bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase I-II trial of a monoclonal anti-tumor necrosis factor alpha antibody for the treatment of refractory severe acute graft-versus-host disease
Authors: Herve P , Flesch M , Tiberghien P , Wijdenes J , Racadot E , Bordigoni P , Plouvier E , Stephan JL , Bourdeau H , Holler E
Published: October 2019

In a multicenter pilot study, 19 patients with severe acute graft- versus-host disease (aGVHD) refractory to conventional therapy and serotherapy with a monoclonal anti-interleukin-2 receptor antibody were treated by in vivo infusion of a monoclonal anti-tumor necrosis factor alpha (TNF alpha) antibody (B-C7). Ten patients were grafted from a genotypically identical sibling, five from an HLA-mismatched family member, and four from an HLA-matched unrelated donor. Before B-C7 treatment, 15 patients had grade IV and four had grade III GVHD. In all cases, patients received cyclosporine/methotrexate as aGVHD prophylaxis. Patients were administered increasing doses of antibody (from 0.1 to 0.4 mg/kg). The antibody was infused in bolus daily for 4 days and then every other day twice (6 doses). No side effects were observed during treatment regardless of the dose level used. Changes in peripheral blood cell counts occurred in 8 of the 19 patients and appeared to be unrelated to B-C7. No truly complete response was observed; eight patients achieved a very good partial response (42.6%) and six a partial response (31.5%). The treatment was ineffective in five patients (26.4%). When present, the response occurred early (less than 3 days). In the 14 responding patients, gut lesions responded best (100%), followed by skin (85%) and liver (35.7%) lesions. In 9 of 11 evaluable patients (81%), GVHD recurred when treatment was discontinued in a median delay of 3 days (range, 2 to 120 days). All except one died from aGVHD. Two patients did not experience GVHD recurrence and are still alive 13 and 18 months post-bone marrow transplantation. This pilot study shows that a monoclonal anti-TNF alpha antibody may be of benefit to some patients with severe refractory aGVHD, but is ineffective to prevent GVHD recurrence in the majority of cases.
Continuous treatment with all-trans retinoic acid causes a progressive reduction in plasma drug concentrations: implications for relapse and retinoid "resistance" in patients with acute promyelocytic leukemia [published erratum appears in Blood 1992 Aug 1;80(3):855]
Authors: Muindi J , Frankel SR , Miller WH Jr , Jakubowski A , Scheinberg DA , Young CW , Dmitrovsky E , Warrell RP Jr
Published: October 2019

Although all-trans retinoic acid (RA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), all groups have described clinical relapses despite continued RA treatment. This finding suggests that resistance to the cytodifferentiating effects of the retinoid had been acquired. To investigate potential mechanisms of clinical resistance to RA, we serially evaluated the clinical pharmacology of the drug in APL patients treated with this agent. Leukemic cells from patients relapsing from RA treatment were cultured in the presence of RA and examined for evidence of morphologic maturation. We also studied messenger RNA expression of the newly described gene product of the (15;17) translocation in APL, PML/RA receptor-alpha (PML/RAR-alpha). Serial pharmacokinetic studies showed that continuous daily RA treatment was associated with a marked decrease in plasma drug concentrations at the time of relapse compared with the initial day of therapy. Doubling the RA dose in six patients failed to reinduce response at the time of relapse and also failed to significantly augment plasma RA concentrations. However, leukemic cells obtained at the time of relapse from four patients retained in vitro sensitivity to the differentiating activity of RA (10(-6) mol/L). No change was observed in the pattern of PML/RAR-alpha expression assessed by Northern blot analysis at the time of relapse compared with pretreatment in two patients who were tested. These results indicate that clinical relapse and “resistance” to continuous treatment with all- trans RA in APL is associated with progressive reduction of plasma concentrations, potentially to levels below those that sustain differentiation of leukemic cells in vivo. Long-term success of this treatment will require the development of strategies that circumvent this pharmacologic phenomenon.
Iron chelation with desferrioxamine B in adults with asymptomatic Plasmodium falciparum parasitemia [see comments]
Authors: Gordeuk VR , Thuma PE , Brittenham GM , Zulu S , Simwanza G , Mhangu A , Flesch G , Parry D
Published: October 2019

To determine if iron chelation therapy has activity against human malaria, we administered desferrioxamine B in amounts of 100 mg/kg per day by continuous 72-hour subcutaneous infusions to 28 volunteers with asymptomatic Plasmodium falciparum infection in a randomized, double- blind, placebo-controlled crossover trial. Peripheral blood concentrations of P falciparum ring forms were determined at 12-hour intervals in all subjects and serum concentrations of desferrioxamine B + ferrioxamine (the iron complex of desferrioxamine B) were measured in 26 subjects. Geometric mean concentrations of asexual intraerythrocytic parasites decreased with both chelator and placebo treatment, but the decrement with desferrioxamine B was significantly greater than that with placebo (P less than .006) during both the initial and crossover periods. Compared with placebo, desferrioxamine B treatment was associated with an almost 10-fold enhancement of the rate of parasite clearance during both phases of the trial (P less than .007). Mean +/- SEM steady state concentrations of desferrioxamine B + ferrioxamine were 6.90 +/- 0.60 mumol/L at 36 hours and 7.72 +/- 0.68 mumol/L at 72 hours; in vitro, the ID50 has been reported to be approximately 4 to 20 mumol/L. No drug toxicity was detected. Parasitemia recurred in 19 of 24 participants followed-up over 1 to 6 months. We conclude that desferrioxamine B enhances the clearance of P falciparum parasitemia and that iron chelation may provide a new strategy to be developed for the treatment of malaria.
Characterization of glycoprotein IIb/IIIa-positive cells in human umbilical cord blood: their potential usefulness as megakaryocyte progenitors
Authors: Zucker-Franklin D , Yang JS , Grusky G
Published: October 2019

