bluuebunny/crossref_metadata_2025 huggingface dataset:
Blunted erythropoietin production and decreased erythropoiesis in early pregnancy [see comments]
Authors: Beguin Y , Lipscei G , Thoumsin H , Fillet G
Published: October 2019

After decreasing in the first trimester of pregnancy, the total red blood cell mass increases in the second and third trimesters to peak at term at about 120% to 125% of nonpregnant values, but how this is brought about by changes in the rate of erythropoiesis is not known. We evaluated erythropoiesis by measuring serum transferrin receptor (TfR) levels in 406 women during normal pregnancy (N = 317), at delivery (N = 63), or in the early postpartum (N = 27). Despite the presence of the placenta and the frequent occurrence of iron deficiency, TfR levels remained low in the first two trimesters and increased in the third trimester and at delivery. To explain why erythropoiesic activity was relatively low in early pregnancy, we also measured serum immunoreactive erythropoietin (Epo) in relation to the degree of anemia. There was a very strong correlation between serum TfR and Epo levels in the entire group (r = .59, P less than .0001) as well as in each period of pregnancy. Epo levels remained low for the degree of anemia and did not correlate with hematocrit in the first two trimesters, but recovered afterwards. In the early postpartum, Epo production and erythropoiesis were normal. We conclude that: (1) erythropoiesis is decreased in the first part of pregnancy but increases afterwards; and (2) blunted Epo production in early pregnancy could be responsible for that observation.
Complement activation in cancer patients undergoing immunotherapy with interleukin-2 (IL-2): binding of complement and C-reactive protein by IL-2-activated lymphocytes
Authors: Vachino G , Gelfand JA , Atkins MB , Tamerius JD , Demchak P , Mier JW
Published: October 2019

Plasma samples from cancer patients undergoing immunotherapy with high- dose recombinant interleukin-2 (IL-2) were obtained over a 5-day course of treatment and assayed by radioimmunoassay or enzyme-linked immunosorbent assay for the complement degradation products, C3a, iC3b, Ba, Bb, C4d, and SC5b-9. In the majority of patients, pretreatment C3a, Ba, Bb, and SC5b-9 plasma levels were comparable with those measured in normal donor plasma. However, by the end of the 5-day treatment course, C3a levels had increased 15.6-fold. In several patients, peak concentrations of C3a were as high as those reported in patients with sepsis or burn injury. Plasma levels of alternative pathway components Ba and Bb also increased, 8.0- and 5.0-fold, respectively, during IL-2 treatment. Likewise, levels of one of the terminal complexes, SC5b-9, increased 5.0-fold and the plasma C4d and iC3b concentrations increased 4.8- and 2.9-fold, respectively, by the fifth day of treatment. To determine whether activated lymphocytes participate in IL-2-induced complement activation, peripheral blood mononuclear cells (PBMC) obtained from IL-2 recipients before and 5 days after beginning therapy were reacted with monoclonal antibodies (MoAbs) against C3c and the terminal complement complex SC5b-9. Dual-color cytofluorographic analysis showed that within the CD3(+) population, the percentage of cells binding the anti-C3c and anti-SC5b-9 MoAbs increased 6.2-fold and 5.1-fold, respectively, by day 5. The anti-C3c MoAb also bound to CD3(+) cells stimulated in vitro with IL-2 and then exposed to serum. Moreover, fluid-phase iC3b was generated from purified C3 by PBMC activated in vitro with IL-2, but not by unstimulated cells. Serum levels of C-reactive protein (CRP) are markedly elevated in patients undergoing IL-2 immunotherapy. This hepatic acute phase reactant has been shown to activate the classical pathway when bound to cell surfaces. Because levels of the classical component C4d increase markedly during IL-2 treatment, we sought to determine if CRP became bound to PBMC during IL-2 treatment and found that during therapy, the percentage of CD3(+) cells reactive with an anti-CRP MoAb increased from less than 2% to greater than 18%. When PBMC were activated with IL- 2 in vitro and then exposed to exogenous CRP, greater than 20% of the CD3(+) cells reacted with the anti-CRP MoAb.(ABSTRACT TRUNCATED AT 400 WORDS)
Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse
Authors: Ploemacher RE , van der Sluijs JP , van Beurden CA , Baert MR , Chan PL
Published: October 2019

We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal “feeders” depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.
Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils
Authors: Yamaguchi Y , Suda T , Ohta S , Tominaga K , Miura Y , Kasahara T
Published: October 2019

