bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoglobulins from normal sera bind platelet vinculin and talin and their proteolytic fragments
Authors: Reid DM , Jones CE , Luo CY , Shulman NR
Published: October 2019

Our previous finding that normal serum immunoglobulins bind to internal platelet proteins on Western blots led us to further identify these proteins and determine the possible significance of autoantibodies against them. A 95-Kd protein reactive with immunoglobulins in most normal sera and easily confused with gpIIIa was shown to be a fragment of vinculin generated by calpain proteolysis. Identity was established by peptide sequencing of the protein purified from platelets stored without specific protease inhibitors. Normal immunoglobulins bound intact vinculin (117 Kd) and metavinculin (152 Kd), and their 105-, 95- , and 80- to 85-Kd proteolytic fragments. IgG in 89%, and IgA and IgM in 100% of normal sera reacted in titers of 10 to 1,000 with purified vinculin. In addition, IgG in 79%, and IgA and IgM in 93% of normal sera reacted in titers of 10 to 5,000 with talin (235 Kd), another cytoskeletal protein, and its 200-Kd proteolytic fragment. IgGs in sera from animals of several different phylogenetic classes also reacted with human vinculin and talin on Western blots. Frequency of occurrence, titers, and classes of antivinculin and antitalin autoantibodies in patients with thrombocytopenia did not differ discernibly from those of normal individuals. These antibodies had no effect on platelet aggregation or clot retraction, and no apparent pathogenic significance, but their widespread presence and the variability in extent of proteolysis of platelet preparations used for Western blots can complicate interpretation of patterns obtained with sera from patients with presumed immune-mediated thrombocytopenias.
Effect of uremia on the membrane transport characteristics of red blood cells
Authors: Langsdorf LJ , Zydney AL
Published: October 2019

Even though there is extensive evidence that uremia affects the fragility and deformability of red blood cells (RBCs), essentially all data on the RBC membrane permeability have been obtained with nonuremic blood. Permeability data were obtained for creatinine and uric acid, two metabolites of interest in hemodialysis, using a stirred ultrafiltration device with direct cell- and protein-free sampling. Experiments examined the effects of temperature and suspending phase on solute transport for both normal and uremic blood cells. Creatinine and uric acid transport from normal RBCs at 37 degrees C were characterized by saturation half-times of 40 +/- 10 minutes and 54 +/- 12 minutes, respectively. The corresponding half-times for uremic cells were significantly longer, 94 +/- 26 minutes and 180 +/- 38 minutes. Data indicated that the slower rate of creatinine transport in uremic blood was caused by an alteration in the RBC membrane, while the reduction in uric acid transport was associated with alterations in the uremic plasma. The temperature dependence of the RBC permeability was also much less pronounced for uremic cells for both solutes. These results provide important insights into the effects of uremia on the RBC membrane permeability, and have important implications for dialysis.
9-cis-retinoic acid: effects on normal and leukemic hematopoiesis in vitro
Authors: Sakashita A , Kizaki M , Pakkala S , Schiller G , Tsuruoka N , Tomosaki R , Cameron JF , Dawson MI , Koeffler HP
Published: October 2019

Retinoic acid exhibits effects on the proliferation and differentiation of many hematopoietic cells. Cellular responsiveness to retinoic acid (RA) is conferred through two distinct classes of nuclear receptors, the RA receptors (RARs) and the retinoid X receptors (RXRs). The RARs bind to both 9-cis- and all-trans-RAs, but 9-cis-RA alone directly binds and activates RXR. This suggested that 9-cis-RA could have expanded hematopoietic activities as compared with all-trans-RA. We compared the abilities of 9-cis- and all-trans-RAs to induce differentiation and inhibit proliferation of three acute myelogenous leukemia (AML) cell lines and fresh leukemic cells from 28 patients and found that: (1) 9-cis-RA in general was more potent than all-trans-RA in suppressing the clonal growth of two AML cell lines and 17 AML samples from patients, including four from individuals with acute promyelocytic leukemia (APL). Eleven leukemic samples, including three from patients with chronic myelogenous or chronic myelomonocytic leukemia, were relatively refractory to both retinoids. (2) The range of activities of both retinoids was similar except that the clonal growth of samples from three AML patients were inhibited by 9-cis-RA, but not by all-trans-RA. (3) Both retinoids inhibited the clonal proliferation of leukemia cells without necessarily inducing their differentiation; in fact, the only fresh AML cells that were able to undergo differentiation were from patients with APL and one individual with M2 AML. (4) Both retinoids enhanced myeloid and erythroid clonal growth from normal individuals, and 9-cis-RA showed slightly more stimulation of the myeloid clonal growth than did the all-trans-RA. Our study suggests that 9-cis-RA is worthy of further study for the treatment of selected individuals with AML.
Transferrin synthesis by mouse lymph node and peritoneal macrophages: iron content and effect on lymphocyte proliferation
Authors: Djeha A , Perez-Arellano JL , Brock JH
Published: October 2019