A need for hematopoietic stem cells, particularly cells destined to enter the megakaryocyte (MK) series, prompted phenotypic analysis of mononuclear leukocytes in human cord blood. To this end, immunohistochemical, flow cytometric, and ultrastructural techniques were used. The immunogold silver enhancement method (IGS) for the detection of the MK-specific glycoprotein (GP) IIb/IIIa epitopes combined with a monocyte-specific stain for alpha-naphthyl butyrate esterase proved to be superior to flow cytometry (FACS) for precise quantitation of cell types in each sample. Immunoelectron microscopy afforded a description of distinctions between precursors bearing GPIIb/IIIa epitopes and other stem cells of the myeloid series. The number of presumed MK progenitors was surprisingly high, averaging 1.8% +/- 1.3% (range, 0.2% to 4.6%) by IGS and 4.1% +/- 3.0% (range, 0.2% to 9.3%) by FACS analysis. The occurrence of GPIIb/IIIa-positive denuded MK nuclei in cord blood was of interest, but was too small to affect these data. These observations should advocate a greater use of cord blood for restitution of MK/platelet-lineage-depleted patients as well as for experimental studies concerned with MK differentiation.
Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells
Authors: Avraham H , Vannier E , Cowley S , Jiang SX , Chi S , Dinarello CA , Zsebo KM , Groopman JE
Published: October 2019

The kit ligand (KL), also termed stem cell factor (SCF), is a recently discovered hematopoietic growth factor that augments response of early progenitor cells to other growth factors and supports proliferation of continuous mast cell lines. Histological studies suggest that the receptor for SCF/KL, the c-kit proto-oncogene product, is present in bone marrow megakaryocytes. We studied the effects of SCF/KL on immortalized human megakaryocytic cell lines (CMK, CMK6, and CMK11–5) and on isolated human marrow megakaryocytes. Human SCF/KL alone or in combination with the hematopoietic growth factors, interleukin-3 (IL- 3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL- 6, stimulated proliferation of these megakaryocytic cell lines. SCF/KL treatment did not alter expression of gpIb, gpIIb/IIIa, LFA-1, ICAM-1, or GMP-140 in CMK cells. No effect on ploidy was observed. Furthermore, human SCF/KL induced expression of IL-1 alpha, IL-1 beta, IL-2, and IL- 6 in CMK cells. In a fibrin clot system, SCF/KL modestly potentiated megakaryocyte colony formation when added alone to cultures containing CD34+, DR+ bone marrow cells. Addition of SCF/KL with IL-3 or GM-CSF to these cultures resulted in a more marked marrow megakaryocytic cells. SCF/KL may directly affect megakaryocytopoiesis, as well as secondarily modulate hematopoiesis through induction of cytokines in target cells.
Endotoxin enhances the expression of monocyte prothrombinase activity
Authors: Robinson RA , Worfolk L , Tracy PB
Published: October 2019

Thrombin is generated on the surface of mononuclear cells (MNCs) through the assembly and function of the prothrombinase complex consisting of the enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate membrane surface for proper assembly of the protein constituents. Assays performed in the presence of factors Va and Xa indicated that endotoxin significantly enhanced the prothrombinase activity (1.5- to 2.5-fold; P less than .001) expressed by MNCs in a dose- and time-dependent manner. Monocytes present in the MNC suspensions were responsible for this increased activity through processes resulting in both enhanced cellular activity and the enhanced release of membranous vesicles. Endotoxin was without effect on the expression of lymphocyte prothrombinase activity. Scanning electron microscopy techniques indicated that endotoxin resulted in extensive membrane blebbing of the monocytes present in the MNC suspensions with no effect on the morphology of the lymphocytes. Within 5 hours, endotoxin maximally enhanced the prothrombinase activity expressed by the monocyte membrane surface 2.8-fold, whereas 8 hours was required to maximally enhance the activity associated with the released vesicles by twofold. The observed increase in activity expressed by the monocyte membrane surface was due solely to endotoxin, since the activity expressed by the unstimulated monocyte membrane surface remained unaltered over time. In contrast, cell vesiculation, which occurred in the absence of any stimulus, was further enhanced by endotoxin. The increase in activity associated with the released vesicles from both stimulated and unstimulated cells paralleled an increase in the vesicle number as determined by flow cytometric analyses. The vesicle released from both unstimulated and stimulated monocytes were indistinguishable in size as determined by image analysis and ranged between 0.05 and 0.3 microns in diameter. 2-Deoxy-D-glucose (2DG) significantly enhanced the prothrombinase activity expressed by the monocyte membrane surface, as well as the released vesicle fraction, when used alone or in addition to endotoxin. The enhanced activity associated with the vesicle fraction again was attributed to the release of more vesicles. In contrast, cycloheximide decreased the prothrombinase activity expressed by the monocyte membrane surface, as well as the activity associated with vesicles released from both stimulated and unstimulated cells. These data suggest that the expression of monocyte prothrombinase activity can be significantly enhanced by endotoxin through processes that alter the monocyte membrane surface and augment the vesiculation process. Both processes appear to be regulated by protein synthesis and adenosine triphosphate (ATP)-dependent mechanisms.
Lipoprotein-associated coagulation inhibitor (LACI) is a cofactor for heparin: synergistic anticoagulant action between LACI and sulfated polysaccharides
Authors: Wun TC
Published: October 2019