We and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5-mediated survival. In the absence of human IL-5 (hIL-5) mature eosinophils succumbed after 4 days, while in the presence of hIL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hIL-5, while no significant DNA fragmentation was observed in the culture with hIL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hIL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hIL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis.
Cloning of the gene encoding the human erythropoietin receptor
Authors: Maouche L , Tournamille C , Hattab C , Boffa G , Cartron JP , Chretien S
Published: October 2019

The genomic and complementary DNAs of the human erythropoietin receptor (hEpo-R) have been isolated and characterized from a genomic placental library and from two cDNA libraries prepared from bone marrow and fetal liver. The five different partial cDNAs isolated were aberrant in the predicted reading frames as compared with the Epo-R protein sequence, because all retained insert sequences that may represent splicing intermediates (three clones), cloning artifact (one clone), or a new sequence at a splice junction (one clone) of the gene. The cDNAs were used to isolate several genomic clones encompassing the complete hEpo-R gene. This gene, which encodes a 508-amino acid polypeptide chain of predicted M(r) 55,000, is organized into eight exons spread over 6 kb of DNA and exhibited a high degree of sequence homology (81.6% in the coding region) and structural organization with its murine counterpart. Primer extension analysis indicated that the transcription initiation site is located 141 bp upstream of the initiation codon. Sequence homology 320 bp upstream of the cap site was significantly lower (60%) and diverged completely further upstream as compared with the murine gene. Similarly, the human and murine sequences were largely divergent downstream of the stop codon, indicating that a strong conservation during evolution was restricted to the coding sequence of the Epo-R protein. The 320-bp region upstream of the cap site does not contain the typical TATA or CAAT boxes present in many tissue-specific genes, but does include potential binding sites for the ubiquitous Sp1 and the erythroid-specific GATA-1 trans-activating factors. These boxes are well conserved in sequence and position relative to the cap site within the promoter region of the human and murine genes, but the CACCC boxes present in the murine gene are absent in the human gene.
Inhibition of human erythroid colony-forming units by gamma interferon can be corrected by recombinant human erythropoietin [see comments]
Authors: Means RT Jr , Krantz SB
Published: October 2019

Tumor necrosis factor (TNF), interleukin-1 (IL-1), and gamma interferon (gamma IFN) inhibit erythropoiesis in vivo and in vitro, and have been implicated in the pathogenesis of the anemia of chronic disease. Anemia in patients with rheumatoid arthritis and in animals exposed chronically to IL-1 and TNF can be corrected by the administration of recombinant erythropoietin (Epo). We exposed highly purified human erythroid colony-forming units (CFU-E) cultured from peripheral blood burst-forming units-erythroid (BFU-E) and unpurified human marrow CFU-E to recombinant human gamma IFN and showed inhibition of colony formation in vitro. This inhibition was reversed by increased concentrations of Epo. The mechanisms by which this effect occurs are unknown at present. Epo may cause a downregulation of gamma IFN receptor expression on CFU-E or, alternatively, gamma IFN may cause a downregulation of Epo receptor expression. A full understanding of these mechanisms awaits a more complete comprehension of the regulation of erythropoiesis; however, the effect of Epo in vitro is similar to its ability to correct the anemia of chronic disease in vivo.
Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking [see comments]
Authors: Coller BS , Seligsohn U , West SM , Scudder LE , Norton KJ
Published: October 2019

To assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the alpha v beta 3 vitronectin receptor to platelet levels of fibrinogen and vitronectin, we analyzed the platelets from two groups of Glanzmann thrombasthenic patients: Iraqi- Jews, whose platelets lack both receptors, and Arab patients in Israel, whose platelets lack GPIIb/IIIa, but have normal or increased numbers of alpha v beta 3 vitronectin receptors. The platelets from both thrombasthenic groups had profound deficiencies of fibrinogen, but the defect in the Iraqi-Jewish patients' platelets appeared to be slightly more severe. This finding indicates that GPIIb/IIIa is the major determinant of platelet fibrinogen, presumably acting by receptor- mediated uptake, and that the alpha v beta 3 vitronectin receptor plays little or no role. Arab patients' platelets have normal amounts of platelet vitronectin, whereas Iraqi-Jewish patients' platelets have nearly five times as much vitronectin as control or Arab patients' platelets. To account for these data, we propose a working hypothesis in which vitronectin is synthesized in megakaryocytes and the alpha v beta 3 vitronectin receptor is involved in transport of the protein out of megakaryocytes and/or platelets. Collectively, these observations suggest that in addition to their recognized roles in cell adhesion and in the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets.
Establishment of idiotype bearing B-lymphocyte clones from a patient with monoclonal gammopathy
Authors: Osterborg A , Steinitz M , Lewin N , Bergenbrant S , Holm G , Lefvert AK , Mellstedt H
Published: October 2019