Transferrin is an essential requirement for lymphocyte proliferation, because it supplies activated lymphocytes with iron needed for cell proliferation. However, during inflammation or an immune response, the iron content of circulating transferrin, which is of hepatic origin, decreases. It is hypothesized that activated lymphocytes may therefore obtain transferrin-iron from an alternative source, and we have investigated the possibility that transferrin is synthesized locally in lymphoid tissues. It was found that lymph node cells from mice stimulated in vivo with Freund's complete adjuvant were able to synthesize transferrin, and this was because of the macrophage rather than the lymphocyte population. Transferrin synthesized by mouse lymph node or peritoneal macrophages contained iron and was able to promote mouse lymphocyte proliferation. Peritoneal macrophages activated in vivo synthesized more transferrin, released more transferrin-bound iron, and were more effective than resident macrophages at enhancing lymphocyte proliferation. These results suggest that transferrin synthesized by macrophages acts in a paracrine manner to support lymphocyte proliferation, thus eliminating possible detrimental effect of hypoferremia on the immune system.
The major regulatory element upstream of the alpha-globin gene has classical and inducible enhancer activity
Authors: Ren S , Luo XN , Atweh GF
Published: October 2019

A major positive regulatory element has recently been identified 40 kb upstream from the human zeta 2-globin gene. This regulatory element increases the expression of a linked alpha-globin gene in mouse erythroleukemia cells and in transgenic mice. This element has been shown to share many of the structural and functional features of the locus control region (LCR) of the beta-globin gene cluster. We have examined the activity of a small fragment from this regulatory domain (alpha LCR) in a transient expression system. We show that this element is active as an enhancer in the erythroid environment of K562 cells. It is somewhat less effective as an enhancer in the nonerythroid environment of HeLa cells. This alpha LCR fragment does not exhibit promoter specificity because it can activate both the promoter of its endogenous target gene and the heterologous promoter of the SV40 early genes. Although the major activity of this element is mediated by its interaction with the promoter of the alpha-globin gene, some increase in activity is seen when structural elements from the 5′ end of the alpha-globin gene are included with the target promoter. In addition, we show that the enhancing activity of the alpha LCR is potentiated by hemin-induction of K562 cells. Whereas phorbol esters that induce megakaryocytic differentiation of K562 cells markedly decrease alpha- globin messenger RNA accumulation, they do not seem to have a negative effect on the activity of the alpha LCR. These studies suggest a role for the alpha LCR in the basal activity of the alpha-globin gene in erythroid cells and in its increased expression seen with erythroid differentiation. The mechanism of negative regulation of alpha-globin gene expression in phorbol-differentiated K562 cells does not appear to be mediated through the action of the alpha LCR.
Quantitative assessment of erythropoiesis and functional classification of anemia based on measurements of serum transferrin receptor and erythropoietin
Authors: Beguin Y , Clemons GK , Pootrakul P , Fillet G
Published: October 2019

We evaluated the quantitative value of a simple model of erythropoiesis, based on the basic assumptions that the red blood cell (RBC) mass determines erythropoietin (Epo) production, which in turn stimulates erythropoietic activity. The RBC mass was quantitated by direct isotopic measurement (RCM), Epo production by serum Epo levels, and erythropoiesis by the ferrokinetic measurement of the erythron transferrin uptake (ETU), the serum transferrin receptor (TfR) level, and the reticulocyte (retic) index, and was completed by an evaluation of overall marrow erythron cellularity. We studied a total of 195 subjects, including 31 normal individuals, 38 patients with polycythemia, and 126 patients with various forms of anemia. Instead of only quantitating Epo and erythropoiesis in absolute terms, we also evaluated them in relation to the degree of anemia or polycythemia, and expressed the results as a ratio of observed values to values predicted from the regression equations between hematocrit (Hct) on the one hand, and Epo, TfR, and ETU on the other, obtained in a carefully selected subpopulation. The slope of the regression of TfR (as well as ETU) versus Hct was very similar to the slope of the regression of Epo versus Hct. Average EPO and TfR (as well as ETU) values predicted from the regression equations were quite comparable to observed values in most groups of subjects, with exceptions predictable from knowledge of the pathophysiology of these hematologic disorders. We identified four major patterns of erythropoiesis, ie, normal, hyperdestruction (with variants of hemolysis or ineffective erythropoiesis), intrinsic marrow hypoproliferation, and defective Epo production. Dissecting out groups of patients showed much greater heterogeneity than when patients were analyzed by group. This was particularly true in the case of a hypoproliferative component being combined with hyperdestruction, giving what we called a “mixed disorder of erythropoiesis.” We conclude that the pathophysiology of anemia can be assessed by a simple measurement of Hct, retic index, Epo, and TfR levels, with Epo and TfR being more informative when expressed in relation to the degree of anemia. The model is particularly useful for detecting the presence of multiple mechanisms of anemia in the same patient. However, it has limitations inherent to the relative invalidity of TfR in iron deficiency, the imprecision of a retic count, and the difficulty in distinguishing hemolysis from ineffective erythropoiesis in some patients and in recognizing a component of hyperdestruction in hypoproliferative anemia.
Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11- dUTP
Authors: Yang G , Ulrich PP , Aiyer RA , Rawal BD , Vyas GN
Published: October 2019