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits tissue factor (TF)/factor VIIa-induced coagulation in a factor Xa-dependent manner. The roles of endogenous plasma LACI and exogenously added LACI and heparin, in the regulation of coagulation, initiated via the intrinsic and extrinsic pathways, were studied using the activated partial thromboplastin time (APTT) and the modified prothrombin time (PT) assays, respectively. Both LACI- depleted plasma and normal plasma have identical APTTs and similar prolongations of the APTT in response to heparin; both are fully anticoagulated (arbitrarily defined as clotting times of greater than 1 hour) at similar concentrations of heparin. These results indicate that heparin is an effective anticoagulant when coagulation is initiated by the intrinsic pathway and that endogenous LACI is not significantly involved in the regulation of this pathway. The PT of normal plasma is only marginally longer than that of LACI-depleted plasma in the absence of heparin, suggesting that endogenous plasma LACI has a very limited capacity to inhibit TF-induced clotting. However, in the presence of heparin, the PTs of LACI-depleted plasma and normal plasma are different. Prolongation of the PT occurred only moderately and linearly with increasing concentrations of heparin in LACI-depleted plasma. In contrast, normal plasma showed a greater extent of PT prolongation in response to heparin and the plasma became fully anticoagulated at a certain threshold concentration of heparin. These results suggest that LACI serves as a cofactor for heparin and thus greatly enhances the inhibition of TF-induced coagulation. LACI-depleted plasma was supplemented with purified recombinant LACI and/or heparin and the effects on TF-induced clotting were studied. A combination of LACI and heparin greatly enhanced anticoagulation compared with LACI or heparin alone. Many sulfated polysaccharides were also found to enhance the LACI-dependent inhibition of TF-induced clotting. By weight, the relative potencies of these compounds are: low molecular weight heparin (mean Mr, 5,100) greater than unfractionated heparin greater than low molecular weight heparin (mean Mr, 3,700) greater than pentosan polysulfate greater than dermatan sulfate greater than dextran sulfate greater than heparan sulfate. Based on the above results, it is concluded that LACI is a cofactor for heparin in the inhibition of TF- induced clotting and that LACI and sulfated polysaccharides act synergistically in whole plasma.
Clinical significance of multidrug resistance P-glycoprotein expression on acute nonlymphoblastic leukemia cells at diagnosis [see comments]
Authors: Campos L , Guyotat D , Archimbaud E , Calmard-Oriol P , Tsuruo T , Troncy J , Treille D , Fiere D
Published: October 2019

To evaluate the clinical value of the expression of multidrug resistance P-glycoprotein (P-170) on the surface of acute nonlymphoblastic leukemia (ANLL) cells, we analyzed specimens from 150 newly diagnosed patients for staining with MRK16, a monoclonal antibody (MoAb) that binds to an external epitope of P-170. Other surface markers (CD13, CD14, CD15, and CD34) were studied by the same technique. A marker was considered positive when 20% or more cells were stained. Of 150 samples, 71 were P-170-positive. These cases did not differ from P-170-negative cases with regard to age, sex, initial white blood cell (WBC) counts, or French-American-British (FAB) type (except for M3 ANLL, which were more frequently negative). However, leukemias arising from previous myelodysplastic syndrome (MDS) and therapy- induced leukemias were more frequently P-170-positive. CD34 and P-170 expression were significantly associated. All patients were treated by intensive chemotherapy. Complete remission (CR) rates were significantly lower in P-170-positive (23/71, 32%) than in P-170- negative cases (64/79, 81%) (P less than 10(-5)). CD34 positivity was also associated with a low remission rate (P less than 10(-5)). Survival was shorter for P-170- and CD34-positive patients (P less than 10(-5)). The prognostic value of both markers was confirmed in multivariate analysis. CR duration was also shorter for P-170-positive cases, but the difference is less significant (P = .05). It is concluded that P-170 analysis may be an important tool for predicting the outcome of intensive chemotherapy in ANLL patients.
Differential expression of cytokines in human blood monocyte subpopulations [see comments]
Authors: Ziegler-Heitbrock HW , Strobel M , Kieper D , Fingerle G , Schlunck T , Petersmann I , Ellwart J , Blumenstein M , Haas JG
Published: October 2019

Cytokine expression was analyzed in CD14++ regular monocytes and in the novel subset of CD14+/CD16+ small monocytes. Biologic activity for tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 in the supernatant of elutriator-enriched, cell sorter-purified small monocytes was about 10-fold lower compared with regular monocytes when stimulated with lipopolysaccharide (LPS) for 12 hours. In CD14++ regular monocytes levels were 1,157 U x 10(-3)/mL, 158 U/mL, and 1,337 U/mL for TNF, IL-1, and IL-6, respectively. By contrast, CD14+/CD16+ small monocytes exhibited 137 U x 10(-3)/mL, 14 U/mL, and 60 U/mL for TNF, IL-1, and IL-6, respectively. Additional treatment with interferon- gamma enhanced production of TNF in both subsets, but CD14+/CD16+ small monocytes still exhibited lower levels. Stimulation of the monocyte subsets by platelet-activating factor gave the same pattern of results. Hybridization with 32P-labeled oligonucleotides specific for the respective cytokine messenger RNAs (mRNAs) showed a 10-fold lower prevalence of transcripts for TNF, IL-1, and IL-6, as well. By contrast, the constitutive expression of Glyceraldehyde-3-phosphate- dehydrogenase mRNA was 1.7-fold higher in the CD14+/CD16+ small monocytes. These data indicate that the novel subset of small monocytes is selectively suppressed in the expression of the cytokines TNF, IL-1, and IL-6, suggesting that these cells may comprise a deactivated type of cell. The expression of class II transcripts in the small monocytes is, however, similar to the regular monocytes, and the cell surface expression of class II protein about threefold increased. Thus, the novel subset of small monocytes appears to be a functionally distinct type of cell.
Selection of histocompatible apheresis platelet donors by cross- matching random donor platelet concentrates
Authors: O'Connell BA , Lee EJ , Rothko K , Hussein MA , Schiffer CA
Published: October 2019

