bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of a heparin-like anticoagulant from the plasma of a patient with metastatic bladder carcinoma
Authors: Tefferi A , Owen BA , Nichols WL , Witzig TE , Owen WG
Published: October 2019

A 73-year-old woman with metastatic transitional cell carcinoma of the bladder developed vaginal bleeding a few days after undergoing radical cystectomy. She had no other signs of mucocutaneous bleeding. Coagulation studies revealed a markedly prolonged thrombin time (greater than 600 seconds), a slightly prolonged reptilase time (20 seconds), and mildly elevated fibrinogen (4.39 g/L), and fibrin D-dimer (200 to 500 ng/mL) levels. Treatment of the patient's plasma in vitro with protamine or barium sulfate normalized the thrombin time. The anticoagulant activity corresponded to 0.15 heparin U/mL when measured by a thrombin time assay using normal plasma as substrate and standardized with porcine heparin. The anticoagulant was quantitatively bound to and subsequently eluted with 1 mol/L NaCl from quaternary aminoethyl (QAE) Sephadex, and then isolated by affinity chromatography on immobilized antithrombin III. The isolated anticoagulant was shown to be sensitive to heparinase digestion. Therefore, the inhibitor has functional and chemical properties similar to those of high-affinity heparin. Thus far, this is the only anticoagulant of this type isolated from the plasma of a patient bearing a tumor other than plasma cell myeloma.
Effect of recombinant human granulocyte-macrophage colony-stimulating factor on myelopoiesis in patients with refractory metastatic carcinoma
Authors: Phillips N , Jacobs S , Stoller R , Earle M , Przepiorka D , Shadduck RK
Published: October 2019

The effect of human recombinant GM-colony-stimulating factor (CSF) was evaluated in ten patients with refractory metastatic carcinoma. Initially they received an intravenous (IV) bolus injection of 5 or 25 micrograms/m2 for assessment of acute responses. Six days later, continuous IV infusions of 100 or 500 micrograms/m2 were initiated for a 14-day treatment course. All patients developed profound leukopenia within five to 30 minutes of the bolus injection. This appeared to result from increased expression of an adhesion-promoting glycoprotein (GP) on neutrophils and monocytes as judged by increased reactivity to the Mo1 monoclonal antibody (MoAb). Leukocyte counts returned to normal levels within two hours as cells were released from marrow stores. With the continuous infusion, leukocyte counts increased by 24 hours; peak values of 22,960 and 75.900/microL were achieved after ten to 14 days of treatment with the two dose levels of GM-CSF. This leukocytosis was due to an increase in virtually all cell types. At the high dose level, there was a striking increase in neutrophils (49,400/microL) and eosinophils (20,905/microL) with a sixfold increase in monocytes and two- to threefold increase in lymphocytes. Leukocyte counts declined promptly after cessation of the infusion but remained above baseline for as long as 2 weeks in some patients. These results suggest that GM- CSF may be useful as an adjuvant therapy by stimulating myelopoiesis in cancer patients.
Characterization of murine bone marrow and spleen-derived stromal cells: analysis of leukocyte marker and growth factor mRNA transcript levels
Authors: Gimble JM , Pietrangeli C , Henley A , Dorheim MA , Silver J , Namen A , Takeichi M , Goridis C , Kincade PW
Published: October 2019

Stromal cells are believed to regulate lympho-hematopoiesis through direct cell-cell interactions and the release of growth factors. Many questions remain, however, about their lineage derivation and functional heterogeneity. We previously prepared a panel of stromal cell lines from murine spleen and bone marrow and characterized them based on their ability to support lymphocyte growth in long-term cultures. These cells are now compared with respect to their expression of various immunoglobulin superfamily and cytokine genes by Northern blot analysis. These results indicate that although stromal cells appear to be mesodermal in origin, they are not closely related developmentally to the hematopoietic progenitor cells they support. The potential production of at least six cytokines was demonstrated. All clones constitutively expressed mRNA for macrophage colony stimulating factor, interleukin-6, transforming growth factor beta and neuroleukin. The most potent lymphocyte supporting clones also made interleukin 7 constitutively. Previous findings had suggested that these clones responded to exogenous stimuli and this has now been demonstrated in terms of induced expression of IL-6 and G/M-CSF mRNA. Interleukin 6 mRNA levels were markedly upregulated by exposure of cells to LPS, TNF, IL-1, IL-6, IL-7, and EGF. G/M-CSF mRNA levels were “superinduced” by the combination of LPS and cycloheximide, a protein synthesis inhibitor. These responses are similar to ones documented by investigators working with endothelial cells and fibroblasts. Together, these data suggest that stromal cells are a multifunctional component of the lymphopoietic microenvironment and may be active participants in a complex, cytokine-mediated regulatory network.
High-dose intravenous immunoglobulin modifies complement-mediated in vivo clearance
Authors: Basta M , Langlois PF , Marques M , Frank MM , Fries LF
Published: October 2019

