bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of multiple quinine-dependent antibodies in a patient with episodic hemolytic uremic syndrome and immune agranulocytosis
Authors: Stroncek DF , Vercellotti GM , Hammerschmidt DE , Christie DJ , Shankar RA , Jacob HS
Published: October 2019

A 23-year-old woman experienced six distinct episodes of severe combined neutropenia and thrombocytopenia. At least one of the episodes was accompanied by hemodialysis-requiring acute renal failure and fragmentation hemolysis (hemolytic uremic syndrome [HUS]). In retrospect, all episodes were probably associated with the ingestion of quinine. Quinine-dependent antibodies to platelets, neutrophils, T lymphocytes, and red blood cells (RBCs) were detected in the patient's serum. By a monoclonal antibody antigen capture assay, the patient's serum contained IgG antibodies, which in the presence, but not absence, of quinine reacted with platelet glycoprotein (GP) complexes Ib/IX and IIb/IIIa, but not Ia/IIa. By immunoprecipitation assay, the serum, after addition of quinine, reacted strongly with an 85-Kd neutrophil membrane protein and weakly with 130- and 60-Kd moieties. Serum adsorbed with RBCs in the presence of quinine continued to react with platelets and neutrophils, and serum that was absorbed with platelets continued to react with neutrophils and RBCs, indicating that the antigenic targets were different on platelets, neutrophils, and RBCs. Since platelets and endothelial cells share some antigens, we tested patient serum for antibodies to human umbilical vein endothelial cells (HUVEC); no quinine-dependent antibodies to HUVEC were detected. However, her quinine-dependent antibodies not only bound to platelets and neutrophils, but also activated neutrophils. Thus, the patient's serum with quinine aggregated neutrophils, but neither agent alone caused activation. Moreover, the patient's serum with quinine (but not without) augmented the adherence of neutrophils to HUVEC. Treatment of the patient's serum with staphylococcal protein A removed the quinine neutrophil aggregation cofactor, suggesting it was due to IgG. In both neutrophil aggregation and adherence assays, decomplementation of the patient's serum markedly blunted its effect. Furthermore, the patient's serum failed to aggregate formalin-inactivated neutrophils, suggesting neutrophil activation, probably by activated complement, was necessary for aggregation and adhesivity to endothelium. We conclude that our patient's neutropenia, thrombocytopenia, lymphopenia, and anemia were due to quinine-dependent antibodies, and that these antibodies recognized epitopes that were different in the three target cell populations. We further suggest that HUS was likely secondary to the activation and adhesion of neutrophils to endothelium.
Improved isolation of normal human reticulocytes via exploitation of chloride-dependent potassium transport
Authors: Sorette MP , Shiffer K , Clark MR
Published: October 2019

Studies on normal human reticulocytes have been limited by a lack of methods for effective reticulocyte enrichment. This study shows a convenient new approach for selective enrichment of reticulocytes from normal blood samples. We have developed a modified arabinogalactan density gradient that contains high potassium levels, approximating the internal cation composition of red blood cells (RBC). The low-density populations from this gradient are enriched in reticulocytes, and the highly selected lowest density fraction shows a much higher reticulocyte enrichment than that obtained with high sodium chloride arabinogalactan density gradients, or other previously reported density gradient methods. We found that this improved isolation is caused by suppression of potassium loss and reticulocyte dehydration via chloride (KCI) cotransport. When the low-density fraction of RBC from a high- potassium gradient was subsequently incubated in high sodium chloride medium and reseparated on a sodium chloride density gradient, the reticulocytes dehydrated and were recovered in high-density fractions. The highest-density fractions from this secondary gradient yield 95% to 99% reticulocytes. We anticipate that this method will benefit investigators who require reticulocyte enriched populations for a wide variety of applications.
Circulating stem cells in mice treated with cyclophosphamide
Authors: Craddock CF , Apperley JF , Wright EG , Healy LE , Bennett CA , Evans M , Grimsley PG , Gordon MY
Published: October 2019

Chemotherapy has been used clinically to mobilize hematopoietic progenitor cells into the peripheral blood so that they can be harvested for autologous transplantation. In humans, this is demonstrated by the presence of circulating granulocyte-macrophage colony-forming cells (CFU-GM) and CD34-positive cells, but it has not been possible to confirm the presence of marrow-repopulating stem cells. In this study, we treated mice with 200 mg/kg cyclophosphamide (CY) and measured the numbers of white blood cells, day 12 CFU-S (CFU- S12), and CFU-GM in the peripheral blood. There was a peak in the numbers of CFU-S12 and CFU-GM 8 days after treatment with cyclophosphamide. Peripheral blood cells taken at this time rescued lethally irradiated mice and engraftment of donor cells was confirmed after 140 days in sex mismatched recipients using a Y chromosome- specific probe. In vitro culture of the blood cells harvested after cyclophosphamide showed that they proliferated in suspension cultures for at least a year in the presence of interleukin-3. The cultured cells rapidly lost their abilities to rescue irradiated mice and to form colonies in vitro, but they did not become leukemic. Also, CY- treated mice were irradiated with a leukemogenic dose of x-rays to coincide with peak circulating cell numbers but these animals did not develop an excess of leukemias over mice given irradiation alone.
Recombinant human tissue plasminogen activator for the treatment of established severe venocclusive disease of the liver after bone marrow transplantation
Authors: Bearman SI , Shuhart MC , Hinds MS , McDonald GB
Published: October 2019