Blood donations are routinely screened by multiple serologic assays for antigens/antibodies associated with infection by blood-borne viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1 and HIV-2), and human T-cell lymphotropic virus (HTLV-I and HTLV-II). A direct detection of these viruses would be more effective for the prevention of transfusion- transmitted infections than the indirect measurement of the variable host immune response to these agents. Because the polymerase chain reaction (PCR) for viral gene amplification offers the most sensitive and direct means of detecting viruses in blood, we have developed a nonisotopic PCR procedure for the detection of HBV, chosen as a prototype. The problems, common to previously described PCR methods, of nucleic acid extraction and inhibition of the PCR by plasma proteins were overcome by isolation of HBV from plasma by means of 450-microns polystyrene beads covalently coated with monoclonal antibody to the Pre- S1 region of the viral envelope protein. Detergent lysis and proteinase K digestion of the immunocaptured virions isolated from plasma released the HBV DNA. A modified PCR-amplification protocol, incorporating digoxigenin-labeled dUTP in the amplified gene products followed by hybridization with a specific biotinylated oligonucleotide probe bound to streptavidin-coated 2.8-microns magnetic beads, allowed flow cytometric analyses of HBV-specific PCR products by means of antibodies to digoxigenin labeled with fluorescein isothiocyanate. The endpoint serial dilutions of pedigreed human plasma samples containing chimpanzee infectious dose (CID50) of 10(7) for adw and CID50 of 10(7.5) for the ayw subtypes were compared in repeated testing of PCR products by our immunoreactive bead (PCR-IRB) assay. HBV DNA was consistently detected in a 5 x 10(-10) dilution of each sample. In testing 20 coded specimens of blood donors, with or without serologic markers of HBV infection, the PCR-IRB was specific and more sensitive than the PCR analyses by slot blot hybridization with radioactive probe. The PCR-IRB assay can be adapted for simultaneous detection of multiple blood-borne viruses by an automated flow cytometric analysis system.
Bone marrow transplantation corrects the splenic reticuloendothelial dysfunction in sickle cell anemia
Authors: Ferster A , Bujan W , Corazza F , Devalck C , Fondu P , Toppet M , Verhas M , Sariban E
Published: October 2019

In sickle cell anemia (SCA), the loss of reticuloendothelial function is the result of vasoocclusive events occurring in the spleen. Such asplenia occurs early in the course of the disease and is considered to be permanent in late childhood. In this report, three patients 10, 11, and 14 years of age suffering from severe SCA and found to be asplenic were treated by bone marrow transplantation (BMT). Before transplantation, all three patients had loss of reticuloendothelial splenic function, as assessed by the presence of abundant Howell-Jolly bodies on blood smears and absence of technetium 99m (99mTc) splenic uptake. After BMT, Howell-Jolly bodies disappeared from blood smear, whereas 99mTc isotopic scan found normal isotope uptake. Our data indicate that BMT can correct “permanent asplenia” in SCA patients. However, it remains to be determined if such treatment can also correct other SCA-related organ dysfunctions.
Interleukin-11 acts as an autocrine growth factor for human megakaryoblastic cell lines
Authors: Kobayashi S , Teramura M , Sugawara I , Oshimi K , Mizoguchi H
Published: October 2019

The cytokine interleukin-11 (IL-11) promotes normal human megakaryocytopoiesis in vitro. However, its role in abnormal megakaryocytopoiesis is not well known. Accordingly, we studied its effects on human megakaryoblastic cell lines CMK and Meg-J. IL-11 stimulated the proliferation of CMK and Meg-J in a dose-dependent manner with maximal growth being achieved at the concentration of 50 and 500 ng/mL, respectively. The growth of the cells was inhibited by anti-IL-11 antibody and IL-11 antisense oligonucleotides. IL-11 transcripts were detected in these two cell lines using a reverse transcriptase-polymerase chain reaction assay. These findings indicate that IL-11 might be an autocrine growth factor for megakaryoblastic cells. IL-11 transcripts also existed in other leukemia cell lines: HL- 60, U937, and K562. However, the growth of these cells was not stimulated by IL-11, and was not inhibited by IL-11 antisense oligonucleotides. These results suggested that IL-11 might regulate malignant cells of the megakaryocytic lineage, in part by an autocrine loop.
Maternal administration of granulocyte colony-stimulating factor improves neonatal rat survival after a lethal group B streptococcal infection
Authors: Novales JS , Salva AM , Modanlou HD , Kaplan DL , del Castillo J , Andersen J , Medlock ES
Published: October 2019

Maternally administered recombinant human granulocyte colony- stimulating factor (rhG-CSF) has been shown to cross the placenta and induce a peripheral neutrophilia and increases in the marrow and spleen neutrophil storage pools in fetal and newborn rats. In the present study, we have used this model system to investigate the efficacy of prenatally administered rhG-CSF on neonatal defense to a lethal challenge with Group B-beta hemolytic Streptococcus (GBS). Pregnant rats were injected with rhG-CSF twice daily beginning 6 days before parturition. At birth, all pups were infected with a dose of GBS that is lethal for 90% of infected pups (LD90). Survival was monitored daily for 5 days. Survival of infected pups from saline-treated mothers beyond 60 hours after infection was 10%. No difference in survival was observed among pups from mothers treated 2 and 4 days before parturition. In contrast, we determined that survival was 82.5% among infected pups from mothers treated for 6 days before parturition with rhG-CSF. Our results demonstrate that maternal administration of rhG- CSF augments neonatal defenses against a lethal bacterial challenge.
Human multipotential progenitor cells (CFU-GEMM) have extensive replating capacity for secondary CFU-GEMM: an effect enhanced by cord blood plasma
Authors: Carow CE , Hangoc G , Broxmeyer HE
Published: October 2019