The mechanism of effect of high-dose intravenous immunoglobulin (IVIG) therapy in immune cytopenias is incompletely known. One of the leading theories ascribes the short-term effects of IVIG to the competition of infused IVIG for Fc receptors, thereby inhibiting IgG-mediated clearance. Using a system independent of IgG-Fc receptor interactions, we examined another potential mechanism of IVIG action. Guinea pigs were infused with a human IVIG preparation at 600 mg/kg/day for two consecutive days. Parallel groups of animals were treated with the same volume and/or concentration of saline and albumin. Clearance of IgM- sensitized guinea pig erythrocytes, which is wholly complement dependent, was significantly retarded in animals treated with high-dose IVIG. The effect was specific for IVIG, since human albumin (as a second foreign protein) failed to change the clearance of IgM- sensitized guinea pig erythrocytes. Experiments in which IVIG-treated animals were subjected to pre- and posttreatment clearance studies revealed heterogeneity among individual animals in respect to their response to IVIG infusions. Decrease of available plasma complement components did not account for the effect, since both C3 and CH50 values remained unchanged after IVIG treatment, despite rising levels of IVIG in sera of treated animals. The results of in vitro C3 uptake studies and the effect of IVIG on clearance of preopsonized cells suggest that IVIG produces a kinetic depression of C3 uptake and modifies the process of complement fragment deposition on erythrocytes. A generalized effect on mononuclear phagocytes is less likely but cannot be wholly ruled out. These studies establish another potential mechanism of IVIG action and suggest extension of its use to other complement-mediated diseases.
Antigen-specific recognition of autologous leukemia cells and allogeneic class-I MHC antigens by IL-2-activated cytotoxic T cells from a patient with acute T-cell leukemia
Authors: Fisch P , Weil-Hillman G , Uppenkamp M , Hank JA , Chen BP , Sosman JA , Bridges A , Colamonici OR , Sondel PM
Published: October 2019

Culturing of leukemic blood lymphocytes from a patient with acute T- cell lymphoblastic leukemia (T-ALL) with interleukin-2 (IL-2) yielded T- cell line AK-1 with a remarkable cytotoxic specificity. This line mediated strong lysis of tumor target lines expressing major histocompatibility complex (MHC) class I antigens, such as Raji, CEM, and Molt-4 cells, but no killing of K562 and Daudi cells, which are deficient in MHC class I. In contrast, lymphokine-activated killer (LAK) cells from normal donors destroyed all these tumor targets, without MHC restriction. Line AK-1, originating from residual normal T cells present in the leukemic blood, lysed autologous leukemic blasts and peripheral blood lymphocytes (PBL) from many but not all allogeneic individuals but failed to kill autologous remission lymphocytes. Destruction of the autologous leukemic targets by AK-1 could be inhibited by unlabeled competitor target cells that were lysed by AK-1, but not by target cells that were not lysed. This suggests that AK-1 specifically recognized an alien determinant on the autologous ALL cells, crossreactive with allogeneic MHC class I antigens. This reactivity with some degree of tumor specificity may be a leukemic equivalent to responses reported for populations of tumor infiltrating lymphocytes (TIL) seen in some solid tumors.
Establishment and characterization of a childhood T-cell acute lymphoblastic leukemia cell line, PER-255, with chromosome abnormalities involving 7q32-34 in association with T-cell receptor- beta gene rearrangement
Authors: Kees UR , Lukeis R , Ford J , Garson OM
Published: October 2019

A human leukemia cell line, PER-255, was established from the bone marrow of a 5-year-old boy with features typical of lymphomatous T- acute lymphoblastic leukemia (T-ALL). The leukemic origin of cell line PER-255 is indicated by its cytochemical and immunologic similarity to the patient's fresh leukemic cells, which correspond to immature cortical thymocytes. Southern blot analysis showed that the IgJH genes were in germline configuration, whereas both alleles of the T-cell receptor-beta (TCR-beta) gene were rearranged in PER-255 cells, with identical rearrangements present in the patient's leukemic cells. Cytogenetic analysis of the cell line revealed a single abnormal clone with the karyotype 46,XY,t(7;10)(q32–34;q24),t(9;12) (p22;p12–13). Reciprocal translocations involving chromosome bands 7q32–36, containing the gene for the TCR-beta chain, have been reported for a number of tumors of T-cell origin. Translocations involving the 7q32–36 region appear to be nonrandomly associated with childhood T-ALL, whereas abnormalities of 9p and 12p have been reported to be nonrandomly involved in ALL but not specifically associated with the T- cell phenotype.
Nonrandom abnormalities of chromosome 9p in childhood acute lymphoblastic leukemia: association with high-risk clinical features
Authors: Murphy SB , Raimondi SC , Rivera GK , Crone M , Dodge RK , Behm FG , Pui CH , Williams DL
Published: October 2019

To assess the frequency and significance of nonrandom abnormalities of chromosome 9p in childhood acute lymphoblastic leukemia (ALL), we analyzed our experience with 398 consecutive cases with completely banded karyotypes. Forty cases (10%) with abnormalities of 9p were identified: 26 with deletions, nine with unbalanced translocations resulting in the loss of 9p material, and five with apparently balanced reciprocal translocations. As compared with children with ALL lacking 9p abnormalities, these 40 cases were significantly older, had higher initial circulating WBC counts, more “lymphomatous” disease characteristics (including presence of a mediastinal mass in 15%. T- cell phenotype in 26%, splenomegaly greater than 8 cm in 25%), an increased failure rate in the first 2 to 3 years after diagnosis, and a higher incidence of extramedullary relapse. Conversely, lymphomatous ALL cases were twice as likely (19% v 8%) to have an abnormality of chromosome 9p than ALL cases lacking lymphomatous features (P = .01). The finding of an abnormal chromosome 9p, however, was not specific for lymphomatous ALL or T-cell lineage, because most cases were neither lymphomatous nor T-cell, and the overall Kaplan-Meier distribution of treatment failures for abnormal 9p cases was not statistically significantly different from control ALL cases receiving the same treatment who lacked abnormalities of 9p (P = .06, by log-rank test). We conclude that nonrandom abnormalities of chromosome 9p, especially a breakpoint in 9p21–22, occur with increased frequency in childhood ALL in association with some high-risk clinical features. Despite this association, the chromosome anomaly is nonspecific in its syndrome delineation and confers no major adverse consequence on long-term survival of childhood ALL treated with modern therapy. However, due to an apparently increased hazard of involvement of the CNS (eight of 17 failures), it may be inadvisable to lessen the intensity of CNS preventive therapy for this group of patients.
Histologic typing of non-Hodgkin's lymphomas by in situ hybridization with DNA probes of oncogenes
Authors: Hamatani K , Yoshida K , Kondo H , Toki H , Okabe K , Motoi M , Ikeda S , Mori S , Shimaoka K , Akiyama M
Published: October 2019

