), as in chronic myeloid leukemia (CML), and those outside it (bcr-). These encode different c-abl messenger RNAs (mRNAs), p210 and p190, respectively. It has been suggested that one class of Ph+ ALL (bcr+) may be a variant of CML arising in a multipotent stem cell, the other (bcr-) de novo ALL initiated in a lymphoid-committed progenitor. Thirty-two cases of ALL (12 Ph1+, ten chromosomally normal, and ten non- mitotic cases) were investigated for bcr involvement. Breakpoints were found within five Ph1+ and in one normal case. There was no difference in clinical features, common ALL antigen (CALLA) positivity, cytogenetics, or response to treatment between the 6 bcr+ and 7 Ph1+ bcr- patients. Myeloid antigen expression was found in 2 bcr+ cases. Bcr rearrangement appeared to be restricted to the lymphoblastic component of marrow or blood in at least four bcr+ cases. In one case, separated myeloid and lymphoid cell fractions were both bcr+. Potential heterogeneity of the Ph1+ target cell, as seen in this study, may be more important in determining disease outcome than the precise location of the Ph breakpoint.
Correlated flow cytometric analysis of H-ras p21 and nuclear DNA in multiple myeloma
Authors: Tsuchiya H , Epstein J , Selvanayagam P , Dedman JR , Gallick G , Alexanian R , Barlogie B
Published: October 2019

Correlated analysis of the H-ras oncogenes product (p21) and of nuclear DNA content was performed by flow cytometry (FCM) in patients with DNA- aneuploid multiple myeloma (MM). Bone marrow cells from normal donors and MM patients in remission served as controls. Seventy-four percent of 23 patients with active MM had higher p21 fluorescence in aneuploid tumor cells than were observed in normal donor or myeloma remission bone marrows; 39% of the 23 patients also showed high H-ras p21 expression in diploid cells. There was an inverse relationship between p21 levels and the presence of trisomy 11; especially high p21 levels were noted in patient without trisomy 11. The frequent elevation of p21 protein in aneuploid plasma cells suggests the involvement of the H-ras oncogene in the pathophysiology of MM, which is further supported by a shorter survival among patients with high p21 levels.
Interleukin-6 enhances growth factor-dependent proliferation of the blast cells of acute myeloblastic leukemia
Authors: Hoang T , Haman A , Goncalves O , Wong GG , Clark SC
Published: October 2019

The effects of recombinant interleukin-6 (IL-6) on the proliferation of blast precursors present in the peripheral blood of patients with acute myeloblastic leukemia (AML) was investigated. IL-6 had little effect by itself; however, it synergized with granulocyte macrophage colony- stimulating factor (GM-CSF) and interleukin-3 (IL-3) in the stimulation of AML blast colony formation. Responsiveness of blast progenitors to IL-6 was heterogeneous. On normal bone marrow cells the same synergy was observed on granulocyte and monocyte precursors (GM-CFC), while there was no significant effect on erythroid and multipotential precursors.
Use of recombinant human granulocyte-macrophage colony-stimulating factor in autologous marrow transplantation for lymphoid malignancies
Authors: Nemunaitis J , Singer JW , Buckner CD , Hill R , Storb R , Thomas ED , Appelbaum FR
Published: October 2019

The use of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) following autologous marrow transplantation for lymphoid malignancies was explored in a phase I/II dose escalation study. rhGM-CSF given as a 2-hour infusion daily for 14 days was well tolerated at doses up to 240 micrograms/m2/day. When compared with 86 disease-matched and treatment-matched historical controls, patients receiving greater than or equal to 60 micrograms/m2/day rhGM-CSF recovered neutrophil and platelet counts more rapidly, had fewer days with fever, and were discharged from the hospital sooner.
Malignant histiocytosis: a specific t(2;5)(p23;q35) translocation? Review of the literature
Authors: Benz-Lemoine E , Brizard A , Huret JL , Babin P , Guilhot F , Couet D , Tanzer J
Published: October 2019

In this paper, the investigators report a well documented case of malignant histiocytosis (MH) with a t(2;5)(p23;q35) translocation. A breakpoint in 5q35 appears to be specific, either for the disease or for a subclass of the disease. Additional cases of MH with cytogenetics are needed. This will help to determine if one class of MH or several subclasses can be defined by cytogenetic anomaly(ies).
The role of fibrinogen A alpha chains in ADP-induced platelet aggregation in the presence of fibrinogen molecules containing gamma' chains
Authors: Amrani DL , Newman PJ , Meh D , Mosesson MW
Published: October 2019