It can be impossible to identify compatible platelet donors for alloimmunized patients whose HLA type cannot be determined or who have uncommon HLA types. We have previously shown that histocompatible donors can be rapidly identified by “mass screening” of platelet concentrates (PC), which are readily available in all blood banks, using a solid-phase adherence platelet cross-matching technique. Compatible PC were given to five alloimmunized patients with multispecific HLA antibodies refractory to random donor (RD) PC and selected single-donor platelet transfusions. After transfusions which produced satisfactory responses, we identified the original whole blood donors to serve as apheresis donors. Thus, the donors selected were compatible in vitro by cross-matching, and in vivo by transfusion. Only 3% to 13% of PC cross-matched for these alloimmunized patients were potentially compatible and it was necessary to screen large numbers (65 to 205 U) of PC per patient. Eighteen of 22 PC-selected transfusions produced satisfactory increments, allowing selection of 12 donors, all of whom were willing to undergo apheresis. Ten of 12 of these single- donor transfusions were successful; the two unsuccessful transfusions were infused 2 weeks after the initial PC cross-match and were still compatible with the original serum, but incompatible with more recent serum, demonstrating a change in antibody reactivity. The HLA types of the successful single donors selected by PC cross-matching differed widely from the patients' HLA types and, therefore, these donors would not have been selected by standard approaches using HLA typing. Cross- matching large numbers of RD PC for the identification of apheresis donors is helpful in the management of the alloimmunized patient and may be of particular utility for blood centers that do not have access to HLA-typed donor pools.
The t(15;17) breakpoint in acute promyelocytic leukemia cluster within two different sites of the myl gene: targets for the detection of minimal residual disease by the polymerase chain reaction
Authors: Chang KS , Lu JF , Wang G , Trujillo JM , Estey E , Cork A , Chu DT , Freireich EJ , Stass SA
Published: October 2019

The retinoic acid receptor alpha (RAR alpha) and the myl gene are involved in the translocation breakpoint t(15;17)(q22;q21) in acute promyelocytic leukemia (APL). The majority of the breakpoint sites have been mapped within the second intron of the RAR alpha gene; however, the breakpoint sites on the myl gene are variable. Using primer sets derived from exon 2 or exon 3 of the RAR alpha gene and a primer derived from the myl cDNA, we were able to amplify the breakpoint sites of the fusion transcripts of all six APL RNA samples by the reverse transcriptase-polymerase chain reaction (RT-PCR). A DNA fragment of 290 bp (breakpoint A) was amplified using RNA samples from three patients, whereas two DNA fragments of 630 and 774 bp (breakpoint B) were amplified using RNA samples from the other three APL patients. DNA sequence analysis of the amplified fragments suggests that the APL breakpoints clustered within two different introns of the myl gene. Northern blot analysis demonstrated that fusion transcripts RAR alpha/myl and myl/RAR alpha of varying sizes were detected in patients with different breakpoint sites on the myl gene. In addition, we analyzed five APL samples in complete remission and detected t(15;17)- positive cells. We conclude that the t(15;17) breakpoints in APL can be amplified by PCR using a single primer set and that minimal residual disease can be demonstrated in APL using RT-PCR.
Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase I trial of daily bolus infusion
Authors: Grossbard ML , Freedman AS , Ritz J , Coral F , Goldmacher VS , Eliseo L , Spector N , Dear K , Lambert JM , Blattler WA
Published: October 2019

Anti-B4-blocked Ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody (MoAb) and the protein toxin “blocked ricin.” The anti-B4 MoAb is directed against the B-lineage-restricted CD19 antigen expressed on more than 95% of normal and neoplastic B cells. Blocked ricin is an altered ricin derivative that has its nonspecific binding eliminated by chemically blocking the galactose binding domains of the B chain. In vitro cytotoxicity studies demonstrate that the IC37 of Anti-B4-bR is 2 x 10(-11) mol/L compared with 4 x 10(-12) mol/L for native ricin. A phase I dose escalation clinical trial was conducted in 25 patients with refractory B-cell malignancies. Anti-B4-bR was administered by daily 1-hour bolus infusion for 5 consecutive days at doses ranging from 1 microgram/kg/d to 60 micrograms/kg/d. Serum levels above 1 nmol/L were achieved transiently in the majority of patients treated at the maximum tolerated dose of 50 micrograms/kg/d for 5 days for a total dose of 250 micrograms/kg. The dose-limiting toxicity was defined by transient, reversible grade 3 elevations in hepatic transaminases, without impaired hepatic synthetic function. Minor toxicities included transient hypoalbuminemia, thrombocytopenia, and fevers. Human antimouse antibody and human anti-ricin antibody were detected in nine patients. One complete response, two partial responses, and eight mixed or transient responses were observed. These results show the in vitro and in vivo cytotoxicity of Anti-B4-bR and indicate that this immunotoxin can be administered as a daily bolus infusion for 5 days with tolerable, reversible toxicity.
Induced differentiation of HL-60 promyelocytic leukemia cells to monocyte/macrophages is inhibited by hydroquinone, a hematotoxic metabolite of benzene
Authors: Oliveira NL , Kalf GF
Published: October 2019