Expression of six proto-oncogenes (fos, myc, myb, Ki-ras, Ha-ras, and N- ras) in 43 cases of non-Hodgkin's lymphoma was analyzed by means of in situ hybridization. Biotinylated DNA probes of the six oncogenes and those of immunoglobulin H (IgH) gene and T-cell receptor beta (TCR beta) chain gene were used. The results of in situ hybridization performed under blind conditions by IgH gene and TCR beta chain gene probes were compatible with those of typing by cell surface markers. The nuclear protein-related proto-oncogenes, fos, myc, and myb, were expressed in about 70% to 80% of all cases regardless of phenotype, histology, or histologic grade. On the contrary, genes of ras family were expressed in more limited numbers of cases except for the Ki-ras gene, which was more frequently expressed by cases of the T-cell immunophenotype with a high malignancy grade. The results of dot hybridization with RNA extracted from some cases were compatible with those of in situ hybridization, further demonstrating the specificity of in situ hybridization.
Therapy of chronic graft-v-host disease in a rat model
Authors: Vogelsang GB , Hess AD , Friedman KJ , Santos GW
Published: October 2019

Chronic graft-v-host disease (GVHD) is the most frequent late complication of allogeneic bone marrow (BM) transplant. To test different treatments, we used a rat model of chronic GVHD which clinically, histologically, and immunologically resembles the disease occurring after human marrow transplant. The following treatments were administered: azathioprine 50 mg/kg/day plus prednisone 1 mg/kg/every other day; Cyclosporine (CsA) 30 mg/kg/day; CsA 30 mg/kg/day plus prednisone 1 mg/kg/every other day; thalidomide 50 mg/kg/day; thalidomide 10 mg/kg/day; CsA 10 mg/kg/day; and thalidomide 10 mg/kg/day plus CsA 10 mg/kg/day. All drugs were administered by gavage. Half of the animals (six of 12) treated with azathioprine plus prednisone every other day responded to treatment. The majority of animals (seven of 12) treated with CsA died early. The addition of prednisone every other day did not improve survival. The surviving animals treated with CsA (with or without prednisone) responded to treatment. Half of the animals treated with each of the above regimens had recurrent or progressive disease after therapy was discontinued. Thalidomide treatment was successful in the majority of animals (16 of 18). The addition of low-dose CsA to low-dose thalidomide resulted in a faster rate of response (12.1 +/- 1.8 v 28.2 +/- 1.6 days). We conclude that both thalidomide and thalidomide plus CsA appear promising in this model as treatment for chronic GVHD.
Successful HLA nonidentical bone marrow transplantation in three patients with the leukocyte adhesion deficiency
Authors: Le Deist F , Blanche S , Keable H , Gaud C , Pham H , Descamp-Latscha B , Wahn V , Griscelli C , Fischer A
Published: October 2019

Three consecutive patients with the severe phenotype of leukocyte adhesion deficiency characterized by a defective expression of LFA-1, Mac-1 (CR3), and p150.95 on leukocytes have received HLA partially incompatible bone marrow transplantation (BMT). The degree of HLA incompatibility between related donors and recipients was 2 HLA antigens in one and one full haplotype in the two others. Graft-v-host disease (GVHD) prophylaxis consisted in T-cell depletion of the bone marrow inoculum and a 60-day course of cyclosporin A. A first attempt led to autologous recovery in one patient. The second transplant in this patient and the first transplant in the two others led to stable partial engraftment of lymphocytes and phagocytic cells, as shown by expression of adhesion molecules (LFA-1, Mac-1) on leukocytes and by HLA typing and restriction fragment-length polymorphism studies using minisatellite probes. Although the level of mixed chimerism was lower in one patient (7% to 30% donor cells) and greater than 50% in the two others, recovery of lymphocyte and phagocytic cell functions was sufficient enough to allow the patient to lead a normal life, infection free in the three cases. These patients, now 57, 32, and 19 months post- transplant, are in good condition without any therapy. These results lead us to propose that the LFA-1 molecule plays a role in HLA- incompatible graft rejection, probably by mediating adhesion of cytotoxic T and non-T lymphocytes to their targets.
Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates
Authors: Lin L , Wiesehahn GP , Morel PA , Corash L
Published: October 2019

Transmission of viral diseases through blood products remains an unsolved problem in transfusion medicine. We have developed a psoralen photochemical system for decontamination of platelet concentrates in which platelets are treated with long wavelength ultraviolet radiation (UVA, 320–400 nm) in the presence of 8-methoxypsoralen (8-MOP). Bacteria, RNA viruses, and DNA viruses ranging in genome size from 1.2 x 10(6) daltons, encompassing the size range of human pathogens, were inoculated into platelet concentrates and subjected to treatment. This system inactivated 25 to 30 logs/h of bacteria Escherichia coli or Staphylococcus aureus, 6 logs/h of bacteriophage fd, 0.9 log/h of bacteriophage R17 and 1.1 logs/h of feline leukemia virus (FeLV) in platelet concentrates maintained in standard storage bags. Platelet integrity and in vitro function before, immediately following photochemical treatment, and during prolonged storage after treatment, were evaluated by measuring: (1) extracellular pH; (2) platelet yields; (3) extracellular lactate dehydrogenase (LDH) levels; (4) platelet morphology; (5) platelet aggregation responsiveness; (6) thromboxane beta-2 (TXB-2) production; (7) dense body secretion; and (8) alpha granule secretion. These assays demonstrated that this photochemical inactivation system inactivated bacteria and viruses in platelet concentrates with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.
Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness
Authors: Park LS , Waldron PE , Friend D , Sassenfeld HM , Price V , Anderson D , Cosman D , Andrews RG , Bernstein ID , Urdal DL
Published: October 2019