B-lymphocyte clones bearing an idiotypic Ig structure, which could also be found on the serum M-component, have been isolated from peripheral blood of a patient with monoclonal gammopathy (MG) using an immune- rosetting technique and limiting dilution. The majority of the cells had the surface phenotype id+/CD19+/CD20+/CD21+ or id+/CD19+/CD20+/CD21- . Some of the clones originated from spontaneous outgrowth of lymphocytes and some were derived from in vitro Epstein-Barr virus- infected lymphocytes. Three of the B-lymphocyte clones and the patient's bone marrow plasma cells appeared to have clonal identity as shown by the same Ig heavy chain gene rearrangements in Southern blot analysis using a JH probe. The study further supports the notion that the peripheral blood of patients with MG contains B lymphocytes that belong to the tumor clone.
Treatment with interleukin-3 plus granulocyte-macrophage colony- stimulating factors improves the selectivity of Ara-C in vitro against acute myeloid leukemia blasts
Authors: Bhalla K , Holladay C , Arlin Z , Grant S , Ibrado AM , Jasiok M
Published: October 2019

Hematopoietic growth factors (HGFs) interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) individually have been shown to increase the percentage of acute myeloid leukemia (AML) blasts in S phase and enhance the cytotoxic effects of Ara-C against these blasts in culture. We compared in vitro the effects of a combined treatment with GM-CSF (10 ng/mL) plus IL-3 (10 ng/mL) on the metabolism and cytotoxicity of Ara-C in normal bone marrow mononuclear cells (NBMMC) and AML blasts. NBMMC from six healthy volunteers and AML blasts from 10 patients were incubated for 20 hours with or without IL- 3 plus GM-CSF, followed by a concurrent treatment with Ara-C for 4 additional hours. Exposure to the HGFs and Ara-C produced significantly higher intracellular Ara-CTP levels as well as higher Ara-CTP/dCTP pool ratios in AML blasts as compared with NBMMC. Treatment with HGFs resulted in [3H] Ara-C DNA incorporation that was significantly higher in AML blasts versus NBMMC. This selective improvement of Ara-C metabolism in AML blasts was associated with an enhanced Ara-C-mediated leukemia colony-forming unit (CFU) growth inhibition. In contrast, exposure to HGFs resulted in an improved colony growth of normal CFU granulocyte-monocyte and CFU-granulocyte, erythroid, monocyte, megakaryocyte. These in vitro studies indicate that a combined treatment with IL-3 plus GM-CSF may improve the selectivity of Ara-C against AML blasts.
Eosinophils from patients with blood eosinophilia express transforming growth factor beta 1
Authors: Wong DT , Elovic A , Matossian K , Nagura N , McBride J , Chou MY , Gordon JR , Rand TH , Galli SJ , Weller PF
Published: October 2019

The infiltration of eosinophils into tissues during pathologic responses is often associated with extracellular matrix alterations such as fibrosis. Transforming growth factor-beta 1 (TGF-beta 1) is a well-characterized multifunctional cytokine known to exert potent effects on the extracellular matrix. In this report, we showed the production of TGF-beta 1 by human eosinophils from patients with blood eosinophilia. Northern blot analysis using RNA isolated from eosinophils purified from a patient with the idiopathic hypereosinophilic syndrome (HES) detected the 2.5-kb TGF-beta 1 transcript. In situ hybridization and immunohistochemistry of leukocytes from two patients with HES and two patients with blood eosinophilia localized TGF-beta 1 messenger RNA (mRNA) and protein to eosinophils. No other cell type contained TGF-beta 1 mRNA by in situ hybridization, whereas other leukocytes contained detectable TGF-beta 1 protein by immunohistochemistry. Eosinophils from four normal donors contained little or no detectable TGF-beta 1 protein by immunohistochemistry, whereas eosinophils from two of these four normal donors labeled weakly for TGF-beta 1 mRNA by in situ hybridization. These results show that eosinophils in the peripheral blood of patients with blood eosinophilia can express TGF-beta 1, but that eosinophils in the blood of normal donors contained little or no TGF-beta 1.
Relationship of clinical severity to packed cell rheology in sickle cell anemia
Authors: Phillips G Jr , Coffey B , Tran-Son-Tay R , Kinney TR , Orringer EP , Hochmuth RM
Published: October 2019