Chronic exposure of humans to benzene has been shown to have a cytotoxic effect on hematopoietic progenitor cells in intermediate stages of differentiation, which can lead to aplastic anemia and acute myelogenous leukemia. We studied the effect of hydroquinone (HQ), a toxic metabolite of benzene found in the bone marrow, on the human promyelocytic leukemia cell line (HL-60), which can be induced to differentiate to both monocyte and myeloid cells, and thus has been used as a surrogate for a granulocyte/macrophage progenitor cell. Exposure of HL-60 cells to noncytotoxic concentrations of HQ for 3 hours before induction with phorbol myristate acetate (TPA) caused a dose-dependent inhibition of the acquisition of characteristics of monocytic differentiation, such as adherence, nonspecific esterase (NSE) activity, and phagocytosis, but had no effect on cell proliferation. HQ appeared to be affecting maturation beyond the monoblast/promonocyte stages. HQ also prevented differentiation induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]; however, the block occurred after the acquisition of adherence. HQ at concentrations that inhibited monocytic differentiation had no effect on differentiation to granulocytes, suggesting that the block in the differentiation of these bipotential cells is a step unique to the monocytic pathway. HQ was unable to prevent differentiation induced by the macrophage-derived cytokine, interleukin (IL)-1, a differentiation factor for cells of the monocytic lineage.
Role of c-kit ligand in the expansion of human hematopoietic progenitor cells
Authors: Brandt J , Briddell RA , Srour EF , Leemhuis TB , Hoffman R
Published: October 2019

To test the hypothesis that the c-kit ligand plays an important role in the regulation of early events occurring during human hematopoiesis, we determined the effect of a recombinant form of c-kit ligand, termed mast cell growth factor (MGF), on the high-proliferative potential colony-forming cell (HPP-CFC) and the cell responsible for initiating long-term hematopoiesis in vitro (LTBMIC). MGF alone did not promote HPP-CFC colony formation by CD34+ DR- CD15- marrow cells, but synergistically augmented the ability of a combination of granulocyte- monocyte colony-stimulating factor (GM-CSF) interleukin (IL)-3 and a recombinant GM-CSF/IL-3 fusion protein (FP) to promote the formation of HPP-CFC-derived colonies. MGF had a similarly profound effect on in vitro long-term hematopoiesis. Repeated additions of IL-3, GM-CSF, or FP alone to CD34+ DR- CD15- marrow cells in a stromal cell-free culture system increased cell numbers 10(3)-fold by day 56 of long-term bone marrow culture (LTBMC), while combinations of MGF with IL-3 or FP yielded 10(4)- and 10(5)-fold expansion of cell numbers. Expansion of the number of assayable colony-forming unit-granulocyte-monocyte (CFU- GM) generated during LTBMC was also markedly enhanced when MGF was added in combination with IL-3 or FP. In addition, MGF, IL-3, and FP individually led to a twofold to threefold increase in HPP-CFC numbers after 14 to 21 days of LTBMC. Furthermore, the effects of these cytokines on HPP-CFC expansion during LTBMC were additive. Throughout the LTBMC, cells receiving MGF possessed a higher cloning efficiency than those receiving IL-3, GM-CSF, or FP alone. These data indicate that the c-kit ligand synergistically interacts with a number of cytokines to directly augment the proliferative capacity of primitive human hematopoietic progenitor cells.
Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow
Authors: Terstappen LW , Huang S , Picker LJ
Published: October 2019

Using multidimensional flow cytometry we have defined and quantified the human T-cell differentiation pathway, focusing on those events occurring among the most immature thymocytes and putative bone marrow (BM) T-precursors. Early thymocytes were found to express the CD34 antigen and consisted of a mean 1.2% of cells within human pediatric (n = 9) and 2.0% in fetal thymi (n = 4). All CD34+ thymocytes were atypical blast by morphology, expressed intracytoplasmatic, but not cell surface, CD3, and were cell surface CD2+, CD5+, CD7+, CD38+, CD45+, CD45RA+, CD49d+, and LECAM-1(Leu8)high. CD34high thymocytes lacked surface expression of CD4 and CD8, but as CD34 expression diminished there was a coordinate increase in CD4 levels, followed by the appearance of CD8. The expression of CD1 and CD10 also increased concomitant with the loss of CD34, whereas expression of LECAM-1 diminished with CD34 downregulation. The differential expression of these antigens on early thymocytes (as well as the number of thymocytes displaying these patterns) was highly reproducible among the nine pediatric and four fetal specimens examined, suggesting a precise, stereotyped regulation of early differentiation events. Cell populations with antigen expression patterns suggestive of pluripotent stem cell (CD34high, CD38-), or non-T-lineage committed stem cells (CD34+, CD33+ or CD34+, CD19+) were not identified in either fetal or pediatric thymi (sensitivity = 1/10(4)). The presence of cells with the antigenic profile of the earliest CD34+ thymocytes was explored in human BM. Putative BM T-cell precursors with the appropriate phenotype (CD34+, CD7+, CD5+, CD2+, LECAM-1high) were readily identified in fetal specimens (constituting +/- 2% of the CD34+ population), but could not be reliably detected in adults. In contrast with thymi, only 13% of these cells expressed cytoplasmatic CD3, suggesting the presence of the immediate precursor of the putative prothymocyte population. This was further supported by the detection of CD34bright, CD7+, CD2-, CD5-, LECAM-1moderate cells in fetal specimens. Our results document the flow of cell surface differentiation during T-lymphopoiesis and suggest that T-lineage features are first acquired in the BM. The ability to reproducibly identify and isolate T-cell precursor populations of precisely defined maturational stage in marrow and thymus by multiparameter flow cytometry will facilitate characterization of the molecular events controlling T-lineage differentiation.
Elevated serum levels of interleukin-5 in patients with the syndrome of episodic angioedema and eosinophilia
Authors: Butterfield JH , Leiferman KM , Abrams J , Silver JE , Bower J , Gonchoroff N , Gleich GJ
Published: October 2019