The replating capability of human umbilical cord blood (CB) multipotential (CFU-GEMM) progenitors was assessed in vitro as an estimate of self-renewal using erythropoietin (Epo), steel factor (SLF), and either fetal bovine serum (FBS) or CB plasma. This study found a much higher replating efficiency for CB CFU-GEMM than previously reported, in terms of the percentage of colonies that could be replated, the number of secondary colonies per replated primary colony, and the size of secondary colonies. Moreover, the majority of secondary colonies were CFU-GEMM-derived. Although the percentages of bone marrow CFU-GEMM that replate was similar to that for CB CFU-GEMM and the sizes of secondary bone marrow and CB CFU-GEMM were also similar, replated CB CFU-GEMM gave rise to far greater numbers of secondary colonies. No tertiary colonies were observed when secondary CFU-GEMM were replated. Detection of extensive secondary replating potential was enhanced by the addition of CB plasma to the cultures. This activity was not found in either adult blood (PB) plasma, umbilical cord vein endothelial cell-conditioned medium (ECCM), FBS plus ECCM, or FBS plus the combination of interleukin-1 (IL-1), IL-3, IL-6, IL-11, granulocyte colony-stimulating factor, and granulocyte- macrophage colony-stimulating factor. Whether the CB plasma-enhancing activity for CFU-GEMM replating capacity is attributable to a novel factor or factors, or represents effects of other known cytokines, alone or in combination, remains to be determined. Of particular relevance, these studies suggest that human CFU-GEMM have some degree of stemness and perhaps should be classified as a subset of stem cells.
Red blood cell regeneration induced by subcutaneous recombinant erythropoietin: iron-deficient erythropoiesis in iron-replete subjects [see comments]
Authors: Brugnara C , Chambers LA , Malynn E , Goldberg MA , Kruskall MS
Published: October 2019

Limited red blood cell (RBC) regeneration often prevents collection of sufficient blood from autologous donors. We studied the effects of subcutaneous recombinant erythropoietin (rEPO) in subjects making frequent blood donations. Six healthy iron-replete male subjects took rEPO (200 U/kg) subcutaneously daily, and donated blood (450 mL) twice a week for 3 weeks. During a control study, these subjects also attempted twice-weekly blood donations without rEPO. Four other males given rEPO, including one with idiopathic hemochromatosis, waited until day 8 to begin blood donations. All healthy subjects took oral ferrous sulfate. Subcutaneous rEPO given with blood donations resulted in a marked reticulocytosis (mean peak value 568 +/- 159 x 10(9)/L v 235 +/- 77 x 10(9)/L, control study; P < .05), and enhanced RBC production at 28 days (1,208 +/- 227 mL v 719 +/- 161 mL, P < .05). rEPO in advance of blood donations was slightly less effective in normal subjects (941 +/- 139 mL, P < .05); however, the subject with hemochromatosis produced substantially more RBCs (1,764 mL) than any normal subject. rEPO-treated normal subjects (but not the rEPO-treated patient with hemochromatosis or untreated controls) produced iron-deficient RBCs with elevated zinc protoporphyrin levels and low hemoglobin content. These cells appeared within 1 week of rEPO administration and before laboratory confirmation of depleted iron stores. Thus, subcutaneous rEPO is an effective stimulant of erythropoiesis in nonanemic blood donors. However, in addition to eventual depletion of iron stores, early functional iron deficiency affects response to the drug.
Changes in hematopoiesis-supporting ability of C3H10T1/2 mouse embryo fibroblasts during differentiation
Authors: Nishikawa M , Ozawa K , Tojo A , Yoshikubo T , Okano A , Tani K , Ikebuchi K , Nakauchi H , Asano S
Published: October 2019

To investigate the functional change of stromal cells along with differentiation, we used a differentiation-inducible mouse embryo fibroblast cell line, C3H10T1/2 (10T1/2). Stably determined preadipocyte and myoblast cell lines were established after a brief exposure of 10T1/2 cells to 5-azacytidine. These cell lines terminally differentiated into adipocytes and myotubes, respectively, under appropriate conditions. The hematopoiesis-supporting ability of each 10T1/2-derived cell line was examined by coculture with FACS-sorted murine hematopoietic stem cells (Thy-1lo c-kit+ Lin-). The number of granulocyte-macrophage progenitors (CFU-GM) was slightly reduced after 7 days of culture with parent 10T1/2 fibroblasts, whereas a marked increase in CFU-GM number was observed when the cells were cultured on preadipocytes. Mature adipocytes and myogenically determined cell lines, on the other hand, did not support CFU-GM growth. Further, Northern analysis showed that the preadipocyte cell line acquired the ability to produce a significant amount of stem cell factor (SCF), interleukin-6 (IL-6), leukemia inhibitory factor, and macrophage colony- stimulating factor mRNAs in response to IL-1 or lipopolysaccharide stimulation. Terminal adipocytic differentiation resulted in reduced ability to express these cytokine mRNAs. Similarly, highest IL-6 activity was detected in the supernatant of preadipocyte culture, whereas adipocytes did not secrete IL-6 even after IL-1 stimulation. Interestingly, hematopoiesis-nonsupporting myoblasts and myotubes also expressed abundant SCF mRNA, suggesting that SCF, per se, may not be sufficient for stem cell growth and survival. The 10T1/2-derived cell lines could provide a valuable tool to aid in the analysis of stromal cell development and the search for novel stromal factors.
Lack of CD24 antigen expression in B-lineage acute lymphoblastic leukemia is associated with intrinsic radiation resistance of primary clonogenic blasts
Authors: Uckun FM , Song CW
Published: October 2019