There is wide variation in the clinical manifestations of sickle cell disease (SCD) from one affected individual to another. Many investigators have sought to discern parameters that would explain this variability. In the present studies we have attempted to correlate the frequency of painful events and the extent of end organ failure in SCD with rheologic properties of packed suspensions of sickle cells, using a magneto-acoustic ball microrheometer developed in our laboratory. Using this device we have measured the steady-state viscosity, and the viscous and elastic moduli of cell suspensions in 16 individuals with hemoglobin SS disease who were untransfused and in their steady state. The rheologic parameters were then correlated with clinical parameters. The clinical parameters measured were emergency department visits, hospitalizations, hemoglobin, reticulocyte count, age, and end organ failure (nephropathy, avascular necrosis of bone, stroke, retinopathy, resting hypoxemia after acute chest syndrome(s), leg ulcer, and priapism with impotence). The P value for the correlation between the steady state viscosity and end organ failure was .001 with a correlation coefficient (R value) of .73. The P value for the correlation between the viscous modulus of viscosity and end organ failure was .00006 with an R value of .83. The P value for the correlation between the elastic modulus of viscosity and end organ failure was .0006 with an R value of .76. However, there was no significant correlation between any component of packed cell rheology and emergency department visits or hospitalizations for pain.
CD34 expression by stromal precursors in normal human adult bone marrow
Authors: Simmons PJ , Torok-Storb B
Published: October 2019

Normal bone marrow cells were isolated by fluorescence-activated cell sorting (FACS) on the basis of CD34 antigen expression and then assayed in vitro for colonies of fibroblastic cells (fibroblast colony-forming units [CFU-F]). Greater than 95% of detectable CFU-F were recovered in the CD34+ population, while their numbers were markedly depleted in the CD34- population. Additional experiments showed that the majority of CFU-F exhibited high forward and perpendicular light scatter and low- density CD34 antigen. Growth of sorted cells in medium optimized for long-term marrow culture (LTMC) produced a complex mixture of adherent stromal elements including fibroblasts, adipocytes, smooth muscle cells, and macrophages. Monoclonal antibody STRO-1, which identifies bone marrow stromal cells, reacted with approximately 5% of CD34+ cells, which included all CFU-F and stromal precursors in LTMC. Experiments using soybean agglutinin (SBA) further showed that these stromal elements were restricted to a population of bone marrow cells with the phenotype CD34+/SBA+. These properties of stromal precursors are quite distinct from those of primitive hematopoietic progenitors, showing that although the precursors of the hematopoietic and stromal systems share expression of CD34, they are otherwise phenotypically distinct cell types.
Effects of thymidine and hydroxyurea on the metabolism and cytotoxicity of 1-B-D arabinofuranosylcytosine in highly resistant human leukemia cells
Authors: Bhalla K , Swerdlow P , Grant S
Published: October 2019

We have examined the effect of the ribonucleotide reductase inhibitors thymidine (dThd) and hydroxyurea (HU) on the metabolism and cytotoxicity of high concentrations (10 to 100 mumol/L) of cytosine arabinoside (Ara-C) in a deoxycytidine kinase-deficient, highly Ara-C- resistant human promyelocytic leukemic cell subline (HL-60/Ara-C). Administration of dThd or HU (0.5 to 3 mmol/L) in conjunction with high concentrations of Ara-C either sequentially or simultaneously lead to enhanced (and in some cases a supra-additive) inhibition of HL-60/Ara-C suspension culture growth and soft agar colony formation. In addition, exposure of cells to 0.5 mmol/L dThd or HU for 48 hours significantly increased the percentage of HL-60/Ara-C in S-phase. In contrast to previous studies involving low-dose Ara-C administration, we were unable to detect dThd- or HU-mediated increments in Ara-CTP formation in HL-60/Ara-C cells treated with high-dose Ara-C despite reductions in intracellular dCTP pools as great as 65%. However, dThd or HU did increase Ara-C incorporation into newly synthesized DNA. These studies show that administration of dThd or HU in conjunction with high concentrations of Ara-C results in an enhanced antiproliferative effect of Ara-C against Ara-C-resistant leukemic cells deficient in deoxycytidine kinase.
Duration of the preclinical phase of chronic myelogenous leukemia: a case report
Authors: Berman E , Strife A , Wisniewski D , Desai S , Gulati S , Jhanwar S , Clarkson BD
Published: October 2019