Seven patients were treated with recombinant human tissue plasminogen activator (tPA) for severe hepatic venocclusive disease (VOD) that developed after bone marrow transplantation for hematologic malignancy. Recombinant human tPA (10 mg/d x 2 days) and heparin (1,000 U bolus followed by continuous intravenous infusion of 150 U/kg/d x 10 days) were begun a median of 9 days (range, 4 to 18 days) posttransplant. The median total serum bilirubin and percent weight gain from baseline were 19.4 mg/dL (range, 14.6 to 34.9 mg/dL) and 9.1% (range, 1% to 18.5%), respectively, at the start of tPA administration. Five patients responded to therapy with prompt reduction in total serum bilirubin within 96 hours of starting tPA. Three patients are alive 178 to 379 days posttransplant without evidence of VOD. No patient had significant hemorrhagic complications with tPA. We conclude that recombinant human tPA can be administered to patients with severe VOD at the dosage described. Whereas preliminary data suggests that recombinant human tPA can alter the natural history of severe VOD, further study is necessary to determine its efficacy.
Favorable outcome of B-cell acute lymphoblastic leukemia in childhood: a report of three consecutive studies of the BFM group
Authors: Reiter A , Schrappe M , Ludwig WD , Lampert F , Harbott J , Henze G , Niemeyer CM , Gadner H , Muller-Weihrich S , Ritter J
Published: October 2019

In 1981 the BFM group introduced a new treatment strategy for B-cell acute lymphoblastic leukemia (B-ALL). A cytoreductive prephase (prednisone/cyclophosphamide) was followed by eight 5-day courses of chemotherapy. Fractionated cyclophosphamide, methotrexate (MTX) 0.5 g/m2 (24-hour infusion), and MTX intrathecally were administered at each course and cytosine arabinoside (ARA-C)/teniposide (VM-26) was given alternately with doxorubicin. In study ALL-BFM-83, central nervous system (CNS) chemotherapy was intensified by adding dexamethasone, while MTX/ARA-C was administered intraventricularly. Therapy duration was reduced to six courses. In study ALL-BFM-86, MTX 0.5 g/m2 was replaced by high-dose (HD) MTX, 5 g/m2 (24-hour infusion), and MTX/ARA-C/prednisolone intrathecal therapy was introduced. Doses of ARA-C and VM-26 were increased and fractionated, cyclophosphamide was partially replaced by ifosfamide, and vincristine was added. CNS irradiation was 24 Gy for prevention and 30 Gy for overt disease in studies ALL-BFM-81 and -83, but was omitted in ALL-BFM-86. In all, 87 patients were enrolled, 22 (8 CNS-positive) in study All-BFM-81, 24 (7 CNS-positive) in study ALL-BFM-83, and 41 (0 CNS-positive) in study ALL- BFM-86. The estimated 5-year duration of event-free survival (EFS) was 43% in study ALL-BFM 81, 50% in study ALL-BFM-83, and 78% in study ALL- BFM-86 (minimal follow-up, 25 months). Nineteen of 24 relapses occurred while on therapy or shortly thereafter. In study ALL-BFM 81, the CNS was the most frequent site of failure. In ALL-BFM-83, there were no isolated CNS relapses, but more bone marrow (BM) relapses occurred. In ALL-BFM-86, localized manifestations were the predominant site of failure, no isolated BM relapses occurred, and only one CNS relapse was diagnosed. No single parameter exerted a consistent influence on outcome with one exception. The presence of residual disease after the first two courses was correlated with an increased risk of therapy failure. We conclude that an intensive, short-pulse therapy delivered within a 4-month period is highly effective in the treatment of B-ALL. In addition to fractionated cyclophosphamide/ifosfamide, a 24-hour infusion of HD MTX 5 g/m2 in conjunction with an i.th. therapy is an important component for prevention of both systemic and CNS relapses. CNS irradiation is not needed for CNS-negative patients.
Roles of erythropoietin, insulin-like growth factor 1, and unidentified serum factors in promoting maturation of purified murine erythroid colony-forming units
Authors: Boyer SH , Bishop TR , Rogers OC , Noyes AN , Frelin LP , Hobbs S
Published: October 2019