The radiation sensitivity of primary clonogenic blasts from 27 children with immunologically classified CD2-CD5-CD7-CD19+slg- B-lineage acute lymphoblastic leukemia (ALL) was analyzed using leukemic progenitor cell (LPC) colony assays. Radiation survival curves of primary clonogenic blasts (ie, LPC) were constructed for each patient using computer programs for the single-hit multitarget as well as the linear quadratic models of cell survival. The D0 values ranged from 49 to 891 cGy (median, 239 cGy; mean +/- SE, 307 +/- 44 cGy) and the alpha values ranged from 0.000 to 2.047 Gy-1 (median, 0.156 Gy-1; mean +/- SE, 0.284 +/- 0.078 Gy-1). Patients were divided into groups according to sex, age, white blood cell count (WBC) at diagnosis, plating efficiency of primary bone marrow blasts, and immunophenotype. Patient sex, age, WBC at diagnosis, or in vitro plating efficiency was not associated with radiation resistance. Notably, freshly isolated primary clonogenic blasts from patients with a CD19+CD24-CD34+ composite immunophenotype (stage I B-cell precursor [BCP]) had 2.2-fold higher D0 values (P = .005) and 3.4-fold lower alpha values (P = .05) than those from patients with a CD19+CD24+CD34+ (stage II BCP) or CD19+CD24+CD34- (stage III BCP) immunophenotype. The relative values for CD24- primary clonogenic blasts signify greater intrinsic radiation resistance. Furthermore, the CD19+CD24-CD34+ immunophenotype had a larger radiation resistant fraction. Whereas only 60% of CD19+CD24+CD34+ and 33% of CD19+CD24+CD34- cases had clonogenic blasts with alpha < or = 0.2, 100% of CD19+CD24-CD34+ cases had clonogenic blasts with alpha < or = 0.2. Furthermore, clonogenic blasts from established CD19+CD24- B-lineage ALL cell lines were significantly more radiation resistant than CD19+CD24+ B-lineage ALL cell lines. Notably, radiation doses sufficient to induce apoptosis of CD24- B-lineage ALL cells were higher than those capable of inducing apoptosis in CD24+ B-lineage ALL cells. Thus, the lack of CD24 surface antigen expression in B-lineage ALL is associated with intrinsic radiation resistance at the level of primary clonogenic blasts.
Expression of multidrug resistance P-glycoprotein on peripheral blood mononuclear cells of patients with granular lymphocyte-proliferative disorders
Authors: Yamamoto T , Iwasaki T , Watanabe N , Oshimi K , Naito M , Tsuruo T , Kobayashi Y
Published: October 2019

Some patients with granular lymphocyte-proliferative disorders (GLPD) have been reported to have an aggressive clinical course with a poor prognosis and to be refractory to chemotherapy. In this study, expression of multidrug resistance P-glycoprotein on peripheral blood mononuclear cells (PBMC) of 11 patients with GLPD was examined by staining with MRK 16, a monoclonal antibody that binds to an external epitope of P-glycoprotein, and with the dye rhodamine, a known substance to be excreted from the cells through P-glycoprotein. Among those tested, the PBMC of six of eight patients with T-cell-type GLPD as well as those of three of three patients with natural killer cell- type GLPD expressed P-glycoprotein significantly. Furthermore, PBMC of two of two patients were also poorly stained with the dye rhodamine, and the treatment of PBMC with either verapamil or quinidine, multidrug resistance-reversing agents, led to their increased staining, suggesting that these PBMC actively release chemotherapeutic agents.
Interleukin-4 protects double-negative and CD4 single-positive thymocytes from dexamethasone-induced apoptosis
Authors: Migliorati G , Nicoletti I , Pagliacci MC , D'Adamio L , Riccardi C
Published: October 2019

Glucocorticoid hormones (GCH) and anti-CD3 monoclonal antibodies (MoAbs) induce in mouse thymocytes and T-cell tumor lines an active process of cell death called apoptosis. Interleukins (IL), including IL- 1 and IL-2, have been reported to inhibit such apoptosis. In this study we show that IL-4 also reduced the DNA fragmentation characteristic of dexamethasone (DEX)-induced apoptosis in thymocytes. This effect, studied in both time-course and dose-response experiments, was also detected at low IL-4 concentrations (1 U/mL) and against high DEX levels (10(-7) mol/L). The effect of IL-4 was blocked by an anti-IL-4 but not by an anti-IL-1 alpha MoAb, and was thus both specific and direct. Phenotypic analysis showed that IL-4 protects predominantly CD4- CD8- and CD4+CD8- cells. Our findings suggest that intrathymic T-cell development may be influenced by IL-4.
Establishment and characterization of a new granulocyte-macrophage colony-stimulating factor-dependent and interleukin-3-dependent human acute myeloid leukemia cell line (GF-D8)
Authors: Rambaldi A , Bettoni S , Tosi S , Giudici G , Schiro R , Borleri GM , Abbate M , Chiaffarino F , Colotta F , Barbui T
Published: October 2019