The molecular events that allow for clonal expansion of the malignant population in chronic myelogenous leukemia (CML) are poorly understood. Recent experiments in transgenic mice suggest a close temporal relationship between expression of the aberrant protein and manifestation of a hematologic neoplasm that resembles CML; tracing the same phenomenon in humans has not been possible. We studied a patient who underwent autologous bone marrow harvest after completion of chemotherapy and radiation therapy for advanced stage Hodgkin's disease. At the time of harvest his peripheral blood counts and bone marrow were morphologically normal. Sixteen months later he developed the clinical manifestations of CML. Detailed molecular evaluation of the harvested marrow showed that a small number of cells contained the Philadelphia chromosome. The time interval required for expansion of the malignant clone, as suggested by this particular patient, was at least 16 months although it is recognized that this figure may be variable.
Bone marrow from children in relapse with pre-B acute lymphoblastic leukemia proliferates and disseminates rapidly in scid mice
Authors: Kamel-Reid S , Letarte M , Doedens M , Greaves A , Murdoch B , Grunberger T , Lapidot T , Thorner P , Freedman MH , Phillips RA
Published: October 2019

Bone marrow samples from patients with pre-B acute lymphoblastic leukemia (pre-B ALL), either at diagnosis or at relapse, were transplanted into scid mice to determine whether these freshly obtained leukemic cells could proliferate in vivo and whether there were any differences in their in vivo growth characteristics. Cells from three patients who relapsed within 13 months of diagnosis proliferated rapidly in the murine bone marrow, spleen, and thymus, invaded peripheral organs, and resulted in morbidity and mortality of the animals within 4 to 16 weeks. Cells from two patients who relapsed 3.5 years after diagnosis grew much slower than the early relapse samples, taking up to 30 weeks to infiltrate the bone marrow of recipient mice. In contrast, leukemic cells were absent or were detected at low numbers in scid mice transplanted with cells obtained at diagnosis from three patients who have not yet relapsed. These results show an increased ability of leukemic cells from patients with aggressive lymphoblastic leukemia of poor prognosis to proliferate in scid mice.
Deficiency of alpha-spectrin synthesis in burst-forming units-erythroid in lethal hereditary spherocytosis
Authors: Whitfield CF , Follweiler JB , Lopresti-Morrow L , Miller BA
Published: October 2019

A child diagnosed in utero with hydrops fetalis and a hematocrit of 6.4% was studied to determine the etiology of the anemia. Fetal red blood cells (RBCs) obtained during in utero transfusion had extremely abnormal osmotic fragility. A maternal history of mild autosomal dominant hereditary spherocytosis was present, and the father, who was hematologically normal, had a slightly abnormal osmotic fragility test. The patient was transfusion dependent after birth, with circulating nucleated RBCs but less than 1% reticulocytes. The patient's anemia failed to respond to splenectomy. Because mature RBCs of the patient were not available for study, progenitor-derived erythroblasts grown in culture were investigated. Immunodot assays of the patient's progenitor- derived cells showed a total cell spectrin content 26% of normal. Immunoprecipitation of whole burst-forming units-erythroid-derived cells and solubilized membranes from cells pulse-labeled with 35S- methionine showed a severe deficiency in alpha-spectrin synthesis and a markedly reduced amount of alpha- and beta-spectrin on cell membranes. No alpha-spectrin degradation products were found within the cells or were produced during membrane preparation. Ankyrin content and band 3 synthesis were not different from control. Inheritance of two genetic defects causing severely reduced alpha-spectrin synthesis is proposed as the cause of the lethal anemia, resulting in cell fragmentation during precursor enucleation or during egress from bone marrow.
Autosomal dominant erythrocytosis caused by increased sensitivity to erythropoietin
Authors: Juvonen E , Ikkala E , Fyhrquist F , Ruutu T
Published: October 2019

We describe here a family with autosomal dominant erythrocytosis. In in vitro cultures, performed using the methyl cellulose assay, the number of erythroid colonies was normal or marginally increased when a standard concentration of erythropoietin (Epo) was used, but at lower Epo concentrations, the investigated persons formed more colonies than the controls. The difference was generally greater the lower the Epo concentration became. Some erythroid colony growth was seen even in the absence of any added Epo (apart from the minute concentration found in fetal calf serum), a phenomenon not seen in the controls. This finding indicates that the erythrocytosis in this family is caused by hypersensitivity of erythroid progenitors to Epo. The serum Epo concentration was low or low normal in all of the investigated family members, which is in good accordance with hypersensitivity to Epo. The erythrocytosis has not had any obvious effect on the health or life- span of the affected individuals. Many of them have reached an advanced age, and one of the affected family members has won several Olympic gold medals and world championships in endurance sports.
Increased life span and correction of metabolic defects in murine mucopolysaccharidosis type VII after syngeneic bone marrow transplantation
Authors: Birkenmeier EH , Barker JE , Vogler CA , Kyle JW , Sly WS , Gwynn B , Levy B , Pegors C
Published: October 2019