Recombinant human granulocyte-macrophage (GM) colony-stimulating factor (GM-CSF), G-CSF, and interleukin-3 (IL-3) labeled with 125I were used to study the characteristics and distribution of receptors for these factors on in vitro cell lines and on cells from patients with acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). Receptors for GM-CSF and G-CSF were restricted to a subset of myelomonocytic cell lines whereas IL-3 receptors were also found on pre- B- or early B-cell lines. Receptors for all three CSFs were broadly distributed on ANL cells, with considerable variability in levels of expression. Measurement of the colony-forming ability of ANL cells in response to the CSFs showed that there was no direct correlation between the ability of the cells to respond to a growth factor and the absolute number of receptors expressed for that growth factor. Binding of radiolabeled IL-3 and GM-CSF to ANL cells produced complex biphasic curves. Further analysis showed that both IL-3 and GM-CSF were able to partially compete for specific binding of the heterologous radiolabeled ligand to cells from several ANL patients, suggesting that heterogeneity may exist in human CSF receptors. These results provide new insights into the complex role that CSFs may play in ANL.
Novel vitamin D analogs that modulate leukemic cell growth and differentiation with little effect on either intestinal calcium absorption or bone mobilization
Authors: Zhou JY , Norman AW , Lubbert M , Collins ED , Uskokovic MR , Koeffler HP
Published: October 2019

Induction of terminal differentiation of leukemic and preleukemic cells is a therapeutic approach to leukemia and preleukemia. The 1 alpha, 25- dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active form of vitamin D3, can induce differentiation and inhibit proliferation of leukemia cells, but concentrations required to achieve these effects cause life-threatening hypercalcemia. Seven new analogs of 1,25(OH)2D3 were discovered to be either equivalent or more potent than 1,25(OH)2D3 as assessed by: (a) inhibition of clonal proliferation of HL-60, EM-2, U937, and patients' myeloid leukemic cells: and (b) induction of differentiation of HL-60 promyelocytes. Furthermore, these analogs stimulated clonal growth of normal human myeloid stem cells. The most potent analog, 1,25-dihydroxy-16ene-23yne-vitamin D3, was about fourfold more potent than 1,25(OH)2D3. This analog decreased clonal growth and expression of c-myc oncogene in HL-60 cells by 50% within ten hours of exposure. Effects on calcium metabolism of these novel analogs in vivo was assessed by intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Each of the analogs mediated markedly less (10 to 200-fold) ICA and BCM as compared with 1,25(OH)2D3. To gain insight into the possible mechanism of action of these new analogs, receptor binding studies were done with 1,25(OH)2–16ene-23yne-D3 and showed that it competed only about 60% as effectively as 1,25(OH)2D3 for 1,25(OH)2D3 receptors present in HL-60 cells and 98% as effective as 1,25(OH)2D3 for receptors present in chick intestinal cells. In summary, we have discovered seven novel vitamin D analogs that are more potent than the physiologic 1,25(OH)2D3 as measured by a variety of hematopoietic assays. In contrast, these compounds appear to have the potential to be markedly less toxic (induction of hypercalcemia). These novel vitamin D compounds may be superior to 1,25(OH)2D3 in a number of clinical situations including leukemia/preleukemia; they will provide a tool to dissect the mechanism of action of vitamin D seco-steroids in promoting cellular differentiation.
Effects of interleukin-1 on hematopoietic progenitors: evidence of stimulatory and inhibitory activities in a primate model
Authors: Gasparetto C , Laver J , Abboud M , Gillio A , Smith C , O'Reilly RJ , Moore MA
Published: October 2019

The effects of recombinant human interleukin-1 rhIL-1 beta (rhIL-1b) on hematopoietic recovery following chemotherapy in a primate model were investigated. Cynomolgus monkeys received 1 microgram/kg/day rhIL-1b intravenously for 2, 7, and 14 days following 5-Fluorouracil (5-FU) treatment (75 mg/kg x 2 days). Compared with controls, a significantly shortened time to achieve an absolute neutrophil (ANC) count over 500/microL was observed in animals receiving 2- and 7-day courses of rhIL-1b (17 v 30 days), while animals receiving a 14-day course of rhIL- 1b achieved an ANC over 500/microL by 23 days. Concomitantly, a marked increase in granulocyte-macrophage colonies (CFU-GM) was observed at 14 days following 5-FU in animals receiving 2- and 7-day rhIL-1b courses. In animals receiving a 14-day rhIL-1b course, a significant increase in CFU-GM relative to control was not seen until 21 days post 5-FU. Utilizing a serum-free colony assay system, a 50% inhibition of normal marrow CFU-GM growth was observed with the addition of sera obtained on day 9 post 5-FU from animals receiving rhIL-1b for 14 days. Sera obtained at any time from animals receiving 2- and 7-day rhIL-1b treatment did not show any growth inhibition. Addition of antibodies to TNFa to the coculture assay abrogated the CFU-GM growth inhibition. TNFa levels in sera with the inhibitory activity was relatively high (918 pg/mL). Our data indicate that rhIL-1b enhances hematopoietic recovery following 5-FU if administered for short periods of time (less than 7 days), whereas prolonged administration has a counterproductive effect that is due in part to the induction of TNFa production.
Interleukin-1 stimulates proliferation of acute myeloblastic leukemia cells by induction of granulocyte-macrophage colony-stimulating factor release
Authors: Delwel R , van Buitenen C , Salem M , Bot F , Gillis S , Kaushansky K , Altrock B , Lowenberg B
Published: October 2019