A novel factor-dependent human myeloid leukemia cell line (GF-D8) was established from the peripheral blood of an 82-year-old man suffering from acute myeloblastic leukemia (AML). By morphology, cytochemical staining, and analysis of surface antigens, GF-D8 cells are myeloblasts of immature progenitor origin. The consensus karyotype is 45, XY, -5, 7q-, inv(7) (q31.2q36), 8q+, +8q+, 11q+, 12p-, -15, -17, + marker. The long-term survival and proliferation of GF-D8 cells is dependent on the presence of either granulocyte-macrophage colony-stimulating factor (GM- CSF) or interleukin-3 (IL-3). Weak colony growth was observed after exposure of GF-D8 cells to stem cell factor (SCF) but not after exposure to granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), IL-1 beta, IL-2, IL-5, or tumor necrosis factor-alpha (TNF-alpha). GM-CSF- and IL- 3-induced proliferation is dose dependent, with significant growth observed at concentrations as low as 0.1 ng/mL, but the combination of both factors has no synergistic effect. A significant proliferation is induced by GM-CSF and IL-3 even in serum-deprived cultures, although with a slightly decreased efficiency. GF-D8 cells were shown to express specific messenger RNAs for the alpha chains of the GM-CSF and IL-3 receptors as well as for the beta chain, common to both receptors. Interestingly, despite the absence of biologic response to G-CSF, specific transcripts for the G-CSF receptor gene were similarly identified by reverse polymerase chain reaction analysis. GF-D8 cells represent a useful tool for studying chromosome abnormalities of human AML as well as the regulation of myeloid proliferation and differentiation in vitro.
Alternative mechanisms with and without steel factor support primitive human hematopoiesis
Authors: Sutherland HJ , Hogge DE , Cook D , Eaves CJ
Published: October 2019

As a first approach to defining the molecular requirements for supporting human hematopoietic stem cell maintenance and differentiation in vitro, we have analyzed and compared the ability of various factors to support the maintenance and initial differentiation of human long-term culture-initiating cells (LTC-ICs), a distinct, rare primitive hematopoietic cell type whose progeny after 5 weeks include cells detectable as colony-forming cells. Normal human marrow cells highly enriched in LTC-ICs (approximately 1% pure) were placed in cultures containing either preestablished, irradiated human marrow adherent feeder layers, or feeders consisting of Steel factor-deficient SI/SI, or normal +/+ murine fibroblasts, or no feeders. In some groups, either Steel factor alone, granulocyte colony-stimulating factor (G- CSF) and interleukin-3 (IL-3), or all three factors combined were also added repeatedly. SI/SI murine fibroblasts were equivalent to +/+ controls and to normal human marrow feeders in supporting both LTC-IC maintenance and clonogenic cell output over a 5-week period. Soluble Steel factor alone could, however, effectively substitute for human marrow feeders to support LTC-IC maintenance, although clonogenic cell output was markedly reduced under these conditions. Conversely, soluble Steel factor with G-CSF and IL-3 or with feeders (or all together) did not further enhance (or depress) LTC-IC maintenance, although under these conditions clonogenic cell output was markedly increased. These findings confirm previous evidence that LTC-IC maintenance and clonogenic cell production are differentially regulated and show for the first time that LTC-IC maintenance can be supported by different nonsynergizing factors that may, but need not, include Steel factor.
Purified murine hematopoietic stem cells function longer on nonirradiated W41/Wv than on +/+ irradiated stroma
Authors: Vecchini F , Patrene KD , Boggs SS
Published: October 2019

Mouse bone marrow (BM) was separated into low-density, lineage- negative, wheat germ agglutinin-positive (WGA+), Rhodamine-123 bright (Rhbright) or dim (Rhdim) cells to obtain populations that were highly enriched for committed progenitors (Rhbright cells) or for more primitive stem cells (Rhdim). When 2,500 Rhbright or Rhdim cells were seeded onto 6-week-old irradiated (20 Gy) long-term BM cultures (LTBMC), the nonadherent cell production from Rhbright cells was transient and ended after 5 weeks. Production from Rhdim cells did not begin until week 3, peaked at week 5, and ended at week 8, when the irradiated stroma seemed to fail. Termination of cell production from Rhdim cells did not occur in nonirradiated LTBMC from W41/Wv mice. During peak nonadherent cell production, 25% to 30% of the cells in the nonirradiated LTBMC from W41/Wv mice had donor cell markers. Two approaches were tested to try to enhance the proportion or number of donor cells. Addition of Origen-HGF at the time of seeding Rhdim cells caused a nonspecific increase in both host and donor cell production, but a specific increase in production of donor cells was obtained by seeding the cultures at 2 weeks rather than 6 weeks. Limiting dilution of Rhdim cells gave the same frequency of wells producing cells on both irradiated +/+ and nonirradiated W41/Wv or W/Wv cultures.
Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo
Authors: Quesniaux VF , Graham GJ , Pragnell I , Donaldson D , Wolpe SD , Iscove NN , Fagg B
Published: October 2019

A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.
Expression of perforin and membrane-bound lymphotoxin (tumor necrosis factor-beta) in virus-specific CD4+ human cytotoxic T-cell clones
Authors: Yasukawa M , Yakushijin Y , Hasegawa H , Miyake M , Hitsumoto Y , Kimura S , Takeuchi N , Fujita S
Published: October 2019

