bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a novel self-associating Mr 40,000 platelet glycoprotein [see comments]
Authors: Hildreth JE , Derr D , Azorsa DO
Published: October 2019

A novel platelet glycoprotein has been purified and characterized. This glycoprotein, designated Pltgp40, is an acidic sialylated 40,000-dalton protein that bears both O-linked and N-linked oligosaccharides. Treatment of Pltgp40 with neuraminidase resulted in a 5,000-dalton reduction in its Mr and a 1.5 Unit alkaline shift in the isoelectric point, indicating the presence of a large number of sialic acid residues. A similar size reduction and change in pl were observed after treatment of Pltgp40 with O-glycanase showing that sialic acids are present on O-linked oligosaccharides. Digestion of Pltgp40 with N- glycanase reduced the Mr to approximately 20,000 daltons but did not affect the isoelectric point, suggesting that Pltgp40 contains six to seven nonsialylated N-linked carbohydrate chains. High Mr proteins were observed in affinity purified Pltgp40 and were identified as detergent- stable protein oligomers consisting of multiple 40,000-dalton monomers. Immunodepletion and direct binding studies indicated that Pltgp40 was not equivalent to Ig Fc receptor type II, another 40,000-dalton glycoprotein expressed on platelets. However, Pltgp40 copurified with Fc receptor type II when platelet extracts were loaded onto human IgG affinity columns, raising the possibility that Pltgp40 may associate with Fc receptors or Fc receptor-lg complexes. Amino acid sequence analysis of the N-terminus of Pltgp40 was performed and confirmed that Pltgp40 is a novel platelet glycoprotein. Epitopes on Pltgp40 appear to be widely expressed because monoclonal antibodies against Pltgp40 also reacted with a variety of myeloid, lymphoid, and epithelial cells. Pltgp40 was detected on activated but not resting platelets, indicating that Pltgp40 is a platelet activation marker.
T-cell receptor delta gene recombination in common acute lymphoblastic leukemia: preferential usage of V delta 2 and frequent involvement of the J alpha cluster
Authors: Yokota S , Hansen-Hagge TE , Bartram CR
Published: October 2019

A high frequency (greater than 80%) of acute lymphoblastic leukemias (ALL) exhibit a recombination of the T-cell receptor (TCR) delta chain locus. Interestingly, distinct TCR delta elements are preferentially used in immunologic subtypes. In a recent series of 201 children with common ALL (cALL) we observed a TCR delta rearrangement in 162 patients, 57% of the latter showing a hybridization pattern in Southern blots suggestive of a V delta 2 to D delta 3 recombination. To verify this interpretation and to elucidate in more detail the diversity of this common type of TCR delta recombination we amplified and sequenced the junctional region of nine cALL patients and cell line REH-6 by polymerase chain reaction (PCR). A V delta 2 D delta 3 recombination was confirmed in all cases; convincing evidence for the participation of D delta 1 or D delta 2 elements was not obtained. Eight of nine patients and REH-6 showed complete 5′ D delta 3 boundaries within V delta 2 D delta 3 segments, a limitation of junctional diversity also detected in 50% of peripheral blood cell clones derived from two healthy probands. Notably, sequence identity at the V delta 2 D delta 3 junction was demonstrated for a cALL and one of the control clones. Another group of 35 of 162 cALL patients was characterized by V delta 2 rearrangements and biallelic deletion of J delta and C delta sequences. Using a J alpha consensus primer, PCR-directed sequence analysis demonstrated V delta 2 D delta 3 J alpha recombinations in all four cases analyzed by this approach. The J alpha segments of these patients differed, but were identical or homologous to published J alpha elements. Our data suggest a recombination pathway of the TCR delta/alpha locus leading to chimeric TCR alpha molecules, containing V delta and, remarkably, also D delta sequences.
Human thymic epithelial cells produce interleukin-3
Authors: Dalloul AH , Arock M , Fourcade C , Hatzfeld A , Bertho JM , Debre P , Mossalayi MD
Published: October 2019

Interleukin-3 (IL-3) is a hematopoietic growth factor suggested to be produced by activated T lymphocytes. Meanwhile, supernatants from human thymic stroma could promote the proliferation of myeloid stem cells. Thus, we investigated whether IL-3 accounts for this activity. Therefore, human thymic epithelial cells (TEC), fibroblasts, and adherent cells were isolated, and their culture supernatants assayed for myeloid colony promotion. Only supernatants from thymic epithelial cells supported colony-forming unit growth in semisolid media. This effect decreased following anti-IL-3 monoclonal antibody addition to these cultures. Furthermore, in situ hybridization showed the presence of IL-3 mRNA in epithelial cells. Effect of TEC culture conditions on IL-3 production by these cells was also studied. Together, these data show that IL-3 production is not the exclusive property of human activated T lymphocytes.
Combined therapy with recombinant granulocyte colony-stimulating factor and erythropoietin decreases hematologic toxicity from zidovudine
Authors: Miles SA , Mitsuyasu RT , Moreno J , Baldwin G , Alton NK , Souza L , Glaspy JA
Published: October 2019