Human plasma fibrinogen (Fgn) is heterogenous with respect to the size of its gamma chains, which differ in that residues 408 to 411 of gammaA chains (93% of total) are replaced in gamma' chains by a unique 20 amino acid sequence (gamma408 to gamma427). In this study, we compared the contribution to adenosine diphosphate (ADP)-induced platelet aggregation of the A alpha chains in Fgn molecules containing predominantly (fraction 1–2) or exclusively (peak 1 Fgn) gammaA chains with that of molecules containing approximately 50% gamma' chains (peak 2 Fgn). Using washed human platelets, we confirmed that the number of peak 2 Fgn molecules binding to platelets in the presence of ADP was about half the number of peak 1 Fgn molecules (18,962 +/- 2,298 v 44,366 +/- 16,096 molecules per platelet), and that isolated S- carboxymethylated (SCM) gammaA chains supported ADP-induced platelet aggregation nearly as well as peak 1 Fgn. In contrast, SCM-gamma' chains alone supported aggregation poorly, whereas a mixture of SCM- gammaA and gamma' chains (1:1 ratio) gave intermediate results. Despite the findings with isolated SCM-gamma' chains, we found that peak 2 Fgn supported platelet aggregation nearly as well as peak 1 Fgn. However, peak 2 Fgn from which carboxy (COOH)-terminal A alpha chain segments had been removed by digestion with plasmin showed a markedly decreased platelet aggregation potential. Peak 1 Fgn core fraction from an 88% to 90% coagulable plasmin digest, or Fgn fraction 1–9, which has a high gammaA/gamma' chain ratio (93:7), but lacks COOH-terminal regions of A alpha chains, supported platelet aggregation to the same extent as did intact peak 2 Fgn. These findings indicate that Fgn molecules containing gamma' chains can approach the aggregation potential of Fgn molecules containing predominantly or exclusively gammaA chains only if intact A alpha chains are also present.
Molecular basis and prenatal diagnosis of beta-thalassemia
Authors: Kazazian HH Jr , Boehm CD
Published: October 2019

The molecular characterization of mutations producing beta-thalassemia in world populations is nearing completion. We expect that new rare alleles in thoroughly studied groups and other alleles in less studied groups, eg, inhabitants of New Guinea, Latin America, and certain Pacific Islands, will be found. Knowledge of the molecular basis of the disease and new technology that allows rapid detection of single nucleotide changes in genomic DNA have led to the reality of prenatal diagnosis by direct mutation detection even in the heterogeneous US population. Programs aimed at prevention of beta-thalassemia should be facilitated by these developments.
Regulation of interleukin-6 expression in cultured human blood monocytes and monocyte-derived macrophages
Authors: Bauer J , Ganter U , Geiger T , Jacobshagen U , Hirano T , Matsuda T , Kishimoto T , Andus T , Acs G , Gerok W
Published: October 2019

A culture system that allows human blood monocytes to differentiate into macrophages in vitro was used to study B-cell stimulatory factor- 2/interleukin-6 (interferon-beta 2/26 kd protein) expression in mononuclear phagocytes. Using B-cell stimulatory factor-2 (BSF-2) cDNA and a polyclonal, monospecific antibody directed against human BSF-2, we find that strong interleukin-6 (IL-6) expression is initiated in cultured monocytes on stimulation with endotoxin. Maximally induced monocytic BSF-2/IL-6 synthesis (1% to 2% of total proteins secreted by monocytes) is more than ten times stronger than in terminally differentiated macrophages (approximately 0.1% of total secretory proteins). BSF-2/IL-6 mRNA was detectable as early as one hour after stimulation with endotoxin, reaching maximum levels three hours after stimulus. Interleukin-1 (IL-1) was able to stimulate IL-6 synthesis in monocytes, but not in macrophages. Tumor necrosis factor, interferon- gamma and interleukin-2 (IL-2) had no effect on IL-6 synthesis in monocytes or macrophages. We found five molecular weight forms of BSF- 2/IL-6 to be secreted by monocytes of 21.5 kd, 23.5 kd, 24 kd, 26 kd, and 28 kd apparent molecular weight. The 26 kd and 28 kd forms were found to represent N-glycosylated molecules, which were not detectable on treatment of the cells with the N-glycosylation inhibitor tunicamycin. The 21.5 kd, 23.5 kd, and 24 kd BSF-2/IL-6 forms were unaffected by tunicamycin treatment. We conclude from our data that cells of the mononuclear phagocyte lineage are one of the main sites of BSF-2/IL-6 (interferon-beta 2/26 kd protein/HSF) synthesis.
Heterogeneity of drug-dependent platelet antigens and their antibodies in quinine- and quinidine-induced thrombocytopenia: involvement of glycoproteins Ib, IIb, IIIa, and IX
Authors: Pfueller SL , Bilston RA , Logan D , Gibson JM , Firkin BG
Published: October 2019