The syndrome of episodic angioedema and eosinophilia is characterized by cyclic edema, marked peripheral blood eosinophilia, and eosinophil degranulation in the dermis. Using a sensitive immunoenzymetric method, we measured serum interleukin (IL)-5 levels in four patients with this syndrome. We also determined the percentage of activated T cells in the peripheral blood of a new patient before and during an attack. In the patient presented, IL-5 levels peaked several days before maximal eosinophilia and then declined. This patient's lymphocytes showed an increased percentage, 28% (normal 2% to 3%), of activated T cells staining for both CD3 and HLA-DR 10 days before maximal eosinophilia, but no increase at the time of peak eosinophilia. In serum from three previously reported cases, elevated serum IL-5 levels were found during attacks. After glucocorticoid administration, IL-5 levels became undetectable in three of the four patients. Production of IL-5 is likely an important determinant of the pathophysiology of this syndrome.
Effects on leukocytes after injection of tumor necrosis factor into healthy humans
Authors: van der Poll T , van Deventer SJ , Hack CE , Wolbink GJ , Aarden LA , Buller HR , ten Cate JW
Published: October 2019

Tumor necrosis factor (TNF) has been implicated as a proximal mediator of the septic syndrome. To evaluate the possible role of TNF in leukocyte activation in septicemia, we performed a cross-over saline- controlled study in six healthy men who were intravenously injected with recombinant human TNF (50 micrograms/m2), and analyzed changes in circulating white blood cells and parameters for neutrophil and monocyte activation. TNF elicited a very rapid neutropenia, reaching a nadir after 15 minutes, followed by a neutrophilia. Lymphocytes showed a sustained decrease, whereas monocytes declined transiently. TNF injection was also associated with neutrophil activation, as reflected by a mean fivefold increase in the plasma concentrations of elastase- alpha 1-antitrypsin complexes and a mean sevenfold increase in plasma lactoferrin levels. Serum neopterin, a marker of monocyte activation, was significantly increased 24 hours after the administration of TNF. These changes occurred in the absence of detectable complement activation, as indicated by unchanged C3a-desarg plasma values. Serum interleukin-6 showed a nearly 40-fold increase after TNF injection, whereas interleukin-1 remained undetectable throughout. We conclude that the systemic release of TNF, triggered early after invasive infection, may be involved in the alterations in circulating leukocyte numbers and in the activation of leukocytes, during the development of the septic syndrome.
Immunophenotyping and functional analysis of purified human uterine mast cells
Authors: Guo CB , Kagey-Sobotka A , Lichtenstein LM , Bochner BS
Published: October 2019

Human mast cells have been purified from uterine tissues, and their surface marker profile and function have been evaluated as part of ongoing studies of mast cell heterogeneity. Using a panel of antibodies, purified uterine mast cells (UMC; 81% +/- 7% purity, n = 10) were analyzed by immunofluorescence and flow cytometry for surface expression of various antigens. Consistent with previous analyses of mast cells from other tissues, UMC expressed HLA class I, IgE, c-kit receptor, CD9, CD33, CD43, CD45, and CD54, while CD11a, CD11b, CD14, CD16, CD23, and CD64 were not detected. Unlike other mast cells, UMC expressed CD11c/CD18 (p150,95) and CD32 (Fc gamma RII). Additional antigens not previously studied on mast cells included the selectin LECAM-1 (Leu-8) and several beta 1 and beta 3 integrins; expression of very late activation antigen-4 (VLA-4) (CD49d/CD29), VLA-5 (CD49e/CD29), and the vitronectin receptor (CD51/CD61) was seen. Functional studies showed that treatment of human umbilical vein endothelial cells with interleukin-1 (5 ng/mL for 4 hours) resulted in a twofold to threefold increase in adhesiveness for UMC. Purification procedures did not alter histamine release responses to anti-IgE or the calcium ionophore A23187, and treatment of UMC with an anti-CD32 monoclonal antibody (IV.3) did not induce histamine release or alter anti-IgE-induced release. These data suggest that UMC may possess unique phenotypic characteristics, and support the concept of mast cell heterogeneity.
Reactivity profiles of leukemic myeloblasts with monoclonal antibodies directed to sialosyl-Le(x) and other lacto-series type 2 chain antigens:absence of reactivity with normal hematopoietic progenitor cells
Authors: Muroi K , Suda T , Nojiri H , Ema H , Amemiya Y , Miura Y , Nakauchi H , Singhal A , Hakomori S
Published: October 2019