Twenty-two patients with acquired immunodeficiency syndrome (AIDS) or severe AIDS-related complex and multilineage hematopoietic defects were treated with recombinant granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO) in a phase I/II trial. All patients were neutropenic and anemic after withdrawal of all bone marrow-suppressive drugs. Daily, G-CSF was subcutaneously self-administered until an absolute neutrophil count (ANC) greater than 6,000/microL was achieved and maintained for 2 weeks. Subcutaneous EPO was added to the regimen and the dose increased until an increase of 15 g/L of hemoglobin was observed. Groups of patients were administered increasing doses of zidovudine to determine their tolerance. G-CSF and EPO therapy was continued with dose modification to maintain an ANC greater than 1,500/microL and hemoglobin greater than 100 g/L. The dose of zidovudine was not altered. All 22 patients responded to G-CSF with a mean 10-fold increase in neutrophils occurring in less than 2 weeks. Significant increases in CD4 and CD8 cell number, lymphocyte proliferative response, and bone marrow cellularity were seen. EPO therapy increased hemoglobin in all 20 evaluable patients within 8 weeks. Sixteen patients received 1,000 mg and four patients received 1,500 mg of zidovudine per day. The reinstitution of zidovudine resulted in a decline in reticulocytes and hemoglobin and the reappearance of transfusion requirements in eight of the 20 patients, six of whom had the study medications stopped. No patient had the study medications stopped because of neutropenia or thrombocytopenia. Toxicities were mild and did not require dose modifications. Limiting dilution plasma and lymphocyte co-cultures for HIV as well as serum p24 antigen levels did not change significantly during G-CSF or combined G- CSF and EPO therapy. HIV p24 antigen decreased significantly with zidovudine therapy. Opportunistic infections occurred in 14 patients but were successfully treated with myelosuppressive antimicrobial agents, including ganciclovir, without the development of neutropenia. These results suggest that combined therapy with G-CSF and EPO may improve the neutropenia and anemia of AIDS. Combined therapy may allow the resumption of full-dose zidovudine in most patients intolerant of the hematologic effects of zidovudine without apparent alteration of HIV expression or the efficacy of zidovudine.
Effect of murine mast cell growth factor (c-kit proto-oncogene ligand) on colony formation by human marrow hematopoietic progenitor cells
Authors: Broxmeyer HE , Cooper S , Lu L , Hangoc G , Anderson D , Cosman D , Lyman SD , Williams DE
Published: October 2019

Purified natural (n) and recombinant (r) murine (mu) mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with r human (hu) erythropoietin (Epo), rhu granulocyte-macrophage colony-stimulating factor (rhuGM-CSF), rhuG-CSF, and/or rhuM-CSF for effects in vitro on colony formation by multipotential (colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit erythroid [BFU-E]) and granulocyte- macrophage (CFU-GM) progenitor cells from normal human bone marrow. MGF was a potent enhancing cytokine for Epo-dependent CFU-GEMM and BFU-E colony formation, stimulating more colonies and of a larger size than either rhu interleukin-3 (rhuIL-3) or rhuGM-CSF. MGF, especially at lower concentrations, also acted with rhuIL-3 or rhuGM-CSF to enhance Epo-dependent CFU-GEMM and BFU-E colony formation. MGF had little stimulating activity for CFU-GM colonies by itself, but in combination with suboptimal to optimal amounts of rhuGM-CSF enhanced the numbers and the size of CFU-GM colonies in an additive to greater than additive manner. While we did not detect an effect of MGF on CFU-G colony numbers stimulated by maximal concentrations of rhuG-CSF, MGF did enhance the size of CFU-G-derived colonies. MGF did not enhance the activity of rhuM-CSF. In a comparative assay, maximal concentrations of rmu and rhuMGF were equally effective in the enhancement of human bone marrow colony formation, but rhuMGF, in contrast to rmuMGF, did not at the concentrations tested enhance colony formation by mouse bone marrow cells. MGF effects on BFU-E, CFU-GM, and CFU-GEMM may be direct acting ones as MGF-enhanced colony formation by these cells in highly enriched progenitor cell populations of CD34 HLA-DR+ and CD34 HLA-DR+CD33- sorted cells in which greater than or equal to 1 of 2 cells was a BFU-E plus CFU-GM plus CFU-GEMM. MGF appears to be an early acting cytokine that preferentially stimulates the growth of immature hematopoietic progenitor cells.
Pharmacokinetics of activated protein C in guinea pigs
Authors: Berger H Jr , Kirstein CG , Orthner CL
Published: October 2019

Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of 125I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of 125I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D- phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. The other mechanism involves the direct catabolism of APC by the liver via a pathway that is nonsaturable over a substantial dose range and independent of the active site. This pattern of elimination is distinctly different from that observed with the homologous coagulation enzymes thrombin, factor IXa, and factor Xa.
Granulocyte colony-stimulating factor in cerebrospinal fluid from patients with meningitis
Authors: Shimoda K , Okamura S , Omori F , Mizuno Y , Hara T , Aoki T , Ueda K , Niho Y
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) in the cerebrospinal fluid from patients with meningitis was measured by our modified enzyme- linked immunosorbent assay for G-CSF. The minimal detection level was 20 pg/mL G-CSF. In patients with bacterial meningitis, the G-CSF levels in the cerebrospinal fluid were extremely elevated, showing a mean value of approximately 1,500 pg/mL. On the other hand, G-CSF levels in the cerebrospinal fluid from 67% patients with aseptic meningitis were moderately increased, showing a mean value of about 80 pg/mL, whereas G- CSF levels in 33% samples remained undetectable. The G-CSF levels and neutrophil counts in the cerebrospinal fluid were proven to be related by Spearman's rank correlation coefficient analysis (r = .724). These elevations of G-CSF levels at inflammation sites associated with bacterial meningitis may indicate that G-CSF plays an important role in the combat of bacterial infections.
Mycobacterium avium-intracellulare induces interleukin-6 from human monocytes and large granular lymphocytes
Authors: Blanchard DK , Michelini-Norris MB , Pearson CA , Freitag CS , Djeu JY
Published: October 2019

Mycobacterium avium-intracellulare (MAI) is an opportunistic pathogen commonly found in acquired immunodeficiency syndrome patients, whose immune systems are severely compromised. However, normal responses to this bacterium are apparently sufficient to prevent disseminated infection because disease is rarely found unless an immunocompromised state is present. Because interleukin-6 (IL-6) is an inflammatory cytokine with a multitude of activities, we investigated the potential of MAI to induce IL-6 from normal human leukocytes. Peripheral blood mononuclear cells were fractionated into monocytes (Mo), large granular lymphocytes (LGL), and T cells and stimulated with bacteria. Culture supernatants were collected and assayed for IL-6 activity by bioassay. Mo and LGL, but not T cells, were found to release IL-6 within 12 hours of stimulation, with optimal production occurring by 2 days of culture. Production of IL-6 from human leukocyte subsets was confirmed by Northern blot analysis and by neutralization of biologic function of the culture supernatants with specific antisera. Taken together, these results indicate that production of IL-6 is a key response of Mo and LGL to MAI. The role of IL-6 in MAI infection, therefore, needs to be further investigated.
Correlation of response of aplastic anemia patients to antilymphocyte globulin with in vitro lymphocyte stimulatory effect: predictive value of in vitro test for clinical response
Authors: Abe T , Matsuoka H , Kojima S , Kamachi Y , Tsuge I , Kodera Y , Matsuyama T
Published: October 2019

Therapy with antilymphocyte globulin (ALG) has been shown to be effective in restoring hematopoiesis to some patients with aplastic anemia. It would be useful to have a method for predicting those likely to be responders versus nonresponders. The mode of immunostimulatory action of ALG is of interest in addition to its immunosuppressive action. We examined in vitro the distribution of the proliferative responses of ALG-stimulated peripheral blood mononuclear cells (PBMCs) obtained from 18 patients with aplastic anemia, eight of whom responded to ALG and 10 who did not. We found a significant difference in the proliferative response of PBMCs obtained from the eight responders versus the 10 nonresponders (P less than .01). Two-color flow cytometry analysis of the patients' PBMCs stimulated by ALG in vitro showed that the CD4-positive subsets were activated to a greater extent by ALG than the CD8-positive subsets. Moreover, a positive correlation with the clinical response of patients to ALG with granulocyte-macrophage colony- stimulating factor produced by their PBMCs stimulated by ALG suggests that the immunostimulatory property of ALG has an important role in the treatment of aplastic anemia. Our results suggest that the clinical response to ALG therapy is correlated with its lymphocyte proliferative effect in vitro, and indicates that the assessment of the proliferative response of PBMCs in vitro would be useful in predicting the clinical response to ALG therapy.
Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction
Authors: Torelli G , Marasca R , Luppi M , Selleri L , Ferrari S , Narni F , Mariano MT , Federico M , Ceccherini-Nelli L , Bendinelli M
Published: October 2019

In search of a possible involvement of the human herpesvirus type 6 (HHV-6) in human Hodgkin's and non-Hodgkin's lymphomas, we studied the levels of anti-HHV-6 antibodies in the sera of 94 cases by an immunofluorescence assay, as well as the presence of HHV-6 sequences in the affected tissues of 66 cases by polymerase chain reaction, using one set of primer oligonucleotides. Our results showed higher anti-HHV- 6 antibody titers in human lymphomas than in normal blood donors, but the difference is statistically significant only when normal donors are compared with Hodgkin's lymphoma cases. HHV-6 sequences were detected in 3 of 25 Hodgkin's lymphomas and 0 of the 41 cases of non-Hodgkin's lymphomas studied. The three cases positive for HHV-6 sequences belong to the nodular sclerosis-lymphocyte depletion histologic subtype and share remarkable similarities in their clinical features. Furthermore, Southern blot analysis of total genomic DNA obtained from the neoplastic tissues of two of the three patients showed the same restriction fragment length polymorphism. Our results suggest that: (1) the high level of anti-HHV-6 antibodies in Hodgkin's disease is due to an activation of the immune system not related to the presence of HHV-6 sequences in affected lymph nodes; (2) the presence of HHV-6 sequences in human lymphoid tissues is not a frequent event, rather it is in fact a very rare event in non-Hodgkin's lymphomas, while in Hodgkin's cases it is more frequent than previously reported on the basis of Southern blot analysis; and (3) the presence of HHV-6 sequences in Hodgkin's lymphomas may have a relation with the clinical presentation of the disease.
Functional properties of the beta-globin locus control region in K562 erythroleukemia cells
Authors: Moon AM , Ley TJ
Published: October 2019