In this study, we further established the role of interleukin-1 (IL-1) alpha and IL-1 beta as regulators of proliferation of acute myeloid leukemia (AML) cells. IL-1 stimulated tritiated thymidine (3H-TdR) uptake of AML cells in 13 of 28 cases. Cytogenetic analysis confirmed the leukemic clonality of the IL-1-stimulated cells. Most likely, IL-1 exerted these stimulative effects directly on AML blast cells because IL-1 effectively induced 3H-TdR uptake of CD34-positive AML blasts (separated following cell sorting). Furthermore, adherent cell-depleted AML samples of three patients were more effectively stimulated than nondepleted AML fractions. Cluster and colony formation from adherent cell depleted AML samples could also be stimulated with IL-1, ie, in seven of ten cases analyzed. Subsequent experiments indicated that IL-1 stimulation depended on the release of GM-CSF because (1) induction of DNA synthesis of AML cells by IL-1 could be abrogated with antigranulocyte-macrophage colony-stimulating factor (GM-CSF) antibody, (2) conditioned media (CM) prepared from IL-1 stimulated AML blasts (adherent cell depleted) could stimulate the proliferation of purified normal bone marrow progenitors whereas supernatants from nonstimulated AML blasts did not, and (3) GM-CSF was demonstrated in IL-1/AML-CM with a specific radioimmunoassay, while GM-CSF was not detectable in nonstimulated supernatants. In one case of AML showing significant 3H- TdR uptake in the absence of CSFs, this spontaneous DNA synthesis was found to depend on autocrine IL-1 beta release as it could be suppressed with anti-IL-1 beta antibody or anti-GM-CSF. The blockade by anti-IL-1 beta could be overcome by the addition of high concentrations of IL-1 beta as well as GM-CSF. Thus, in this particular case, endogenously produced IL-1 beta had stimulated the release of GM-CSF which resulted in GM-CSF-dependent proliferation. The results indicate that GM-CSF production by AML blasts is often regulated by IL-1 rather than being constitutive.
Detection of a human CFC with a high proliferative potential
Authors: McNiece IK , Stewart FM , Deacon DM , Temeles DS , Zsebo KM , Clark SC , Quesenberry PJ
Published: October 2019

Colony forming cells (CFC) with high proliferative potential have been detected in nutrient agar cultures of human bone marrow cells containing recombinant human interleukin-3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF). These CFC were detected by the formation of large colonies with diameters greater than 0.5 mm and containing approximately 50,000 cells after 28 days incubation. The incidence of these CFC was only two in 100,000 normal bone marrow cells; however, bone marrow from patients treated with 5-fluorouracil contained up to sevenfold higher numbers of these CFC. The characteristics of these CFC, multifactor-responsive progenitors with high proliferative potential, requiring a prolonged growth period in culture and showing a relative preservation in marrow from individuals pretreated with 5-fluorouracil, are consistent with a human cell type equivalent to the primitive murine progenitor termed HPP-CFC.
Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration
Authors: Koury ST , Koury MJ , Bondurant MC , Caro J , Graber SE
Published: October 2019

In situ hybridization was used to quantitate the cells that produce erythropoietin (EP) in the renal cortices of mice with varying severities of acute anemia and of mice recovering from severe, acute anemia. The number of EP-producing cells in the renal cortex increased in an exponential manner as hematocrit was decreased. Individual EP- producing cells had very similar densities of silver grains in autoradiograms regardless of whether they were from normal mice or from slightly, moderately or severely anemic animals. With increasingly severe anemia, total renal EP mRNA levels and serum EP concentrations showed increases that correlated with the number of renal EP-producing cells. These results indicate that as mice become more anemic, additional cells are recruited to produce EP rather than the cells already producing EP being stimulated to increase their individual production. In mildly and moderately anemic animals, small clusters of EP-producing cells were found in the inner cortex with large areas of cortex containing no EP-producing cells. In severely anemic mice, EP- producing cells were found throughout the inner cortex with only a very few found scattered in the outer cortex and outer medulla. The data indicate that only a subset of total renal interstitial cells produce EP. During recovery from severe, acute anemia, the numbers of EP- producing cells decreased exponentially as hematocrits rose and correlated with decreases in total renal EP mRNA and serum EP concentrations. These results suggest that following an acute blood loss and during the recovery from a blood loss, the capacity to deliver oxygen, as represented by hematocrit, is the major regulator of EP production.
Decreased accessibility of platelet-bound fibrinogen to antibody and enzyme probes
Authors: Peerschke EI
Published: October 2019