The gusmps/gusmps mouse has no beta-glucuronidase activity and develops murine mucopolysaccharidosis type VII (MPS VII). The clinical and pathologic abnormalities are similar to those found in humans with severe MPS VII. Mutant mice are dysmorphic, dwarfed, and have a shortened life span. Pathologic findings include widespread lysosomal storage. To determine whether bone marrow transplantation (BMT) corrects these abnormalities, genetically identical mutant animals were given syngeneic bone marrow transplants using cells from +/+ mice. Initial experiments showed that levels of beta-glucuronidase activity in recipient tissues correlated with the amount of radiation administered before BMT. Two groups of mice given BMT therapy were observed for periods of 1 and 2 years, respectively. These mice were evaluated using a combination of clinical, biochemical, histochemical, and pathologic analyses. Spleen, liver, cornea, and glomerular mesangial cells showed essentially complete correction at all radiation doses. Storage was partially corrected in meninges and perivascular cells in brain, and in renal tubular epithelial cells at the higher radiation doses. Life span in BMT-treated animals was increased approximately three-fold, approaching that seen in normal mice after BMT. These results support the position that BMT has a place in the therapeutic regimen for MPS VII.
Aphidicolin, an inhibitor of DNA replication, blocks the TPA-induced differentiation of a human megakaryoblastic cell line, MEG-O1
Authors: Murate T , Hotta T , Tsushita K , Suzuki M , Yoshida T , Saga S , Saito H , Yoshida S
Published: October 2019

The commitment process of a human megakaryoblastic cell line (MEG-O1) induced with phorbol ester, TPA, was investigated with special reference to glycoprotein (GP) IIb/IIIa expression, multinuclear formation, and DNA replication. TPA (10(-7) mol/L) completely inhibited cellular division in MEG-O1, but did not suppress de novo DNA synthesis. Two days' culture with 10(-7) mol/L TPA was sufficient for MEG-O1 cells to initiate an irreversible commitment process. These cells could not resume cell growth and expressed GP IIb/IIIa antigen; some of them showed multinuclear form and DNA polyploidy even after removal of TPA from the culture medium. DNA histogram analysis showed that, upon treatment with TPA, the percentage of cells whose DNA ploidy was more than 8N was 5 to 10 times higher than that of control cells. Precise analysis using cell size fractionation by centrifugal elutriation method showed that there was strong correlation between the percentage of multinuclear cells and DNA polyploidy in TPA-treated cells. The percentage and staining intensity of GP IIb/IIIa and other megakaryocytic phenotypes such as von Willebrand factor and PAS staining were highest in large multinuclear cell populations, suggesting that these cells are the most differentiated population in this system. In TPA-treated cells, the activity of DNA polymerase alpha, a marker for cell growth, remained at the same level as in control cells. Aphidicolin, a specific inhibitor of DNA polymerase alpha, completely inhibited the differentiation induction of MEG-O1 cells with TPA measured by either GP IIb/IIIa expression or multinuclear cell formation. Therefore, DNA replication appears to be involved in the process of phenotypic expression as well as endomitosis in megakaryocyte differentiation of MEG-O1 cells. Aphidicolin was also effective in inhibiting megakaryocytic differentiation of other leukemia cell lines such as human erythroleukemia (HEL) and K562 cell lines induced with TPA, suggesting the close interplay of DNA replication and phenotypic expression in megakaryopoiesis.
Abnormalities of the retinoblastoma gene in the pathogenesis of acute leukemia
Authors: Ahuja HG , Jat PS , Foti A , Bar-Eli M , Cline MJ
Published: October 2019

The retinoblastoma-susceptibility (Rb) gene is an antioncogene that is frequently altered in retinoblastomas, sarcomas, and some epithelial tumors. We examined the structure of the Rb gene by Southern blotting in 215 cases of leukemias and lymphomas of diverse phenotype and in 15 leukemic cell lines. In selected cases Rb protein expression was examined with specific monoclonal antibodies. Structural abnormalities of the Rb gene with absent protein expression were frequent in all types of human acute leukemia, but were particularly common (27% incidence) in M4 and M5 myeloid leukemia with monocytic differentiation and in Philadelphia chromosome (Ph1)-positive leukemia of lymphoid phenotype (11% to 29% incidence). Changes in Rb were observed early in the transition to acute leukemia in cases of myelodysplastic syndrome and in the accelerated phase of chronic myelocytic leukemia in transition to blast crisis. In one case, molecular changes in Rb could be correlated with leukemia remission and relapse. We conclude that the Rb antioncogene is commonly involved in the evolution of human acute leukemias, particularly in those of a monocytic phenotype and in lymphoid leukemia in which there is an antecedent alteration of the Ph1 chromosome.
Clinical and laboratory features of 78 cases of T-prolymphocytic leukemia
Authors: Matutes E , Brito-Babapulle V , Swansbury J , Ellis J , Morilla R , Dearden C , Sempere A , Catovsky D
Published: October 2019