In this report, we compare the function of the human beta-globin locus control region (LCR) in three K562 erythroleukemia cell assays, including (1) a transient transfection assay for “classical' enhancer activity, (2) a colony assay that detects “productive integration events,” and (3) an assay that detects the ability of LCR fragments to confer hemin inducibility on linked, stably integrated gamma-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an A gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named mu locus activation region [mu LAR]) had activity in all three assays; of the individual LCR sites, only site II was highly active in all three assays. One region within site II, consisting of tandem AP-1/NF-E2 consensus elements, had approximately 10% as much colony assay activity as the entire mu LAR. However, this region did not have detectable activity in a transient enhancer assay in uninduced K562 cells, nor was it capable of conferring hemin inducibility on linked gamma-globin promoters in stably transfected cells. Finally, we tested the ability of the mu LAR to activate promoters (beta-globin and cathepsin G) that are not normally expressed in K562 cells. beta-neo was minimally activated by the mu LAR in transient transfection experiments. The mu LAR increased the number of stably transfected colonies produced by beta-neo, but the absolute number of beta-neo colonies, with or without the mu LAR, was approximately 10% to 20% that of gamma-neo. In contrast, a minimal cathepsin G promoter was activated by the mu LAR in K562 cells. Our results suggest that LCR functions are dependent in part on the environments and the promoters with which the LCR is tested.
Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor
Authors: Bernstein ID , Andrews RG , Zsebo KM
Published: October 2019

We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.
Further examination of the effects of recombinant cytokines on the proliferation of human megakaryocyte progenitor cells
Authors: Bruno E , Cooper RJ , Briddell RA , Hoffman R
Published: October 2019

The effect of several recombinant cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL- 6, and IL-1 alpha, on megakaryocyte (MK) colony formation by a normal human bone marrow subpopulation (CD34+ DR+), enriched for the MK colony- forming unit (CFU-MK), was studied using a serum-depleted, fibrin clot culture system. IL-3 and GM-CSF, but not IL-6 or IL-1 alpha, stimulated MK colony formation by CD34+ DR+ cells. However, the addition of IL-1 alpha to CD34+ DR+ cultures containing IL-6 resulted in the appearance of CFU-MK-derived colonies, suggesting that IL-6 requires the presence of IL-1 alpha to exhibit its MK colony-stimulating activity (MK-CSA). Addition of neutralizing antibodies to IL-3 and GM-CSF, but not to IL-6 and IL-1 alpha, specifically inhibited the MK-CSA of IL-3 and GM-CSF, respectively. The addition of either anti-IL-6, anti-IL-1 alpha, or anti-IL-3 antisera to cultures containing both IL-6 and IL-1 alpha totally abolished the MK-CSA of the IL-6/IL-1 alpha combination. However, neither anti-IL-3 nor anti-GM-CSF antisera could totally neutralize the additive effect of the combination of IL-3 and GM-CSF on MK colony formation, indicating that these two cytokines act by affecting distinct effector pathways. These results suggest that while IL-3 and GM-CSF can directly affect CFU-MK-derived colony formation, IL- 1 alpha and IL-6 act in concert to promote de novo elaboration of IL-3 and thereby promote CFU-MK proliferative capacity.
Iron uptake by human reticulocytes at physiologic and sub-physiologic concentrations of iron transferrin: the effect of interaction with aluminum transferrin [see comments]
Authors: Cochran M , Chawtur V , Jones ME , Marshall EA
Published: October 2019

We have studied the interaction, in vitro, between diferric transferrin (FeTr), aluminum transferrin (AlTr), and human reticulocytes harvested from human placental blood. In particular, we aimed to determine the extent to which the kinetics of AlTr and FeTr differed. Using transferrin labeled with either 59Fe or 125I, the association of radiotracer with reticulocytes, as a function both of time and of transferrin concentration, was examined. Under the conditions of the experiments, the data are consistent with a mechanism involving at least three processes. Two early processes acting in parallel behave as a high-affinity saturable receptor and a low-affinity non-saturable receptor, neither of which distinguish between AlTr and FeTr. In a subsequent process, AlTr and FeTr exhibit different kinetics. This third process may be saturated by FeTr but not by AlTr. Interpreted in terms of a current conventional view of metallo-transferrin uptake, we conjecture that the early parallel processes involve cell surface phenomena including classical transferrin-receptor binding, and that the subsequent process represents events, possibly intracellular, involved in metallo-transferrin dissociation or further iron transport. The extent to which AlTr influences the interaction of FeTr with reticulocytes offers insight into both the normal physiology of iron uptake and the potential for toxicity by aluminum.
Anti-MY9-blocked-ricin: an immunotoxin for selective targeting of acute myeloid leukemia cells
Authors: Roy DC , Griffin JD , Belvin M , Blattler WA , Lambert JM , Ritz J
Published: October 2019