The binding of fibrinogen to platelets is a multiphasic process leading to apparently nonreversible associations between fibrinogen and stimulated platelets. To further investigate changes in platelet- fibrinogen interactions, the present study examined the accessibility of platelet-bound fibrinogen and its GPIIb-IIIa receptor to antibody and enzyme probes as a function of time after platelet stimulation with adenosine diphosphate (ADP). Whereas only minimal changes in fibrinogen and 10E5 binding were observed within 60 minutes after platelet stimulation and equilibrium fibrinogen binding, the binding of polyclonal antifibrinogen antibodies decreased significantly (75% +/- 13%, mean +/- SD, n = 9). Similar decreases were noted with rabbit antifibrinogen Fab and F(ab')2 fragments. In addition, plasmin (32 mU/mL) added to platelets five minutes compared with 60 minutes after equilibrium fibrinogen binding dissociated 52% +/- 12% compared with 33% +/- 7% of platelet-bound fibrinogen in five minutes, and 83% +/- 15% compared with 66% +/- 14% of bound fibrinogen in 15 minutes. No difference in plasmin cleavage products was observed, however, by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE). Complete fibrinogen dissociation occurred 30 minutes after plasmin addition, confirming that fibrinogen was not internalized. In contrast, dissociation of platelet-bound fibrinogen by chymotrypsin was less affected by time after equilibrium fibrinogen binding, and minimal changes in antifibrinogen antibody recognition and plasmin-induced dissociation of fibrinogen bound to stimulated but glutaraldehyde-fixed platelets were observed. The data suggest that ADP-induced fibrinogen binding to fresh platelets is accompanied by progressive rearrangements of fibrinogen on the platelet surface.
Megakaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of gamma chain-variant polypeptides
Authors: Haidaris PJ , Francis CW , Sporn LA , Arvan DS , Collichio FA , Marder VJ
Published: October 2019

The gamma chain of human fibrinogen is heterogeneous in length at the C- terminus due to differential RNA processing of the gamma chain-gene primary transcript. We have produced two specific monoclonal antibodies (MoAbs) against the gamma-chain epitopes generated by this alternative processing event: anti-gamma 57.5(408–416) (L2B), which reacts with gamma 57.5 and gamma 55 chains, and anti-gamma 50(337–411) (H9B7), which reacts preferentially with gamma 50 chains. Using these MoAbs we have studied the expression of gamma-chain polypeptides by immunofluorescence microscopy in the tissues of fibrinogen biosynthesis and have determined that gamma 57.5 polypeptide is expressed in hepatocytes but is absent or present in significantly reduced amounts in megakaryocytes. Therefore the gamma 50 chain is found in plasma, platelet, and megakaryocyte fibrinogens, but the gamma 57.5 chain is found only in plasma fibrinogen. The C-terminal amino acid sequence of gamma 55 includes the L2B epitope 57.5(408–416). Using MoAb L2B we have determined that gamma 55, which is a post-translationally modified gamma 57.5 chain, is found only in plasma fibrinogen and is absent or present in markedly reduced amounts in platelet or megakaryocyte fibrinogen. In addition, the conformation of the L2B epitope is preserved in gamma 55, as determined by Western blot analysis. The hepatocyte-specific expression of the gamma 57.5-chain polypeptide and the post-translational modification to gamma 55 result in a compartmentalization of gamma-chain polypeptide expression. This is suggestive of different mechanisms regulating human fibrinogen gamma- chain gene expression in hepatocytes v megakaryocytes that may operate in a tissue-specific manner at the level of 3′ RNA processing events.
Leukemic B-cell precursors constitutively express functional receptors for human interleukin-1
Authors: Uckun FM , Myers DE , Fauci AS , Chandan-Langlie M , Ambrus JL
Published: October 2019

This study analyzes the expression of functional interleukin-1 (IL-1) receptors on leukemic B-cell precursors (BCPs) from B-cell precursor acute lymphoblastic leukemia (BCP ALL) patients. We first investigated the specific binding of 125I-labeled recombinant IL-1 (125I-rIL-1) (4 x 10(17) cpm/mol) to fresh marrow blasts from 11 BCP ALL patients. In five of 11 cases, the binding of 125I-rIL-1 was significantly blocked by excess cold rIL-1. In these five cases, the cell-bound radioactivity ranged from 146 cpm/10(6) cells to 2,412 cpm/10(6) cells (mean +/- SE = 782 +/- 414 cpm/10(6) cells), indicating that 4 to 60 femtomols (mean +/- SE = 20 +/- 10 femtomols) of 125I-rIL-1 specifically bound per 10(7) cells. The estimated number of 125I-rIL-1 molecules bound per cell ranged from 219 to 3,618 (mean +/- SE = 1173 +/- 621). In all five cases, BCP colony formation was stimulated by 10 ng/mL (570 femtomolar) rIL-1, and the background-subtracted colony numbers ranged from 130 to 298 (mean +/- SE = 226 +/- 31). In contrast, no stimulation was observed in six cases that showed no significant 125I-rIL-1 binding. Hence, there was a high correlation between 125I-rIL-1 binding and IL-1 responsiveness, indicating that functional IL-1 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for leukemic BCPs from two IL-1-responsive BCP ALL cases yielded straight linear regression lines, indicating the existence of a single class of 132 to 154 high affinity IL-1 receptors/cell. The apparent affinity constants (Ka) values ranged from 5.2 x 10(9) mol/L-1 to 1.2 x 10(10) mol/L-1. Notably, the concentrations of IL-1 required for half-maximal receptor occupancy (kd = 83 pmol/L to 190 pmol/L) were approximately three orders of magnitude higher than those needed to elicit a half-maximal proliferative response of leukemic BCPs in colony assays (0.1 to 1.0 ng/mL = 5.7 to 57 femtomolar), indicating that only a small fraction of IL-1 receptors need to be occupied to stimulate leukemic BCPs. Notably, IL-1 unresponsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines (REH, KM-3) did not exhibit any significant IL-1 binding (less than 10 IL-1 binding sites/cell), and two additional IL-1 unresponsive BCP ALL cell lines (NALM-6, HPB-NULL) expressed only 24 to 54 IL-1 binding sites/cell with a Ka of 7.8 to 9.8 x 10(9) mol/L- 1.
Down-modulation of neutrophil production by erythropoietin in human hematopoietic clones
Authors: Christensen RD , Koenig JM , Viskochil DH , Rothstein G
Published: October 2019