In an attempt to clarify the mechanisms of cytotoxicity mediated by CD4+ cytotoxic T lymphocytes (CTL), the expression of perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-beta) in herpes simplex virus (HSV)-specific CD4+ human cytotoxic and noncytotoxic T- cell clones was examined. Three HSV-specific CD4+ human CTL clones that showed HLA-DR-restricted cytotoxicity and proliferative response were established. The cytotoxicity of these clones in 5-hour 51Cr release assays was found to be mediated by the directional target cell lysis and not by the release of cytotoxic soluble factors, ie, “innocent bystander” killing. Northern blot analysis showed that messenger RNAs for perforin and LT, which were both considered to be important mediators for cytotoxicity of CD8+ CTL and natural killer cells, were abundantly expressed in HSV-specific CD4+ CTL clones. Expression of perforin in the cytoplasm of CD4+ CTL clones was also detected by immunohistochemical staining using a monoclonal antibody against perforin. In addition, LT bound to the cell surface of CD4+ CTL clones was detected by flow cytometry. In contrast, little or no expression of perforin and LT was detected in three HSV-specific CD4+ noncytotoxic T- cell clones. Although the cytotoxicity mediated by lymphokine-activated killer cells was partly inhibited by addition of anti-LT antibody, it did not show any effect on the cytotoxicity of HSV-specific CD4+ CTL clones. In addition, it was found that cytotoxicity mediated by these CD4+ CTL clones was Ca2(+)-dependent. These data thus suggest that perforin and membrane-bound LT are both expressed in HSV-specific CD4+ CTL, although perforin might be the more important mediator in short- term culture.
The 8;21 chromosome translocation in acute myeloid leukemia is always detectable by molecular analysis using AML1
Authors: Maseki N , Miyoshi H , Shimizu K , Homma C , Ohki M , Sakurai M , Kaneko Y
Published: October 2019

The AML1 gene was rearranged in leukemic cells with t(8;21)(q22;q22) or its variant, complex t(8;V;21) translocations from 33 acute myeloid leukemia (AML) patients. The AML1 rearrangement was also detected in three AML patients without t(8;21); two had a normal diploid karyotype, and one had a karyotype of 45,X, - X. The AML1 rearrangement in the t(8;21) breakpoint cluster region was not detected in leukemic cells with cytogenetic abnormalities other than t(8;21), or with normal diploidy obtained from 23 AML patients. Because leukemic cells of the five patients with complex t(8;V;21) translocations had a der(8)t(8;21) chromosome with a break in band 8q22 in common, the juxtaposition of the 5' side of AML1 to a predicted counterpart gene located in the breakpoint region of 8q22 may be an essential step in the leukemogenesis of AML with t(8;21). Our findings show that the 8;21 translocation, its variants, and the masked t(8;21) may all be detectable by the Southern hybridization method using the AML1 probes.
Cytogenetic clonality in myelodysplastic syndromes studied with fluorescence in situ hybridization: lineage, response to growth factor therapy, and clone expansion
Authors: Anastasi J , Feng J , Le Beau MM , Larson RA , Rowley JD , Vardiman JW
Published: October 2019

Clonality in myelodysplastic syndromes (MDS) has been studied with various techniques including glucose-6-phosphate dehydrogenase (G6PD) isoenzyme and cytogenetic analyses, and with molecular techniques such as gene deletion studies and the analysis of restriction fragment- length polymorphisms (RFLP) of X-linked genes. In this study, we investigated the use of fluorescence in situ hybridization (FISH) with a chromosome-specific probe to examine cytogenetic clonality in peripheral blood (PB) cells from three patients with MDS. In each case, trisomy 8 was shown by conventional cytogenetic analysis at the time of the initial diagnosis. By using FISH with a probe for the centromere of chromosome 8, we identified the trisomy in individual PB cells from Wright-stained smears. With this technique, we could determine the cell lineage involved by the trisomy, and through serial analyses we could assess the response of the clonal and nonclonal cells to growth-factor therapy, and the expansion of the trisomic clone over time. In each of the three cases, various proportions of granulocytes, monocytes, eosinophils, and basophils showed trisomy 8 by FISH analysis. In none of the cases did we detect trisomy 8 in lymphocytes. By analysis of PB cells before and during therapy with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), we found that GM-CSF stimulated both trisomic and disomic cells. During a 1-year period of sequential study, we detected an abrupt increase in the percentage of trisomic cells in one patient, a stable percentage in another, and a slowly increasing percentage in the third. The abrupt increase in the first patient preceded a transformation to a more acute phase by 2 months. We conclude that FISH analysis of PB cells of patients with MDS offers an additional approach to the study of clonality in this disorder. In some cases this analysis may provide a useful and simple means of assessing response to therapy and progression of disease.
Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins
Authors: Holness CL , Simmons DL
Published: October 2019