The use of immunotoxins (IT) to selectively destroy acute myeloid leukemia (AML) cells in vivo or in vitro is complicated by both the antigenic similarity of AML cells to normal progenitor cells and the difficulty of producing a sufficiently toxic conjugate. The monoclonal antibody (MoAb) anti-MY9 is potentially ideal for selective recognition of AML cells because it reacts with an antigen (CD33) found on clonogenic AML cells from greater than 80% of cases and does not react with normal pluripotent stem cells. In this study, we describe an immunotoxin that is selectively active against CD33+ AML cells: Anti- MY9-blocked-Ricin (Anti-MY9-bR), comprised of anti-MY9 conjugated to a modified whole ricin that has its nonspecific binding eliminated by chemical blockage of the galactose binding domains of the B-chain. A limiting dilution assay was used to measure elimination of HL-60 leukemic cells from a 20-fold excess of normal bone marrow cells. Depletion of CD33+ HL-60 cells was found to be dependent on the concentration of Anti-MY9-bR and on the duration of incubation with IT at 37 degrees C. More than 4 logs of these leukemic cells were specifically depleted following short exposure to high concentrations (10(-8) mol/L) of Anti-MY9-bR. Incubation with much lower concentrations of Anti-MY9-bR (10(-10) mol/L), as compatible with in vivo administration, resulted in 2 logs of depletion of HL-60 cells, but 48 to 72 hours of continuous exposure were required. Anti-MY9-bR was also shown to be toxic to primary AML cells, with depletion of greater than 2 logs of clonogenic cells following incubation with Anti- MY9-bR 10(-8) mol/L at 37 degrees C for 5 hours. Activity of Anti-MY9- bR could be blocked by unconjugated Anti-MY9 but not by galactose. As expected, Anti-MY9-bR was toxic to normal colony-forming unit granulocyte-monocyte (CFU-GM), which expresses CD33, in a concentration- and time-dependent manner, and also to burst-forming unit-erythroid and CFU-granulocyte, erythroid, monocyte, megakaryocyte, although to a lesser extent. When compared with anti-MY9 and complement (C′), Anti- MY9-bR could be used in conditions that provided more effective depletion of AML cells with substantially less depletion of normal CFU- GM. Therefore, Anti-MY9-bR may have clinical utility for in vitro purging of AML cells from autologous marrow when used at high IT concentrations for short incubation periods. Much lower concentrations of Anti-MY9-bR that can be maintained for longer periods may be useful for elimination of AML cells in vivo.
Expression of interleukin-6 and interleukin-6 receptor in Hodgkin's disease
Authors: Jucker M , Abts H , Li W , Schindler R , Merz H , Gunther A , von Kalle C , Schaadt M , Diamantstein T , Feller AC
Published: October 2019

Interleukin-6 (IL-6) is a multipotent lymphokine that can mediate differentiation of B cells into Ig-secreting cells, stimulate the growth of plasmacytomas, hybridomas, and T cells, and induce acute- phase proteins in liver cells. It has been suggested that IL-6 is involved in the pathogenesis of several diseases by autocrine or paracrine pathways. To examine whether IL-6 is possibly involved in the pathophysiology of Hodgkin's disease (HD), we analyzed the expression of IL-6 and IL-6 receptor mRNA and protein in cell lines and primary specimens from patients with HD. IL-6-specific transcripts were detected in three of six HD-derived cell lines by Northern blot analysis. In the culture supernatants of four HD-derived cell lines, IL- 6 was detected by radioimmunoassay. Biologic activity of IL-6 was confirmed by proliferation of an IL-6-dependent cell line. In situ hybridization experiments showed IL-6-specific transcripts in Hodgkin (H) and Reed-Sternberg (RS) cells in primary tissues of two patients. In addition, mRNAs specific for the IL-6 receptor were detected in five HD-derived cell lines. Immunostaining experiments showed expression of IL-6 receptor molecules on H and RS cells in 8 of 16 cases with HD. Thus, our data suggest that IL-6 might be involved in the pathophysiology of HD.
Homing and progression patterns of childhood acute lymphoblastic leukemias in severe combined immunodeficiency mice
Authors: Cesano A , O'Connor R , Lange B , Finan J , Rovera G , Santoli D
Published: October 2019