We have used 75% to 90% pure murine erythroid colony-forming units (CFU- E) to delineate the processes and factors underlying their maturation. These CFU-E form 32 cell colonies and are drawn from what we term generation I of a six-generation long maturation sequence (Landschulz et al, Blood 79:2749, 1992). Applying assays of 59Fe-heme biosynthesis and colony numbers as measures of maturation and analyses of DNA degradation as an index of programmed cell death, we find that (1) erythropoietin (Epo) enhances maturation throughout most of its course; (2) Epo first seems able to forestall DNA degradation when CFU-E reach generation II; (3) the processes that Epo elicits thereafter start to persist when Epo is withdrawn; (4) insulin-like growth factor I (IGF-I) also forestalls DNA breakdown, but later loses effectiveness; (5) IGF-I adds little to maturation when Epo levels are high, but when Epo levels are low, enhances it substantially; and (6) for maturation to be entirely optimal, an unidentified serum factor(s) is probably required when Epo levels are high and is certainly needed when Epo levels are like those in normal animals. Quantitatively, about 40% of optimal in vitro erythropoiesis at normal Epo levels depends on Epo alone, another 30% or less on the addition of IGF-I, and the remaining 30% or more on the addition of unidentified serum factor(s). Applied together, these three or more factors lead to two-thirds of the maximum maturation realized with saturating Epo levels. Because we also find that heme accumulated in CFU-E culture can closely approach levels in red blood cells, we suppose that our conclusions apply as well to CFU-E maturation in vivo.
Release of interleukin-1 and interleukin-6 from human monocytes by antithymocyte globulin: requirement for de novo synthesis
Authors: Rameshwar P , Gascon P
Published: October 2019

Antithymocyte globulin (ATG) is an effective treatment in patients with severe aplastic anemia (SAA). Its mechanism of action remains unclear, although it has been assumed to be immunosuppressive. However, ATG has also been shown by several laboratories to be immunostimulatory. Recently, interleukin-1 (IL-1) production has been found to be decreased in lipopolysaccharide-stimulated peripheral blood monocytes obtained from SAA patients. We have investigated the ability of ATG to function as an immunostimulatory agent via the production of IL-1 and IL-6 by normal human monocytes in vitro. Supernatants from ATG- stimulated monocytes were assayed for biologically active and immunoreactive IL-1 and IL-6. We have found that ATG, via its F(ab')2 fragment is a powerful inducer of IL-1 and IL-6 production. Furthermore, ATG induction of both cytokines from normal monocytes required de novo synthesis, as determined by 35S-methionine incorporation. Because these two cytokines synergize with other cytokines at both the stem cell and progenitor levels, these stimulatory properties of ATG may be relevant to the treatment of SAA. This would favor the hypothesis of a bimodal mechanism for ATG as an inducer of hematopoietic growth factors and as an immunosuppressive agent.
Enhanced expression of the tie receptor tyrosine kinase in endothelial cells during neovascularization
Authors: Korhonen J , Partanen J , Armstrong E , Vaahtokari A , Elenius K , Jalkanen M , Alitalo K
Published: October 2019

We have recently cloned a novel human receptor tyrosine kinase, tie, from human leukemia cells showing megakaryoblastoid differentiation. We report here that the 4.4-kb tie messenger RNA (mRNA) is present in all human fetal and mouse embryonic tissues. By in situ hybridization, the tie mRNA was localized to the endothelia of blood vessels and endocardium of 9.5- to 18.5-day mouse embryos. However, tie was not expressed by endothelial cells of developing hepatic sinusoids. Increased tie mRNA signal was seen in proliferating ovarial capillaries during hormone-induced superovulation. Only a weak tie signal was obtained from adult skin, except during wound healing, when the proliferating capillaries in the granulation tissue contained abundant tie RNA. These results suggest that tie may have a role in neovascularization.
Involvement of nuclear factor-kappa B in induction of the interleukin-6 gene by leukemia inhibitory factor
Authors: Gruss HJ , Brach MA , Herrmann F
Published: October 2019

Recent studies have indicated that the leukemia inhibitory factor (LIF) induces secretion of interleukin-6 (IL-6) in myeloid cells. We here show that synthesis of IL-6 by human mononuclear phagocytes exposed to recombinant human (rh) LIF is preceded by an increase of IL-6 transcript levels as a result of transcriptional activation of the IL-6 gene. Analysis of deleted fragments of the IL-6 promoter indicated that transcriptional activation of the IL-6 promoter was associated with enhanced binding activity of the transcription factor nuclear factor (NF)-kappa B. Binding of activation protein (AP)-1 and NF-IL-6, also known to transcriptionally activate the IL-6 promoter, was not inducible by LIF. Furthermore, introduction of the NF-kappa B sequence into a heterologous promoter construct, but not of AP-1- and NF-IL-6- binding sequences, conferred inducibility by LIF to this promoter. Deletion of the NF-kappa B binding site in the IL-6 promoter was associated with loss of inducibility by LIF, lending further support for the notion that the NF-kappa B binding site is crucial for LIF- mediated induction of the IL-6 promoter. Taken together, our results show that rhLIF induces IL-6 gene expression in mononuclear phagocytes through transcriptional gene activation involving NF-kappa B.
Roles of heme insertion and the mannose-6-phosphate receptor in processing of the human myeloid lysosomal enzyme, myeloperoxidase
Authors: Nauseef WM , McCormick S , Yi H
Published: October 2019