CD68 is a 110-Kd transmembrane glycoprotein of unknown function highly expressed by human monocytes and tissue macrophages. We have isolated cDNA clones encoding CD68 from a U937 cDNA library by transient expression in COS cells and panning with the anti-CD68 monoclonal antibodies (MoAbs) Y2/131, Y1/82A, EBM11, and Ki-M6. CD68 transcripts are constitutively present in the promonocyte cell line U937 and are upregulated by phorbol myristic acid (PMA). By contrast, CD68 transcripts are absent or present at very low levels in many hematopoietic lines including KG1, CEM, and K562, but can be induced by exposure to PMA. The cDNA sequence predicts a type I integral membrane protein of 354 residues with a heavily glycosylated extracellular domain of 298 residues containing nine potential N-linked glycosylation sites and numerous potential O-linked glycosylation sites. The extracellular domain consists of two distinct regions separated by an extended proline hinge: a membrane-distal mucin-like domain containing short peptide repeats and consisting of 54% serine and threonine residues; and a membrane proximal domain that has significant sequence homology to a family of lysosomal/plasma membrane shuttling proteins known as the lamp 1 group. CD68 is a member of a growing family of hematopoietic mucin-like molecules, including leukosialin/CD43, the stem cell antigen CD34, and the lymph node high endothelial ligand for L-selectin GlyCAM-1.
Molecular characterization of a novel form of (A gamma delta beta)zero thalassemia deletion in a Chinese family
Authors: Zhang JW , Song WF , Zhao YJ , Wu GY , Qiu ZM , Wang FN , Chen SS , Stamatoyannopoulos G
Published: October 2019

We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that is associated with elevated fetal hemoglobin in the adult. The propositus is a homozygote from the Yunnan province of China. The deletion spans about 90 kb of DNA and removes the A gamma, delta, and beta-globin genes. The 5' breakpoint of the deletion is located about 0.13 kb upstream from the A gamma-globin gene, whereas the 3' breakpoint is located about 66 kb downstream from the beta-globin gene, about 13 kb upstream from the breakpoint of the Chinese (A gamma delta beta)zero-thalassemia. Heterozygotes for this Yunnanese form of (A gamma delta beta)zero-thalassemia express between 9% and 17% of fetal hemoglobin, whereas the homozygote present with a mild anemia (Hb = 10.7 g/dl). Comparison of the sites of 3' breakpoints of the Yunnanese and the Chinese (A gamma delta beta)zero-thalassemia mutants is compatible with the hypothesis that an enhancer element is located between the 3' breakpoints of these two mutants. Juxta-position to the G gamma gene of this element may be responsible for the efficient gamma-gene expression in the Yunnanese mutant.
Human T-cell leukemia virus type II infection frequently goes undetected in contemporary US blood donors
Authors: Hjelle B , Wilson C , Cyrus S , Bradshaw P , Lo J , Schammel C , Wiltbank T , Alexander S
Published: October 2019

Serologic screening for human T-cell leukemia virus type I (HTLV-I) infection was begun in US blood banks with the licensure of enzyme- linked immunosorbent assays (ELISA) in December 1988. We examined the donation histories of the first 60 Western blot (WB)-confirmed HTLV- I/II positive donors to one blood center and found 8 had made 16 previous donations that scored negative on the screening ELISA. All 16 donations had ELISA absorbance below the cutoff for a positive assay, but still well above that of the average donation (17.6% +/- 5.7% of the cutoff). In a more extensive study, 17 donations from a total of 61,752 at six blood centers were both ELISA-positive and WB-positive for HTLV-I (4) or HTLV-II (13), and 218 samples had ELISA absorbance greater than 50% of the ELISA cutoff. One hundred seventy-eight of the 218 were tested further by WB and 11 were found positive. All 11 positives were confirmed by polymerase chain reaction; 10 had HTLV-II and 1 had HTLV-I. Thus, the HTLV-I-based screening ELISA missed at least 10 of 23, or 43% (95% confidence interval, 23% to 66%), of HTLV- II infections, compared with 1 of 5, or 20%, of HTLV-I infections.
Sequential analysis of hematopoietic reconstitution achieved by transplantation of hematopoietic stem cells
Authors: Okada S , Nagayoshi K , Nakauchi H , Nishikawa S , Miura Y , Suda T
Published: October 2019

We confirmed that murine hematopoietic stem cells express the c-kit molecule but not lymphohematopoietic lineage markers. These lineage marker-negative c-kit-positive (Lin- c-kit+) cells were further divided according to the uptake of rhodamine-123 (Rh-123). Approximately 1,000 Lin- c-kit+ rhodamine-123dull cells, which contained 4.0 +/- 1.3 and 12.5 +/- 1.9 day 8 and day 12 spleen colony-forming units (CFU-S), respectively, rescued the 100% of lethally irradiated mice. One third of these cells formed colonies in the presence of interleukin-3 plus erythropoietin. The time course of the hematopoietic reconstitution of this primitive hematopoietic stem cell fraction was investigated by using Ly-5 congenic mice. Although myeloid cells and B lymphocytes were detected in the peripheral blood 2 to 3 weeks after transplantation, T lymphocytes were not detected until 4 weeks after transplantation. It is generally assumed that myeloid cells and B lymphocytes grow in the bone marrow and that T lymphocytes must pass through the thymus. For the first 2 to 3 weeks after transplantation, donor-type T lymphocytes were not dominant in the thymus, and most donor type cells were CD4/CD8 double-negative or double-positive (including CD4low and CD8low). Four weeks after transplantation, donor-type T lymphocytes were dominant and the ratio of CD4/CD8 cells had recovered to the normal pattern. However, significant numbers of T lymphocytes were detected in the peripheral blood at this stage. Sequential analysis of hematopoietic reconstitution from primitive stem cells demonstrates that myeloid and B-lymphoid lineages occurred earlier than that of the T-lymphoid lineages.