We investigated the expression profiles of lacto-series type 2 antigens in hematopoietic cells and their progenitors, in comparison with leukemic leukocytes. Reactivity profiles of various anti-type 2 chain monoclonal antibodies (MoAbs) with leukemic blasts from 12 patients with acute myeloblastic leukemia (AML) and those from two patients with acute unclassified leukemia (AUL) show that anti-sialosyl-Le(x) MoAb SNH3 reacted strongly with greater than 95% of leukemic blast leukocyte populations from all patients (14 of 14). Another anti-sialosyl-Le(x) MoAb, FH6, showed less reactivity than SNH3 (12 of 14 patients), while anti-Le(y) MoAb AH6 showed reactivity with only 8 of 14 patients. On the other hand, none of the anti-type 2 chain MoAbs reacted with CD34+ normal adult bone marrow (BM) mononuclear cells obtained independently from three healthy volunteers. MoAb SNH3, but not FH6 or AH6, showed complement-mediated cytotoxicity to leukemic blasts from these patients, as well as to myelogenous leukemia cell line HL60. Colony- forming unit granulocyte-macrophage (CFU-GM), but not burst-forming unit-erythroid (BFU-E), was incompletely inhibited by treatment of normal BM mononuclear cells with SNH3 and complement. The absence of type 2 chain antigen expression in hematopoietic progenitor cells and in in vitro hematopoietic colonies (CFU-GM and BFU-E) strongly suggests that application of anti-carbohydrate MoAbs, particularly anti-sialosyl- Le(x) could be useful for elimination of leukemic myeloblasts infiltrating in BM, for purging of leukemic blasts in BM, and for facilitation of autologous BM.
Effect of activin A on globin gene expression in purified human erythroid progenitors
Authors: Shao L , Frigon NL Jr , Young AL , Yu AL , Mathews LS , Vaughan J , Vale W , Yu J
Published: October 2019

The regulatory control of human erythropoiesis through a purified protein, activin A, was examined. Previous studies using mixed populations of bone marrow cells suggested that activin A has an indirect effect on cellular proliferation and DNA synthesis of erythroid progenitors through the mediation of accessory cells. In present studies, the cultures of purified erythroid progenitors were used to examine the effect of activin A on globin gene expression. Human erythroid burst-forming units (BFU-E) were partially purified from peripheral blood, and after 8 days of culture the cells generated consisted mainly of erythroid colony-forming units (CFU-E). It was found that the subsequent 7-day cultures of these purified progenitors yielded similar numbers and size distributions of erythroid colonies, regardless of the presence of activin A in the cultures. In addition, these erythroid progenitor cells were responsive, in terms of stimulation of DNA synthesis, to the addition of erythropoietin, but not to treatment by activin A. Therefore, once the erythroid progenitors are depleted of accessory cells, activin A has little effect on both the proliferation and the DNA synthesis of these progenitors. However, when these purified erythroid progenitors were cultured in the presence of activin A, the levels of all alpha, beta, and epsilon globin transcripts and hemoglobins were significantly increased. In addition, disuccinimidyl suberate was found to chemically cross-link 125I-activin A to cell surface binding proteins (45 to 54 Kd) in both purified erythroid progenitors and K562 cells. The labeling of these binding proteins was specifically inhibited by the presence of unlabeled activin A, but not transforming growth factor-beta. These results suggest that, in addition to its indirect effect on DNA synthesis and cellular proliferation of erythroid progenitors, activin A directly affects the levels of globin mRNAs and hemoglobins in developing human erythroid cells through its specific surface binding receptor(s).
In vivo synergism of recombinant human interleukin-3 and recombinant human interleukin-6 on thrombopoiesis in primates
Authors: Geissler K , Valent P , Bettelheim P , Sillaber C , Wagner B , Kyrle P , Hinterberger W , Lechner K , Liehl E , Mayer P
Published: October 2019

Using a primate model, we examined the effect of recombinant human interleukin-3 (rhIL-3) and rhIL-6 on thrombopoiesis in vivo. Administration of 33 micrograms/kg/d of rhIL-3 for 11 to 14 days increased levels of circulating colony-forming units megakaryocyte (CFU- Mk) by approximately 15-fold in five rhesus monkeys without raising their platelet counts. In contrast, administration of 30 micrograms/kg/d of rhIL-6 for 10 days in four animals did not increase CFU-Mk levels but significantly raised platelet counts from a mean pretreatment value of 460 x 10(3)/microL (range 360 to 610) to a mean maximum of 746 x 10(3)/microL (665 to 790) on day 8. If monkeys were pretreated with rhIL-3 (33 or 100 micrograms/kg/d for 11 days) to expand their CFU-Mk compartment, the thrombopoietic effect of rhIL-6 was synergistically enhanced leading to platelet counts above 1,000 x 10(3)/microL (mean maximum value 1,247) in all three primates studied. The sequential administration of rhIL-3 and rhIL-6 might represent a powerful strategy to stimulate thrombopoiesis in vivo.
Differential roles of stromal cells, interleukin-7, and kit-ligand in the regulation of B lymphopoiesis
Authors: Billips LG , Petitte D , Dorshkind K , Narayanan R , Chiu CP , Landreth KS
Published: October 2019

Newly formed B lymphocytes are a population of rapidly renewed cells in the bone marrow of mammals and their steady state production presumably depends on a cascade of regulatory cells and cytokines. Although considerable information has been forthcoming about the role of interleukin-7 (IL-7) in potentiating pre-B-cell proliferation, few studies have addressed the possibility that multiple cytokines are involved in the progression of early events in cellular differentiation and proliferation in this hematopoietic lineage. Our laboratory previously described pre-B-cell differentiation mediated by the bone marrow stromal cell line S17. In this study, we further delineate the role of stromal cells in differentiation and proliferation of pre-B cells. These experiments show that the stromal cell line S17 potentiates the proliferative effect of IL-7 on B-lineage cells and that this S17-derived potentiator can be replaced with recombinant kit- ligand (KL). Our results further show that pre-B-cell formation from B220-, Ig- progenitor cells and expression of mu heavy chain of immunoglobulin is uniquely dependent on the presence of S17 stromal cells and cannot be reproduced with IL-7, KL, or costimulation with both IL-7 and KL. These data contribute to a rapidly evolving model of stromal cell regulation of B-cell production in the marrow and suggest unique roles for IL-7, KL, and as yet uncharacterized stromal cell- derived lymphokines in this process.
Thrombolytic properties of Desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats
Authors: Witt W , Baldus B , Bringmann P , Cashion L , Donner P , Schleuning WD
Published: October 2019