Biosynthesis of myeloperoxidase (MPO), a myeloid lysosomal hemoprotein critical for the optimal oxygen-dependent microbicidal activity of human neutrophils, is incompletely understood. The primary translation product undergoes cotranslational N-linked glycosylation with subsequent insertion of the Fe-containing prosthetic group into the peptide backbone, thereby converting the enzymatically inactive, heme- free apoproMPO into the peroxidatively active precursor, proMPO. Eventually, proMPO undergoes proteolytic processing into native, lysosomal MPO, with subunits of 59 and 13.5 Kd. We studied three unanswered questions regarding MPO biosynthesis: (1) At what point during MPO biosynthesis is the heme moiety inserted into the apoenzyme? (2) What consequences does heme-insertion have on subsequent processing events? (3) What role does the mannose-6-phosphate receptor (M6PR) system play in the delivery of MPO to the lysosome? Disruption of Golgi by brefeldin A (BFA) produced two major changes in MPO biosynthesis: (1) processing of the 89-Kd precursor to mature MPO was blocked and (2) constitutive secretion of the MPO precursor was inhibited. Inhibition of heme synthesis with succinyl acetone (SA) reduced peroxidase activity and profoundly blocked processing of proMPO to mature MPO. This inhibition of processing was not a generalized effect on all lysosomal enzymes, because the maturation of a non-heme-containing lysosomal enzyme, beta-glucuronidase, was not altered. Electron microscopy showed that, although the normal peroxidase staining of endoplasmic reticulum was absent in SA-treated cells, there were MPO- related peptides in the ER. The role of the M6PR system was assessed by immunoprecipitating fractions obtained from M6PR affinity column chromatography. The 89-Kd proMPO failed to adhere to the M6PR affinity column, whereas the 59-Kd heavy subunit of mature MPO was specifically eluted from the column. We interpret these data to indicate that: (1) processing of proMPO to mature MPO occurs in a post-ER compartment that is itself BFA-sensitive or is distal to a BFA-sensitive compartment and (2) heme insertion into apoproMPO precedes and may be a prerequisite for proteolytic processing to enzymatically active mature MPO. Our analysis of the M6PR system in MPO biosynthesis led to the unanticipated finding that there were phosphomannosyl residues on mature MPO, but none on proMPO. We suggest that the bulk of proMPO at any time is not phosphorylated, but, when generated, the phosphorylated proMPO is quickly processed to the phosphorylated 59-Kd subunit of mature MPO. Thus, if the M6PR is important in the intracellular transport of MPO, it is the phosphorylated mature MPO that is directed to the lysosomal compartment by this system.(ABSTRACT TRUNCATED AT 400 WORDS)
Evidence that sustained growth suppression of intestinal anaerobic bacteria reduces the risk of acute graft-versus-host disease after sibling marrow transplantation
Authors: Beelen DW , Haralambie E , Brandt H , Linzenmeier G , Muller KD , Quabeck K , Sayer HG , Graeven U , Mahmoud HK , Schaefer UW
Published: October 2019

The influence of intestinal bacterial decontamination on the occurrence of grades II to IV acute graft-versus-host disease (GVHD) was retrospectively analyzed in 194 predominantly adult patients treated by genotypically identical sibling marrow transplantation under conditions of strict protective isolation and intestinal antimicrobial decontamination. Forty-five patients (23%) developed acute GVHD and univariate analysis identified four features that significantly increased the risk for this reaction: chronic myeloid leukemia as the underlying disease, as compared with all other disease categories (P < .0001); female marrow donors for male recipients, as compared with other gender combinations (P < .005); ineffective, as compared with sustained growth suppression of intestinal anaerobic bacteria (P < .006); and methotrexate as the sole immunoprophylactic compound, as compared with cyclosporine containing regimens (P < .05). Using the duration of anaerobic growth suppression as a time-dependent explanatory variable, proportional hazards regression analysis confirmed these features as independent predictors for acute GVHD with relative risk estimates of 1.9 (95% confidence interval [CI], 1.3 to 2.7) for the immunoprophylactic regimen (P < .0004), of 1.8 (95% CI, 1.3 to 2.5) for the underlying disease (P < .0005), of 1.7 (95% CI, 1.2 to 2.5) for anaerobic decontamination (P < .002), and of 1.3 (95% CI, 1.1 to 1.6) for the donor/recipient gender combination (P < .008), respectively. Best subset selection modeling also identified the quality of anaerobic decontamination as the third most important predictor for acute GVHD, when all four significant features were included. Estimates of acute GVHD stratified by the quality of anaerobic bacterial growth suppression showed a strong influence of anaerobic decontamination in patients burdened by at least one of the other unfavorable factors (P < .009). In conclusion, this study provides strong evidence that sustained growth suppression of intestinal anaerobic bacteria after clinical sibling marrow transplantation can independently modulate the occurrence of grades II to IV acute GVHD, which is in concordance with previous results from animal transplantation models. Antimicrobial chemotherapy specifically targeted to the intestinal anaerobic bacterial microflora may be complementarily useful in preventing acute GVHD and should be investigated in a prospective trial.
Endocytosis and lysosomal delivery of tissue plasminogen activator- inhibitor 1 complexes in Hep G2 cells
Authors: Underhill DM , Owensby DA , Morton PA , Schwartz AL
Published: October 2019