We describe the clinical and laboratory findings of 78 adult patients with T-prolymphocytic leukemia (T-PLL) studied over the last 12 years. The main disease features were splenomegaly (73%), lymphadenopathy (53%), hepatomegaly (40%), skin lesions (27%), and a high leukocyte count (greater than 100 x 10(9)/L in 75%) with nucleolated prolymphocytes. A variant form with small, less typical cells was recognized in 19%. Membrane markers defined a postthymic phenotype TdT- , CD2+, CD3+, CD5+, CD7+; in 65%, the cells were CD4+ CD8-, in 21%, they coexpressed CD4 and CD8, and, in 13%, they were CD4- CD8+. Serology for human T-cell leukemia/lymphoma virus Type-I (HTLV-I) was negative in the 27 cases investigated. Cytogenetic analysis in 30 cases showed a consistent abnormality of chromosome 14, usually inv (14), with breakpoints at q11 and q32 in 76% of cases. Trisomy 8, including iso8q, was shown in 53%; t (11;14)(q13;q32) was documented in one case; and one had a normal karyotype. The clinical course was progressive with a median survival of 7.5 months. Thirty-one patients were treated with 2′ deoxycoformycin and 15 responded (3 complete remissions and 12 partial remissions); the response rate (48%) increased to 58% in patients with a CD4+ CD8- phenotype. The median survival of responders was 16 months and of nonresponders 10 months; other treatments were less effective. T-PLL is a distinct clinico-pathologic entity with aggressive course and characteristic chromosome abnormalities. A subgroup of patients may benefit from deoxycoformycin.
Hematopoietic reconstitution and prevention of graft-versus-host disease with UVB-irradiated haploidentical murine spleen and marrow cells
Authors: Cohn ML , Cahill RA , Deeg HJ
Published: October 2019

We investigated in a murine model whether UVB irradiation of lymphohemopoietic cells would prevent the development of graft-versus- host disease (GVHD). Preliminary experiments showed that spleen colony (CFU-S) formation by hemopoietic cells was preserved at UVB doses that eliminated lymphocyte proliferation. In a parent into F1 model, UVB irradiation (5 to 15 mJ/cm2) of spleen cells added to normal marrow cells prevented the development of GVHD, whereas all recipients given untreated spleen cells developed GVHD. Syngeneic recipients of marrow exposed to 2.5 to 10 mJ/cm2 of UVB achieved normal hemopoietic reconstitution. Based on these observations, B6D2 F1 (H-2b x H-2d) recipients were given 1,000 cGy of total body irradiation (TBI) followed by transplantation of 5 x 10(6) parental B6 (H-2b) bone marrow cells and 10 x 10(6) B6 spleen cells, either unirradiated or exposed to UVB before infusion. All mice transplanted with cells exposed to 10 or 12.5 mJ/cm2 of UVB survived without GVHD. At 2.5 and 5.0 mJ/cm2, mice showed signs of GVHD, beginning at day 30, and 100% and 80%, respectively, eventually developed chronic GVHD. At 7.5 mJ/cm2, mice had weight loss, from which 60% recovered and survived without GVHD, while 40% died with GVHD. At 15 mJ/cm2, some recipients died from graft failure, while some survived without GVHD. All surviving mice were complete donor-type chimeras. Spleen size and cellularity and in vitro lymphocyte responses correlated inversely with the development of GVHD. Mice without GVHD showed specific tolerance to skin grafts from the second parent strain, while animals with GVHD rejected their skin grafts. Thus, in a murine model UVB irradiation of transplanted hemopoietic stem cells allows for hemopoietic reconstitution and prevents GVHD.
Platelet glycoprotein IIb-IIIa (alpha IIb beta 3 integrin) confers fibrinogen- and activation-dependent aggregation on heterologous cells
Authors: Frojmovic MM , O'Toole TE , Plow EF , Loftus JC , Ginsberg MH
Published: October 2019