In clonogenic assays of hematopoietic progenitors, high concentrations (4 U/mL) of erythropoietin (epo) reduced the formation of granulocyte- macrophage (GM) colonies and diminished the number of granulocytes formed per culture plate. Fetal progenitors were more sensitive to these effects of epo than were progenitors from adults, displaying these reductions at greater than or equal to 1 U epo/mL. The mechanism was investigated by growing fetal progenitors stimulated by recombinant GM-CSF, in the absence of epo, and when eight-cell clones first appeared, mapping their location, then adding epo, and assessing its effect on the subsequent differentiation of the clones. In the absence of epo, the clones developed exclusively into GM colonies. However, if developing clones were presented with epo, 85% matured into GM colonies, but 15% became multilineage or normoblast colonies. In addition, developing clones that were presented with epo produced colonies that contained fewer neutrophils. These effects of epo on neutrophil generation were observed with each of three varieties of recombinant epo, and also with purified human epo, but were not observed using epo that had been neutralized with rabbit anti-epo antiserum.
Spectrin Tunis (Sp alpha I/78), an elliptocytogenic variant, is due to the CGG----TGG codon change (Arg----Trp) at position 35 of the alpha I domain
Authors: Morle L , Morle F , Roux AF , Godet J , Forget BG , Denoroy L , Garbarz M , Dhermy D , Kastally R , Delaunay J
Published: October 2019

Spectrin Tunis (Sp alpha I/78) is an alpha l domain variant that causes asymptomatic elliptocytosis in the heterozygote state. It is manifested by a reduction of spectrin dimer self-association and by the development of a major 78-Kd fragment at the expense of the alpha l 80- Kd fragment upon spectrin-limited digestion. Amino acid sequence analysis, following peptide transfer onto Immobilon membranes, showed that the 78-Kd fragment results from a sensitized cleavage after lysyl residue 10. Using a 13.5-kb genomic alpha-spectrin probe and the Xbal, Pvull, and Mspl polymorphic sites detected with this probe, we concluded that spectrin Tunis is associated with the + - + haplotype (in the above order). Twenty mer oligonucleotides, complementary to genomic segments from introns 2 and 3, respectively, were synthesized. We then performed DNA amplification and sequencing. In the two investigated carriers of spectrin Tunis, we found the C----T base substitution of the codon corresponding to position 35 of the alpha l domain (CGG----TGG; Arg----Trp). The mutation lies in the last part of an alpha helix that extends from residues 9 to 44 of partial repeat alpha 1′ and is comparable with helix 3 of full repeats 1 to 5. The modified proteolytic site, located 25 amino acid residues upstream, occurs at the beginning of the helix.
The plasma cell labeling index: a valuable tool in primary systemic amyloidosis
Authors: Gertz MA , Kyle RA , Greipp PR
Published: October 2019

The plasma cell labeling index (LI) is of value in predicting prognosis in multiple myeloma. Primary systemic amyloidosis (AL) is a plasma cell dyscrasia that shares many features with myeloma. We obtained bromodeoxyuridine LI on 125 patients who presented with AL, 22 of whom also had overt multiple myeloma. Forty-six patients had a plasma cell LI greater than 0%. Of the 46 patients with an elevated LI, 19 (41%) had multiple myeloma as compared with three (4%) of the 79 patients with an LI = 0 (P less than .0001). A response to chemotherapy was seen in 14 (30%) of 46 patients with an LI greater than 0, as compared with ten (13%) of 79 patients with an LI of 0 (P = .015). The median survival of the high LI group was 14.6 months v 29.8 months for the low LI group (P = .02). In the low LI group, 29% are projected to be alive at 60 months, as compared with 20% in the high LI group. When patients with myeloma were excluded from the analysis, the LI did not predict response but continued to indicate a survival disadvantage (P less than .05). The major utility of the LI was in identifying those patients most likely to have multiple myeloma and those AL patients with a poor prognosis (median survival, 14.1 months).
The deamination of adenosine and adenosine monophosphate in Plasmodium falciparum-infected human erythrocytes: in vitro use of 2'deoxycoformycin and AMP deaminase-deficient red cells
Authors: Roth E Jr , Ogasawara N , Schulman S
Published: October 2019

The role of enzymatic deamination of adenosine monophosphate (AMP) and adenosine in the in vitro growth of the malaria parasite Plasmodium falciparum was investigated by means of human red cells deficient in AMP deaminase to which the adenosine deaminase inhibitor 2′- deoxycoformycin was added. Malaria parasites grew normally in red cells lacking one or both of these enzyme activities. As a further probe of adenosine triphosphate (ATP) catabolism, both infected and uninfected RBCs were incubated with NaF (with and without 2′-deoxycoformycin) and the purine nucleotide/nucleoside content was analyzed by high- performance liquid chromatography (HPLC). Uninfected RBCs lacking either AMP or adenosine deaminase were able to bypass the enzyme block and degrade ATP to hypoxanthine. Uninfected RBCs with both deaminases blocked were unable to produce significant quantities of hypoxanthine. On the other hand, infected RBCs were able to bypass blockade of both deaminases and produce hypoxanthine and adenosine. These findings establish that deamination of adenosine and/or AMP are not essential for plasmodial growth. However, further work will be required to elucidate the pathways that permit the parasites to bypass these catabolic steps.
Successful in vitro graft-versus-tumor effect against an Ia-bearing tumor using cyclosporine-induced syngeneic graft-versus-host disease in the rat
Authors: Geller RB , Esa AH , Beschorner WE , Frondoza CG , Santos GW , Hess AD
Published: October 2019