Receptor-mediated endocytosis of tissue-type plasminogen activator (t- PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t- PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.
Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate
Authors: Cattaneo M , Lecchi A , Randi AM , McGregor JL , Mannucci PM
Published: October 2019

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2–3H]-ADP to formalin- fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.
Retroviral gene transfer induced constitutive expression of interleukin- 2 or interferon-gamma in irradiated human melanoma cells
Authors: Gansbacher B , Zier K , Cronin K , Hantzopoulos PA , Bouchard B , Houghton A , Gilboa E , Golde D
Published: October 2019

Cytokines are important modulators of host antitumor responses. Two of these cytokines, interleukin-2 (IL-2) and interferon gamma (IFN-gamma), are produced after antigen-induced activation of helper lymphocytes. The cytokines are released into the immediate vicinity where they either interact with the appropriate receptors on effector cell populations or are rapidly degraded. To mimic this physiologic release of cytokines at the effector-target site, we used retroviral vectors to transduce melanoma cells with the IL-2 or IFN-gamma cDNA. Five melanoma cell lines were transduced with IL-2- or IFN-gamma-containing vectors and secreted IL-2 at 1 to 40 U/mL/10(6) cells/24 h or IFN-gamma 1 to 8 U/mL/10(6) cells/24 h, respectively. After gamma irradiation, these cells continued to secrete cytokines for about 3 to 4 weeks. Secretion of IFN-gamma induced upregulation of major histocompatibility complex class I molecules in a subset of melanoma cell lines. IL-2 production by human melanoma xenografts induced tumor rejection in BALB/c nu/nu mice, showing the in vivo effect of this cytokine. This study shows that (1) human melanoma cells can be stably transduced with cytokine- containing retroviral vectors; (2) cytokines are secreted constitutively by the transduced tumor cells and have the expected biologic effects in vitro and in vivo; and (3) after gamma irradiation, cytokines continue to be secreted for several weeks. These data suggest that irradiated cytokine-secreting allogenic or autologous tumor cells can be used in vaccination protocols for cancer patients.
G19.4(alpha CD3) x B43(alpha CD19) monoclonal antibody heteroconjugate triggers CD19 antigen-specific lysis of t(4;11) acute lymphoblastic leukemia cells by activated CD3 antigen-positive cytotoxic T cells
Authors: Anderson PM , Crist W , Hasz D , Carroll AJ , Myers DE , Uckun FM
Published: October 2019

A highly purified, 300-Kd bispecific monoclonal antibody (MoAb) heteroconjugate was prepared by covalently linking the anti-CD3 MoAb, G19.4, to the anti-CD19 MoAb, B43. Dual-color staining techniques and multiparameter flow cytometry confirmed that this alpha CD3 x alpha CD19 heteroconjugate was able to bind to both CD3+ T cells and CD19+ t(4;11) acute lymphoblastic leukemia (ALL) cells. T-cell-mediated lysis of freshly isolated primary bone marrow blasts from nine newly diagnosed ALL patients with a t(4;11)(q21;q23) chromosomal translocation were studied with 51Cr-release assays. Picomolar concentrations of alpha CD3 x alpha CD19 MoAb heteroconjugate effectively triggered lysis of CD19+ t(4;11) ALL cells by interleukin-2- activated CD3+ peripheral blood T-cell (PBTC) effectors but did not augment the cytolytic activity of the same effectors against CD19- T- ALL cells. In contrast to the alpha CD3 x alpha CD19 heteroconjugate, neither the alpha CD3 x alpha CD3 homoconjugate control nor the alpha CD19 x alpha CD72 heteroconjugate control facilitated the cytolysis of t(4;11) ALL blasts. Occupation of the target CD19 binding sites on t(4;11) ALL blasts by preincubation with excess unconjugated alpha CD19 MoAb abrogated the potentiating effects of the alpha CD3 x alpha CD19 heteroconjugate on PBTC-mediated cytolysis. Thus, the cell type- specific cytolysis of t(4;11) ALL blasts by PBTC effectors is dependent on both the alpha CD19 and alpha CD3 moieties of the alpha CD3 x alpha CD19 heteroconjugate. To our knowledge, this is the first description of an effective bispecific antibody that facilitates the T-cell- mediated lysis of t(4;11) ALL blasts.
T-cell acute lymphoblastic leukemia--the associated gene SCL/tal codes for a 42-Kd nuclear phosphoprotein
Authors: Goldfarb AN , Goueli S , Mickelson D , Greenberg JM
Published: October 2019