The molecular nature of platelet receptors for quinine- and quinidine- dependent antiplatelet antibodies (Q.Ab and Qd.Ab) was studied by immunoblotting. One Q.Ab caused quinine-dependent IgG binding to platelet proteins with molecular weights (mol wts) of 174 Kd and 93 Kd and another to only a 93-Kd protein. A third Q.Ab caused binding to 174- , 140-, 93-, and 57-Kd proteins, while a fourth Q.Ab and a Qd.Ab caused IgG binding to 174- and 18-Kd proteins. Using platelets from patients with Glanzmann's thrombasthenia or Bernard Soulier syndrome and purified GPIIIa, these proteins were shown to be GPIb, GPIIb, GPIIIa, GPIX, and an unidentified 57-Kd protein missing in Bernard Soulier syndrome. Binding to the 93-Kd protein was independent of the PIA1 antigen. Absorption of one Q.Ab with Glanzmann's thrombasthenia platelets revealed different populations of antibodies with different specificities within the one patient. Thus Q.Ab and Qd.Ab are heterogeneous and may be directed toward different epitopes on major platelet glycoproteins.
Lymphocyte depletion of donor bone marrow by counterflow centrifugal elutriation: results of a phase I clinical trial
Authors: Wagner JE , Donnenberg AD , Noga SJ , Cremo CA , Gao IK , Yin HJ , Vogelsang GB , Rowley S , Saral R , Santos GW
Published: October 2019

We report here the results of a phase I clinical trial using counterflow centrifugal elutriation (CCE) for the removal of donor T lymphocytes before allogeneic bone marrow transplantation (BMT). Thirty- eight patients received lymphocyte-depleted allografts from HLA- identical, MLR-nonreactive sibling donors. The patients entered onto the study were either at high risk on the basis of age (median, 39 years) or disease status (acute leukemia in early relapse [ER], chronic myelogenous leukemia [CML] in accelerated phase [AP], or therapy resistant [RES] lymphoma). All patients received a standard lymphocyte dose of 1 x 10(6) morphologic lymphocytes per kilogram ideal body weight (BW) and were maintained on cyclosporine A (CsA) for 170 days after BMT. Prompt engraftment occurred in 37 of 38 patients with a median time to absolute neutrophil count (ANC) greater than 500/microL of 18 days. Although acute graft-v-host disease (GVHD; clinical stage I or greater) was observed in 45%, it was limited to the skin in all but five patients. Survival was related to disease status at the time of BMT. Among patients with acute leukemia in first or second remission, CML in chronic phase (CP) or lymphoma in partial remission (PR), 64% are currently alive, in contrast to 31% of patients with acute leukemia in third remission or early relapse, CML in second CP or AP, or RES lymphoma. Median follow-up for all patients was 351 days (range, 105 to 711 days). We conclude that this procedure is safe and warrants further evaluation in a randomized efficacy trial.
Leukemic cells from a chronic T-lymphocytic leukemia patient proliferated in response to both interleukin-2 and interleukin-4 without prior stimulation and produced interleukin-2 mRNA with stimulation
Authors: Umadome H , Uchiyama T , Onishi R , Hori T , Uchino H , Nesumi N
Published: October 2019

Recently, interleukin-4 (IL-4) has been clarified as having T-cell growth factor activity; therefore, it becomes of interest whether IL-4, as well as interleukin-2 (IL-2), affects the proliferation of leukemic cells derived from mature T cells. In the present study, we describe a case of chronic T-lymphocytic leukemia (T-CLL) with monoclonal proliferation of human T-lymphotropic retrovirus (HTLV)-I or HTLV-II negative CD3(+)4(+)8(-) T cell expressing IL-2 receptors without stimulation. Radiolabeled IL-2 binding assay revealed 750 high-affinity and 6,750 low-affinity binding sites per cell. In accordance with the expression of high-affinity IL-2 receptors, the leukemic cells proliferated in response to exogenous IL-2 without prior stimulation. In addition, exogenous IL-4 also induced their proliferation. Moreover, IL-2 and IL-4 exerted a synergistic effect on the leukemic cell proliferation. Although the expression of IL-2 or IL-4 mRNA was not detected in fresh leukemic cells, the expression of IL-2 mRNA, but not IL-4 mRNA, was induced by phytohemagglutinin stimulation, and the leukemic cells proliferated. These findings suggest that not only IL-2, but also IL-4 are involved in the proliferation of leukemic cells of T- CLL.
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

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

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

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

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

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

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

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

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

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.