The aim of this study was to analyze the homing and progression patterns of childhood acute lymphoblastic leukemias (ALL) in mice with severe combined immunodeficiency (SCID). Upon intraperitoneal (IP) transfer, cells from relapse samples of three children with T-lineage ALL spread hematogenously and infiltrated the non-lymphoid and/or lymphoid organs with a pattern reminiscent of the human clinical disease. These mice either died or were killed in extremis at a mean of 9 weeks. Moreover, cell lines established in vitro from two of these samples manifested identical homing and progression in the SCID mouse as compared with the original patients' cells. Thus, long-term culture of the primary leukemic T cells did not alter their invasive potential and migration pattern. When engrafted IP, three cell lines established from pre-B-ALL cases displayed primarily a lymphatic spread with induction of local tumor masses and kidney/liver nodules. Mice were killed at 11 to 13 weeks, but had not developed imminently fatal leukemia. However, when transferred intravenously, one pre-B ALL cell line was able to spread hematogenously and to infiltrate both lymphoid and non-lymphoid tissues. Overall, these data demonstrate that the SCID mouse provides an efficient and reproducible model to study the pathogenesis of childhood ALL, and may be a suitable system for evaluating therapy.
Use of 8-methoxypsoralen and ultraviolet-A pretreated platelet concentrates to prevent alloimmunization against class I major histocompatibility antigens
Authors: Grana NH , Kao KJ
Published: October 2019

The use of 8-methoxypsoralen (8-MOP) and UV-A irradiation to inactivate contaminating donor leukocytes in platelet concentrates and to prevent primary alloimmunization against donor class I major histocompatibility (MHC) antigens in mice was investigated. CBA/CaH-T6J mice with the H2k haplotype and BALB/cByJ mice with the H2d haplotype were used as donors and recipients, respectively. The mixed leukocyte reaction between these two strains of mice showed that treatment of spleen cells with 500 ng/mL 8-MOP and 5J/cm2 UV-A inhibited 99% of responder and 92% of stimulator function. There was no measurable loss of platelet aggregating activity after the treatment. After two weekly transfusions of platelets without any treatment, 93% of control mice (n = 15) developed anti-H2k antibody. In contrast, only 33% of mice (n = 15) receiving platelets treated with 8-MOP and UV-A became alloimmunized. After six weekly platelet transfusions, all mice became alloimmunized. Nevertheless, the mean titers of anti-H2k antibody in sera of the treated groups were significantly lower than the control groups. One hour posttransfusion recoveries of 51Cr-labeled donor platelets were also higher in mice transfused with the treated platelets. Thus, the pretreatment of platelet concentrates with 8-MOP and UV-A irradiation effectively reduced the alloantigenicity of class I MHC molecules. The implication of this finding in relation to the mechanism by which donor leukocytes allosensitize recipients is discussed.
Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo
Authors: Ley K , Gaehtgens P , Fennie C , Singer MS , Lasky LA , Rosen SD
Published: October 2019

During the inflammatory response, granulocytes and other leukocytes adhere to and emigrate from small venules. Before firm attachment, leukocytes are observed rolling slowly along the endothelium in venules of most tissues accessible to intravital microscopy. The molecular mechanism underlying this early type of leukocyte-endothelial interaction is unknown. Leukocyte rolling was investigated in venules (diameter, 40 microns) of the exposed rat mesentery. Micro-infusion of a recombinant soluble chimera (LEC-IgG) of the murine homing receptor lectin-like cell adhesion molecule 1 (LEC-CAM 1; gp90MEL) into individual venules reduced the number of rolling leukocytes by 89% +/- 2% (mean +/- SEM, n = 20 venules), while a similar CD4 chimera (CD4- IgG) had no effect (inhibition 14% +/- 7%, n = 25). Rolling was also greatly reduced by a polyclonal serum against LEC-CAM 1 (inhibition 84% +/- 3%, n = 35); preimmune serum was ineffective (11% +/- 13% inhibition, n = 28). These findings indicate that LEC-CAM 1 mediates the adhesive interaction underlying leukocyte rolling and thus may play an important role in inflammation and in pathologic conditions involving leukocytes.
Carrier detection in Wiskott-Aldrich syndrome: combined use of M27 beta for X-inactivation studies and as a linked probe
Authors: Goodship J , Carter J , Espanol T , Boyd Y , Malcolm S , Levinsky RJ
Published: October 2019

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with no clinical or immunologic abnormalities in carrier females. The defective gene has been localized to proximal Xp. Carrier females have nonrandom use of the X chromosome in granulocytes, lymphocytes, and monocytes. We have used the probe M27 beta, which detects both a variable number tandem repeat polymorphism and methylation differences between the active and inactive X chromosome, in the investigation of families referred for genetic counseling. M27 beta detects the locus DXS255, which is tightly linked to WAS. As the probe that is used for investigation of X-inactivation patterns is also linked to the disease locus, it is possible to assign phase in families where this could not be done by conventional use of linked probes. The mothers of four isolated male cases had nonrandom use of the X chromosome. A new mutation was identified in one family with two affected males.
Modulation and induction of eosinophil chemotaxis by granulocyte- macrophage colony-stimulating factor and interleukin-3
Authors: Warringa RA , Koenderman L , Kok PT , Kreukniet J , Bruijnzeel PL
Published: October 2019