SCL/tal is a putative oncogene originally identified through its involvement in the translocation t(1;14)(p32;q11) present in the leukemic cell line DU.528. Subsequent studies have shown an upstream deletion activating expression of SCL/tal to be one of the most common genetic lesions in T-cell acute lymphoblastic leukemia (T-ALL). The cDNA sequence of SCL/tal encodes a basic helix-loop-helix (bHLH) protein with regions of marked homology to lyl-1 and tal-2, two other bHLH proteins involved in T-ALL chromosomal translocations. The bHLH motif suggests that the SCL/tal product localizes to the nucleus, binds to specific DNA sequences, and regulates transcription of a specific array of target genes. Our studies directly identify the SCL/tal product as a 42-Kd phosphoprotein that efficiently localizes to the nucleus. Deletion mutagenesis has allowed identification of a region critical for nuclear localization, a region that corresponds to the DNA- binding basic domain within the bHLH motif. Because this domain is shared by lyl-1 and tal-2, these latter putative T-cell oncoproteins probably use a nuclear localization mechanism identical to that of SCL/tal.
Clinical, morphologic, and cytogenetic characteristics of 26 patients with acute erythroblastic leukemia
Authors: Olopade OI , Thangavelu M , Larson RA , Mick R , Kowal-Vern A , Schumacher HR , Le Beau MM , Vardiman JW , Rowley JD
Published: October 2019

We have performed a retrospective analysis of the clinical, morphologic, and cytogenetic findings in 26 patients diagnosed between January 1969 and September 1991 with acute erythroblastic leukemia de novo (EL or AML-M6). Clonal chromosomal abnormalities were found in 20 (77%) patients (95% confidence interval [CI], 61% to 93%). Loss of all or part of the long arm (q) of chromosomes 5 and/or 7 was observed in 17 (65%) patients (95% CI, 47% to 83%). In addition, the karyotypes were often complex, with multiple abnormalities and subclones. Among the remaining nine patients, six had a normal karyotype and one each had trisomy 8, t(3;3), or t(3;5). The overall frequency of abnormalities of chromosomes 5 and/or 7 observed in our M6 patients is similar to that observed in our patients with therapy-related acute myeloid leukemia (t-AML; 99 of 129 patients, 77%), but substantially higher than that noted in our other patients with AML de novo (French- American-British [FAB] subtypes M1-M5: 52 of 334 patients, 16%). Our M6 patients with abnormalities of chromosomes 5 and/or 7 were older and had a shorter median survival (16 v 77 weeks [P = .005]) than did the M6 patients without these abnormalities. We found no correlation between morphologic features and either cytogenetic abnormalities or clinical outcome. Of note was the finding that the percentage of myeloblasts, which may account for only a small fraction of the total marrow elements when the revised FAB criteria are applied, had no bearing on prognosis. We conclude that acute erythroblastic leukemia, when defined by morphologic criteria, consists of two distinctive subgroups: one group tends to be older, has complex cytogenetic abnormalities, especially of chromosomes 5 and/or 7, and shares biologic and clinical features with t-AML; the other group, with simple or no detectable cytogenetic abnormalities, has a more favorable prognosis when treated with intensive chemotherapy.
Granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) enhances high-dose Ara-C-induced programmed cell death or apoptosis in human myeloid leukemia cells
Authors: Bhalla K , Tang C , Ibrado AM , Grant S , Tourkina E , Holladay C , Hughes M , Mahoney ME , Huang Y
Published: October 2019

High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM- CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.
Expression of the novel intermediate filament-associated protein restin in Hodgkin's disease and anaplastic large-cell lymphoma
Authors: Delabie J , Shipman R , Bruggen J , De Strooper B , van Leuven F , Tarcsay L , Cerletti N , Odink K , Diehl V , Bilbe G
Published: October 2019