rDSPA alpha 1 (recombinant Desmodus salivary plasminogen activator alpha 1) is a recombinant protein corresponding to a natural plasminogen activator from the vampire bat Desmodus rotundus. The thrombolytic properties of rDSPA alpha 1 and tissue-type plasminogen activator (t-PA) were compared in a rat model of pulmonary embolism. Whole blood clots, produced in vitro and labeled with 125I-fibrinogen, were embolized into the lungs of anesthetized rats. Thrombolysis was calculated from the difference between initial clot radioactivity and that remaining in the lungs at 60 minutes. Blood was sampled for gamma counting, measurement of hemostatic factors, and plasminogen activator antigen levels. Thrombolysis at 3, 10, 30, and 100 nmol/kg intravenously (10% bolus, 90% over 60 minutes) amounted to 30% +/- 2%, 51% +/- 4%, 85% +/- 4%, 98% +/- 0% for rDSPA alpha 1 and 30% +/- 3%, 41% +/- 3%, 57% +/- 6%, 93% +/- 2% for t-PA (controls: 29% +/- 2%; mean +/- SEM, n greater than or equal to 6). t-PA at 100 nmol/kg significantly decreased fibrinogen, plasminogen, and alpha 2- antiplasmin levels by 33% +/- 7%, 38% +/- 8%, and 61% +/- 9%, whereas rDSPA alpha 1 at 100 nmol/kg only lowered alpha 2-antiplasmin significantly (by 29% +/- 6%). Compared with t-PA, rDSPA alpha 1 is the more potent and more clot selective (fibrin specific) thrombolytic agent. These results suggest that rDSPA alpha 1 may be safer and more efficacious than currently used thrombolytics.
Heme oxygenase is a positive acute-phase reactant in human Hep3B hepatoma cells
Authors: Mitani K , Fujita H , Kappas A , Sassa S
Published: October 2019

The effects of human interleukin-6 (hIL-6), the major acute-phase inducer, on the level of the transcript of microsomal heme oxygenase (HO) were examined in a human hepatoma cell line, Hep3B. Messenger RNAs (mRNAs) encoding HO and haptoglobin (Hpt) increased after hIL-6 treatment in a time- and dose-dependent manner. hIL-6 had no effect on the induction of heat-shock protein 70 (hsp70) mRNA, suggesting that the induction of HO by hIL-6 is regulated by a different mechanism from that which mediates the heat-shock induction of this enzyme. The hIL-6- mediated induction of HO mRNA was completely abrogated by simultaneous treatment of cells with actinomycin D, but not with cycloheximide, suggesting that the induction occurs at the level of transcription. A nuclear factor was shown both in untreated, and in the hIL-6-treated Hep3B cells that binds specifically to the IL-6-responsive element (IL6- RE) of the human HO gene. These findings suggest that HO is a positive acute-phase reactant in this human liver-derived cell line, and that the nuclear factor specific to the IL6-RE may be involved in the activation of the HO gene after hIL-6 treatment.
T-cell receptor beta-chain gene rearrangement and expression during human thymic ontogenesis
Authors: Bonati A , Zanelli P , Ferrari S , Plebani A , Starcich B , Savi M , Neri TM
Published: October 2019

T-cell receptor (TCR) beta-chain proteins appear early (approximately 15th week of gestation) during human thymic ontogenesis. These beta- chain proteins, which appear before terminal deoxynucleotidyl transferase (TdT), could be an expression of a fully rearranged (V-D- J), incompletely rearranged (D-J), or germline TCR beta-chain gene. The aims of this study, performed from the 15th week onward, were the following: (1) to investigate whether or not TCR beta gene rearranges at an early stage during human thymic ontogenesis; (2) to investigate whether complete presumptive functional (1.3 kb) TCR beta gene transcript is present at these early stages of development, or if incomplete (1 kb) or germ-line (1.1 kb) transcripts are expressed; (3) to examine the phenotype of TCR beta-chain+ cells with two-color fluorescence using monoclonal antibody (MoAb) beta F1 and MoAbs that recognize CD1, CD2, CD3, CD4, CD8, CD5, and CD7 antigens (rabbit anti- calf TdT antiserum was used to detect TdT); and (4) to demonstrate whether or not beta gene N-diversity regions are detectable as early as the 15th week and whether or not N-nucleotide insertions correlate to TdT expression. Fifteen- to 22-week fetal thymuses and pediatric thymuses were investigated. We demonstrated that TCR beta-chain gene rearranged as early as the 15th week in human thymus and that a complete functional TCR beta gene transcript was expressed at these early stages of human development. No other analyses to date have investigated TCR beta gene expression in early human thymus using molecular biology techniques. No significant differences were detectable between phenotypic analysis of fetal and pediatric samples, except for TdT expression, which appeared after the 20th week. Essentially all mCD3+ (OKT3+) cells were beta-chain+ at the different weeks investigated. A significant percentage of CD1+ cells were beta- chain+, and the percentage increased along with the age of development. After the 20th week, we identified three main populations: TdT+, cCD3+, beta F-(early thymic precursors); TdT+, CD1+, beta F1+ (intermediate maturity cortical thymocytes); and TdT-, mCD3+, beta F1++ (mature medullary thymocytes). Given these values, we may consider beta-chain expression an ordered process. beta gene N-nucleotide insertions were correlated to TdT expression, since N-regions increased considerably after the 20th week. A further increase of N-nucleotide insertions was detected from the 22nd week to the 32nd week.