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 3 (IL-3), and IL-5. We have investigated the modulatory role of GM-CSF and IL-3 on the platelet-activating factor (PAF)-, neutrophil- activating factor (NAF/IL-8)-, leukotriene B4 (LTB4)-, N-formyl- methionyl-leucyl-phenylalanine (FMLP)-, and human complement factor C5a- induced chemotaxis of eosinophils from normal individuals. These eosinophils show a chemotactic response toward PAF, LTB4, and C5a, but not to NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of GM-CSF caused a significant increase in the response toward LTB4 and induced a significant chemotactic response toward NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of IL-3 also induced a chemotactic response toward NAF/IL-8 and FMLP, and enhanced the PAF-induced chemotaxis response toward C5a was not influenced by both cytokines. Nanomolar concentrations of GM-CSF or IL-3 caused a significant inhibition of the C5a-induced chemotaxis. The LTB4-induced chemotaxis was also significantly inhibited in case of GM-CSF. At these concentrations both GM-CSF and IL-3 acted as chemotaxins for eosinophils were washed after pretreatment with GM-CSF and IL-3 the potentiation of the chemotactic response remained, whereas the inhibitory mode of action disappeared. Our data indicate that at picomolar concentrations the cytokines GM-CSF and IL-3 can modulate eosinophil chemotaxis and at nanomolar concentrations these cytokines can act as chemotaxins for eosinophils.
Regulation of c-myc expression by granulocyte-macrophage colony- stimulating factor in human leukemia cells
Authors: Schwartz EL , Chamberlin H , Brechbuhl AB
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates both the proliferation and functional properties of normal and leukemic myeloid cells via cell surface receptors. The postreceptor mechanisms for these two actions, and the extent to which they represent overlapping biochemical pathways, have not been fully clarified. We have examined the actions of GM-CSF on the expression of c-myc, an early response oncogene associated with the proliferative stimulus of growth factors. GM-CSF reduced the population doubling time of HL-60 leukemia cells from 32 hours to 25 hours, and, at concentrations that were correlated with mitogenicity, induced a rapid twofold increase in the level of c-myc mRNA. Nuclear runoff studies indicated that GM-CSF approximately doubled the transcription rate of c-myc by reversing the transcription attenuation that occurs at the exon 1-intron 1 junction. GM-CSF had no effect on the half-life of c-myc messenger RNA. The biochemical basis for the modulation of c-myc expression by GM-CSF was explored. GM-CSF treatment caused intracellular alkalinization of the cells as measured using the fluorescent probe 2′, 7-bis (2- carboxyethyl)-5(and-6) carboxyfluorescein (BCECF). The sodium channel blocker amiloride prevented the GM-CSF-induced change in pH, but did not affect the stimulation of c-myc transcription by GM-CSF. Agents that increase cellular cyclic adenosine monophosphate (cAMP) levels (prostaglandin E2 and cholera toxin) blocked the actions of GM-CSF on c- myc; however, these agents also reduced the basal level of c-myc expression. GM-CSF caused a rapid (5 minutes) and transient decline in cellular cyclic guanosine monophosphate (cGMP) levels, and a slower (30 minutes) and transient decrease in cellular cAMP levels. These observations are consistent with the hypothesis that the declines in cAMP and cGMP are associated with a stimulation of HL-60 proliferation, while previously reported manipulations that elevate cyclic nucleotides are related to an inhibition of HL-60 proliferation and the potentiation of differentiation.
Human neutrophils express the alpha 1-antitrypsin gene and produce alpha 1-antitrypsin
Authors: du Bois RM , Bernaudin JF , Paakko P , Hubbard R , Takahashi H , Ferrans V , Crystal RG
Published: October 2019

The potent serine protease, neutrophil elastase (NE), is stored in neutrophil azurophilic granules, where it is available to degrade phagocytosed material and can be released by the cell to assist in tissue migration and help clear tissue debris. While neutrophils carry NE, they cannot produce it; the NE gene is expressed only in bone marrow granulocyte precursor cells. Protection of normal tissues from the destructive capacity of NE is provided by alpha 1-antitrypsin (alpha 1 AT), a 52-Kd serine antiprotease produced by hepatocytes and mononuclear phagocytes. In the context of the broad destructive capacity of NE, we evaluated the concept that human neutrophils may be able to modulate the extracellular activity of NE by synthesizing and secreting alpha 1AT. Immunocytochemical analysis demonstrated that the neutrophil contains alpha 1AT. Northern analysis and in situ hybridization with alpha 1AT-specific probes demonstrated the presence of alpha 1AT messenger RNA transcripts within neutrophils. [35S]methionine-labeling of neutrophils followed by immunoprecipitation of the supernatant with an anti-alpha 1AT antibody and sodium dodecyl sulfate-acrylamide gel analysis demonstrated that neutrophils can synthesize alpha 1AT de novo and secrete the synthesized molecule. In the presence of major neutrophil degranulation, the antiprotease effect of neutrophil alpha 1AT is overwhelmed, allowing the NE to act unopposed in the extracellular microenvironment. However, in conditions where small amounts of NE are released by neutrophils, at least some of the secreted newly synthesized alpha 1AT was capable of complexing with NE. Thus, despite the fact that the neutrophil cannot synthesize NE, it can synthesize and secrete alpha 1AT, the inhibitor of NE, ie, the neutrophil is capable, to some extent, of modulating NE activity in the local milieu without the help of antiproteases produced by other cells.
Hereditary erythrocyte adenylate kinase deficiency: a defect of multiple phosphotransferases?
Authors: Lachant NA , Zerez CR , Barredo J , Lee DW , Savely SM , Tanaka KR
Published: October 2019