In this study, the expression of the novel intermediate filament protein Restin in human tissues was analyzed. Restin expression was studied by immunohistochemistry using polyclonal and monoclonal antibodies. Restin was not detected in normal tissues, a range of B- and T-cell non-Hodgkin's lymphomas, and nonlymphoid tumors. However, Restin was present in Reed-Sternberg cells and variants thereof in Hodgkin's disease, with the exception of the lymphocyte-predominant, paragranuloma subtype. Restin was also highly expressed in anaplastic large-cell lymphoma (so-called Ki-1 lymphoma). As expected, Restin was also expressed in Hodgkin cell lines L428, L428KSA, Co, and KM-H2 and the anaplastic large-cell lymphoma cell line Karpas 299, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, as well as Northern blotting. The presence of Restin in both Hodgkin's disease and anaplastic large-cell lymphoma is intriguing and might indicate a role of this structural protein in the pathogenesis of both conditions.
Prevalent skipping of an individual exon accounts for shortened protein 4.1 Presles
Authors: Feddal S , Hayette S , Baklouti F , Rimokh R , Wilmotte R , Magaud JP , Maréchal J , Benz EJ , Girot R , Delaunay J , Morlé L
Published: October 2019

An asymptomatic shortened variant of protein 4.1 (-8.5 Kd) was first recognized in the red blood cells and designated protein 4.1 Presles. We show here that the missing segment belongs to the 22/24 Kd domain. Protein 4.1 cDNA from reticulocytes was amplified, mapped, and sequenced. The truncation appeared to result from the prevalent skipping of an individual and alternatively spliced exon, also called motif II, whereas this motif is preferentially retained under normal conditions. The same phenomenon was observed in lympho-blastoid cells. Sequencing over 80 bp of intronic sequences 5′ and 3′ of motif II failed to reveal any change. A new alternative splice site was incidently found 81 nucleotide downstream of motif II in both normal and truncated 4.1 mRNA.
Bone marrow transplantation for peripheral T-cell lymphoma in children and adolescents
Authors: Gordon BG , Warkentin PI , Weisenburger DD , Vose JM , Sanger WG , Strandjord SE , Anderson JR , Verdirame JD , Bierman PJ , Armitage JO
Published: October 2019

We report nine children with relapsed (n = 8) or high-risk (n = 1) peripheral T-cell lymphoma (PTCL) who underwent autologous (n = 6) or allogeneic (n = 3) bone marrow transplantation (BMT). These children received transplants as part of a prospective phase I/II study of thioTEPA (TT) and total body irradiation (TBI) with escalating doses of VP-16. The median age of these patients at time of BMT was 6.5 years (range 2.5 years to 14 years). Three were transplanted with active disease after failing salvage chemotherapy. Of the other six, one was transplanted in first complete remission (CR) and five in second or subsequent CR. Of these nine patients, eight are free of disease a median of 25 months after BMT (range, 6 to 48 months), with an estimated 2-year relapse-free survival (RFS) of 89%. Six of these eight patients have been followed for 12 or more months after BMT, and in each their current remission exceeds their longest previous remission duration. The toxicity of the TT/TBI +/- VP-16 regimens was significant but manageable, predominantly consisting of severe mucositis. For a comparison, we reviewed retrospective data on the six additional children and adolescents with PTCL who underwent BMT during the 3-year period preceding this phase I/II study. The median age at BMT of these six patients was 19 years (range 15.5 years to 20 years). These patients were prepared for BMT with a variety of other regimens. One had no response to BMT and the other five relapsed at 1.5 to 5 months after BMT (median, 3 months) with an RFS of 0%. Our data suggest that thioTEPA plus TBI, with or without VP-16, is an effective preparative regimen for BMT for young patients with relapsed or high-stage PTCL and leads to prolonged RFS.
Transcription factors, translocations, and leukemia
Authors: Nichols J , Nimer SD
Published: October 2019

The frequent occurrence of TF gene involvement in translocations associated with leukemia is remarkable, although not yet explained. The wide variety of TFs involved in these translocations and the different stages of cellular maturation argue against a unifying mechanism. Recombinases, active during B-cell and T-cell development, have been implicated in gene arrangements involving TCR genes and in the SIL/SCL rearrangement, which involves two genes not normally rearranged. However, other mechanisms must clearly be active in generating these molecular abnormalities and perhaps they relate to the multistep maturation and differentiation processes and continuous cell turnover seen in hematopoietic cells. The difficulties in obtaining human solid tumor samples may make it more difficult to identify translocations involving TF genes in solid tumors. Recently, the cytogenetic analysis of solid tumors has improved and specific cytogenetic abnormalities have been associated with specific types of tumors. With advanced techniques, such as fluorescent in situ hybridization (a technique that does not depend on cell growth) and PCR, abnormalities involving TF genes will be discovered. Abnormalities of TF genes, other than translocations, have been seen in a broad variety of nonhematopoietic malignancies. The p53 protein has been shown to bind DNA in a sequence- specific fashion and interact with a variety of DNA tumor virus oncoproteins. The broad range of cell types that harbor p53 abnormalities suggests that TF abnormalities will likely be implicated in many solid tumors. We have detailed several examples of how gene rearrangements that accompany chromosomal translocations in acute leukemia can alter the expression or activity of cellular TFs. Several translocations generate fusion RNA transcripts and fusion TF proteins with altered functional characteristics. Other translocations result in the expression of a gene not normally detectable in hematopoietic cells or alter the level of its expression, or affect the promoter usage or exon structure of the gene (Table 2). Studies are underway in many laboratories to characterize the biologic activity of these abnormal TFs and it remains to be proven that these molecular abnormalities are directly linked with leukemogenesis. The identification of abnormal fusion transcripts and proteins may allow specific therapies to be directed against “tumor-specific” DNA, mRNA, or protein targets. Therapeutic strategies based on antisense or ribozyme technology may be used to turn off expression of these genes and inhibit leukemia cell growth. Immunologic methods can also be used to direct therapy against the malignant cells.
Effect of stem cell factor on in vitro erythropoiesis in patients with bone marrow failure syndromes
Authors: Alter BP , Knobloch ME , He L , Gillio AP , O'Reilly RJ , Reilly LK , Weinberg RS
Published: October 2019