Lethally irradiated LouM rats reconstituted with syngeneic bone marrow and then treated with cyclosporine (CsA) for 40 consecutive days following transplant developed a graft-v-host disease (GVHD)-like syndrome after CsA cessation. This model of GVHD was used to define and characterize a graft-v-tumor (GVT) effect against a syngeneic plasmacytoma CRL1662 cell line which expresses class II major histocompatibility (MHC) antigen (Ia). Nylon wool-nonadherent spleen cells from animals who developed syngeneic GVHD were capable of significant lysis against chromium-labeled tumor target cells in a four- hour chromium released cell mediated lympholysis assay; maximum lysis occurred five days following cessation of CsA when clinical signs first appeared. Cytolytic activity declined to baseline as GVHD symptoms resolved. Fractionation of splenocytes into lymphocyte subsets demonstrated that cytolytic lymphocytes (CTLs) of the OX8 phenotype (non-helper T) were capable of significant lysis against tumor target cells. Lysis of tumor cells was blocked by preincubation with monoclonal antibodies (MoAb) specific for the rat anti-class II MHC antigen but not with MoAb against class I. Incubation of tumor cells with gamma-interferon increased expression of tumor class II MHC antigens and significantly increased their susceptibility to lysis by nylon wool-nonadherent splenocytes from animals with syngeneic GVHD. These studies have demonstrated an in vitro GVT of syngeneic GVHD against an Ia-bearing tumor; the effector cell is a CTL of the OX8 phenotype specific for the class II MHC antigen.
A monoclonal antibody (LYP18) directed against the blood platelet glycoprotein IIb/IIIa complex inhibits human melanoma growth in vivo
Authors: Boukerche H , Berthier-Vergnes O , Bailly M , Dore JF , Leung LL , McGregor JL
Published: October 2019

A monoclonal antibody (MoAb) (LYP18), generated against human platelet glycoprotein IIb/IIIa (GPIIb/IIIa), immuno-precipitated a IIb/IIIa-like GP complex from a highly tumorigenic human melanoma cell line (M3Dau). The M3Dau melanoma cells specifically bound 125I-labeled LYP18. To study the biologic role of these IIb/IIIa-like glycoproteins, M3Dau melanoma cells were incubated with LYP18 or a control MoAb directed against another melanoma cell-surface antigen and implanted subcutaneously (SC) in nude mice. LYP18 dramatically inhibited the growth of tumor in vivo. LYP18 was not directly cytotoxic to the melanoma cells. These results demonstrate that the IIb/IIIa-like GPs are present on melanoma cells and play a crucial role in tumor cell growth. MoAbs directed against tumor cytoadhesive receptors may represent a novel approach in tumor treatment.
P-glycoprotein expression in plasma-cell myeloma is associated with resistance to VAD
Authors: Epstein J , Xiao HQ , Oba BK
Published: October 2019

Tumor cell-associated expression of multidrug resistance (MDR) was quantitated in 22 patients with DNA-aneuploid myeloma using 2-parameter flow cytometry with monoclonal antibody (MoAb) C-219 for the detection of cytoplasmic p-170 and propidium iodide for nuclear DNA content. The proportion of cells expressing p-170 and the intensity of p-170-related fluorescence were determined for each patient. Among the 14 patients treated with vincristine-adriamycin-dexamethasone (VAD), the proportion of p-170-positive cells distinguished sensitive from resistant disease (P less than .01). Among a subgroup of seven patients with MDR analysis available prior to VAD therapy, two subsequent nonresponders had high proportions of C-219-reactive cells. The presence de novo of high proportions of p-170-expressing cells in another still untreated patient and in a further individual with resistance to dexamethasone and interferon (not associated with MDR) warrants systematic analysis of p-170 expression prior to therapy to determine its clinical implications for response to MDR-associated drugs as combined in the VAD regimen. Concurrent MDR expression by aneuploid tumor cells and cells in the diploid subcompartment may represent involvement of diploid cells in the myeloma disease process.
Enhancement of colony-forming activity of granulocyte-macrophage colony- stimulating factor by monocytes in vitro
Authors: Namiki M , Hara H
Published: October 2019

Human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) stimulated granulocyte-macrophage (GM) colony formation from human marrow mononuclear cells (MMCs) in a dose-dependent manner in methylcellulose culture. When phagocytes were depleted from MMCs, GM colony formation from the phagocyte-depleted (PD) MMCs by hrGM-CSF markedly decreased. Experiments in which PD-MMCs were cultured with hrGM-CSF and adherent cells showed that 94% (on day 7 and day 14) of the colonies from PD-MMCs were dependent on the presence of adherent cells. In contrast, the ability of granulocyte colony-stimulating factor (G-CSF) to form colonies was not affected by phagocyte depletion. To check for the presence or absence of progenitors that could form GM colonies in direct response to hrGM-CSF, single-cell culture of hematopoietic progenitor cell surface antigen (My-10)- positive PD-MMCs was carried out using a flow cytometer and an Autoclone System. In duplicate experiments, 0.7% and 3.5% (day 7) or 3.6% and 3.9% (day 14) of My-10-positive PD-MMCs formed GM colonies in response to hrGM-CSF and 5.1% and 6.0% (day 7) of My-10-positive PD- MMCs formed GM colonies in response to G-CSF. This was clear evidence for the presence of progenitors directly responding to hrGM-CSF. Also observed was a synergistic effect on GM colony formation in which more My-10-positive PD-MMCs stimulated by hrGM-CSF and G-CSF could form GM colonies than the sum of those stimulated by each separately. This enhancing effect of colony-forming activity of hrGM-CSF by adherent cells and the single cell culture experiment were reproduced in serum- free culture system.