Adenylate kinase (AK) modulates the interconversion of adenine nucleotides (AMP + adenosine triphosphate----2 ADP). We evaluated the fifth kindred with hereditary erythrocyte (RBC) AK deficiency. The proband had chronic hemolytic anemia. Her RBC had undetectable AK activity when measured spectrophotometrically, whereas those of her parents had half-normal AK activity. AK electrophoresis showed only AK- 1 in the parents. The activities of pyruvate kinase and phosphoribosylpyrophosphate synthetase were decreased given the young age of the proband's RBC. Despite the absence of spectrophotometric AK activity, the proband's RBC were able to incorporate 14C-adenine into 14C-adenine nucleotides at 50% of the rate expected for her young RBC population, suggesting the possibility of an alternative pathway for the formation of ADP from AMP. Normal hemolysate had AMP:guanosine triphosphate (GTP) phosphotransferase activity, which produced ADP at 8% to 9% of the rate of AK (6.8 +/- 0.8 IU/mL RBC). AMP:GTP phosphotransferase activity was not detectable in the proband's or parent's hemolysates. These additional biochemical defects in the AK- deficient RBC further support the concept that AK deficiency per se may not cause hemolytic anemia. We propose that defects occur in multiple phosphotransferases in the AK-deficient RBC and that these other biochemical defects may produce deleterious lesions that promote the shortened RBC survival in AK deficiency.
Hybridization protection assay: a rapid, sensitive, and specific method for detection of Philadelphia chromosome-positive leukemias
Authors: Dhingra K , Talpaz M , Riggs MG , Eastman PS , Zipf T , Ku S , Kurzrock R
Published: October 2019

The Philadelphia (Ph1) chromosome is present in greater than 90% of patients with chronic myelogenous leukemia (CML) and in 2% to 20% of those with acute leukemias, for which it is an important prognostic marker too. The chimeric BCR-ABL mRNAs resulting from the translocation encode either a 210-Kd or a 190-Kd protein. The techniques used to detect Ph1 chromosome include karyotyping, Southern analysis to demonstrate bcr rearrangement, and polymerase chain reaction to amplify the BCR-ABL transcripts. However, the routine performance of these methods by clinical laboratories is cumbersome, time consuming, and exposes laboratory personnel to radioisotopes. We describe here the clinical application of a new method, the hybridization protection assay (HPA), which uses chemiluminescent acridinium-ester-labeled probes in conjunction with PCR for detection of the amplified BCR-ABL sequences. The method is sensitive, specific, and can reliably distinguish between the transcripts for P190BCR-ABL and P210BCR-ABL. In contrast to the 2 days or longer required for conventional hybridization, HPA analysis can be completed in less than 30 minutes. We have successfully used this method to analyze 60 leukemia samples (34 from Ph1-negative acute leukemias; 6 from Ph1-positive acute leukemias; and 20 from CML) with complete correlation (of BCR-ABL positivity or negativity) with the results of karyotype or Southern Blot analysis of genomic DNA for bcr rearrangement. Therefore, the HPA, in conjunction with PCR, appears to provide a rapid and reliable test for the diagnosis of Ph1-positivity.
Metabolic adaptation during erythropoietin-mediated terminal differentiation of mouse erythroid cells
Authors: Kim HD , Koury MJ , Lee SJ , Im JH , Sawyer ST
Published: October 2019

Metabolic development was examined in erythroid precursor cells, which were isolated from the spleens of mice infected with the anemia- inducing strain of Friend virus (FVA cells). FVA cells undergo differentiation in vitro from the proerythroblast stage through the reticulocyte stage over a 48-hour period in the presence of erythropoietin. Concomitant with marked decreases in cellular size and energy demand, metabolic capacities of both glycolysis and oxygen consumption diminish after 48 hours in culture by 7- and 18-fold, respectively. Because the oxidative capacity decreases more than glycolytic ability does, the metabolic machinery increasingly shifts toward anaerobic metabolism. During the 48-hour period of differentiation, the 2,3-diphosphoglyceric acid (DPG) content per cell and 2,3-DPG mutase activity per cell increased eightfold and threefold, respectively. Freshly harvested FVA cells have adenosine triphosphate (ATP) levels of 7.23 +/- 2.52 mumol/10(10) cells or 3.76 +/- 1.31 mumol/mL cell water which are 12- or 2.3-fold higher, respectively, than the ATP levels of mature red blood cells. In the course of FVA cell differentiation, ATP content per cell decreases by fourfold, but ATP concentration in cell water remains unchanged because of a corresponding decrease in cellular size and water content during differentiation. These studies show that in the face of dramatic decreases in cell size and cellular energy demand, terminally differentiating erythroid cells maintain a constant ATP level by undergoing an involution of their glycolytic machinery as well as by losing their aerobic metabolic capacity.