Stem cell factor (SCF) enhances normal hematopoiesis. We examined its effect in vitro on bone marrow and blood progenitors from patients with inherited bone marrow failure syndromes, including 17 patients each with Diamond-Blackfan anemia (DBA) and Fanconi's anemia (FA), 3 with dyskeratosis congenita (DC), and 1 each with amegakaryocytic thrombocytopenia (amega) and transient erythroblastopenia of childhood (TEC). Mononuclear cells were cultured with erythropoietin (Ep) alone or combined with SCF or other factors. SCF increased the growth of erythroid progenitors in cultures from 50% of normal controls, 90% of DBA, 70% of FA, 30% of DC, and the amega and TEC patients; normal numbers were reached in 25% of DBA studies. Improved in vitro erythropoiesis with SCF in all types of inherited marrow failure syndromes does not suggest a common defect involving kit or SCF, but implies that SCF may be helpful in the treatment of hematopoietic defects of varied etiologies.
Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells
Authors: Irani AM , Nilsson G , Miettinen U , Craig SS , Ashman LK , Ishizaka T , Zsebo KM , Schwartz LB
Published: October 2019

We have previously shown the development in vitro of tryptase+ human mast cells from fetal liver cells cocultured with murine 3T3 fibroblasts. In this study, recombinant human stem cell factor (rhuSCF), the ligand for the c-kit proto-oncogene product called Kit, stimulated the growth and differentiation primarily of mast cells from dispersed fetal liver cells, whereas recombinant human interleukin-3 (rhuIL-3) stimulated the differentiation of basophils along with other cell types. Cultures of fetal liver cells were initiated and maintained in the presence of rhuSCF or rhuIL-3 for up to 6 weeks. Metachromatic cells in cytospins were identified as mast cells primarily on the basis of tryptase expression, and as MCT or MCTC by immunohistochemistry using monoclonal antibodies against tryptase and chymase, whereas basophils were metachromatic, polymorphonuclear, and lacked these proteases. Levels of tryptase and histamine were measured by radioimmunoassay, tryptase and chymase activities by peptide hydrolysis, and cell surface Kit by flow cytometry with the monoclonal antibody YB5.B8. The predominant presence of mast cells occurred only in the cultures supplemented with rhuSCF. The percentage and total number of mast cells increased over time with increasing concentrations of rhuSCF and reached a plateau at 55 ng/mL. At this concentration of rhuSCF, mast cells first appeared by day 7; by day 42, 106% of the starting number of cells were present and 85% of these were tryptase+, 31% being weakly chymase+. These mast cells appeared immature by ultrastructural criteria; most cells were mononuclear, but some had nuclei with deeply divided lobes. DNA synthesis in tryptase+ mast cells at days 21 and 28 of culture with rhuSCF was demonstrated by incorporation of bromodeoxyuridine. Calculated levels of histamine (1.2 pg/mast cell) and tryptase (0.9 pg/mast cell) were similar to those determined previously in coculture experiments with murine 3T3 fibroblasts. Chymase activity was undetectable in most cell extracts. On day 0, 4% to 20% of fetal liver cells expressed cell surface Kit. In the presence of rhuSCF, the percentages and total numbers of Kit+ cells and the apparent concentration of Kit per cell increased along with the number of tryptase+ cells. In the presence of rhuIL-3, toluidine blue+, tryptase- cells first and maximally appeared at day 14 (11% +/- 2.5%). The percentage of these toluidine blue+ cells then declined to about 6% by days 21 and 35, while the total number of positive cells declined over 10-fold. Kit+ cells in the presence of rhuIL-3 declined from 9% on day 3 to 2% on day 35.(ABSTRACT TRUNCATED AT 400 WORDS)
Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation
Authors: Debili N , Issaad C , Masse JM , Guichard J , Katz A , Breton-Gorius J , Vainchenker W
Published: October 2019

Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.