To analyze molecular mechanisms of platelet aggregation, we have studied the aggregation of Chinese hamster ovary (CHO) cells expressing between 1 and 4 x 10(5) recombinant human glycoprotein (GP) IIb-IIIa molecules per cell (A5 cells). These cells aggregated as measured by the disappearance of single cells during rotary agitation. Aggregation was dependent on the presence of extracellular fibrinogen (approximately 500 nmol/L) and divalent cations, and required prior activation of the GPIIb-IIIa. A synthetic peptide (GRGDSP) and monoclonal anti-GPIIb-IIIa antibody (2G12) that block platelet aggregation also blocked aggregation of these cells. Parent CHO cells or those expressing recombinant GPIIb-IIIa containing a point mutation that causes variant thrombasthenia both failed to aggregate when stimulated in the presence of fibrinogen. These data show that GPIIb- IIIa is the only unique platelet surface component required for aggregation.
Interleukin-4 counteracts pyrogen-induced downregulation of thrombomodulin in cultured human vascular endothelial cells
Authors: Kapiotis S , Besemer J , Bevec D , Valent P , Bettelheim P , Lechner K , Speiser W
Published: October 2019

Inflammatory mediators such as tumor necrosis factor (TNF) or interleukin-1 (IL-1) and bacterial lipopolysaccharides (LPS) were shown to shift the hemostatic balance of the endothelial cell (EC) surface in favor of procoagulant activities by inducing tissue factor (TF) expression and downregulation of thrombomodulin (TM). In the present study, the effects of IL-4 on these regulatory mechanisms were investigated using cultured human umbilical vein EC. TM downregulation induced by the pyrogens IL-1 (100 U/mL), TNF (500 U/mL), and LPS (20 micrograms/mL) to less than 50% of TM activity of untreated cells during a 12-hour incubation period was completely neutralized when these mediators were coincubated with IL-4 (100 U/mL). In accordance with TM surface activity, TM messenger RNA was decreased by IL-1, TNF, and LPS to less than 40% of untreated cells; this effect was in part antagonized by IL-4. No influence of IL-4 on EC tissue factor induction by IL-1, TNF, and LPS was found. Binding studies using 125I- radiolabeled IL-4 suggest that EC express a single class of high- affinity binding sites (kd = 3.2 pmol/L; 2,000 to 2,500 receptors per cell). These results show that IL-4, in part, protects the EC surface against pyrogen-induced procoagulant changes. Transcriptional regulatory mechanisms seem to be involved in EC surface TM regulation.
Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model: effects of hirudins, heparin, and calcium chelation
Authors: Badimon L , Badimon JJ , Lassila R , Heras M , Chesebro JH , Fuster V
Published: October 2019

The role of thrombin inhibition in platelet vessel wall interaction and thrombus growth was studied under controlled flow conditions. Natural hirudin and recombinant hirudin (r-hirudin), which are specific thrombin inhibitors, were compared with heparinized blood (1.8 +/- 0.2 U/mL) and Ca(2+)-chelated blood in their potential to inhibit platelet interaction and thrombus growth on two biologic vascular surfaces and one immobilized vessel wall component. The substrates were perfused by flowing blood at shear rates typical of patent and stenosed arteries (212 to 1,690/s) for 5 minutes. Platelet deposition was measured by In- 111-labeled platelets. We found that both natural and r-hirudin have similar effects on platelet-substrate interaction. As compared with heparin, platelet deposition to mildly damaged vessel wall and digested collagen type I was not reduced by hirudin or citrate. However, hirudin and citrate significantly reduced platelet deposition to severely damaged vessel wall (platelets x 10(6)/cm2: 93 +/- 10 in heparinized blood v 50 +/- 7 in blood treated with 100 U/mL r-hirudin). Therefore, thrombus growth on areas of severe wall damage is in part dependent on local thrombin production at the site of vascular damage. We also found that hirudin added to heparinized blood reduced platelet deposition to severely injured wall but not to subendothelium or collagen-coated slides. Hirudin added to citrated blood did not affect platelet deposition. Our study indicates that local thrombin generation at the site of severe injury will induce platelet activation and deposition even in the presence of average therapeutic heparin levels that inhibit blood coagulation.
Peritoneal fluid and plasma levels of human macrophage colony- stimulating factor in relation to peritoneal fluid macrophage content
Authors: Weinberg JB , Haney AF , Xu FJ , Ramakrishnan S
Published: October 2019

The peritoneal fluid (PF) of women with infertility (especially in the presence of endometriosis) contains increased numbers of leukocytes, 90% to 95% of which are macrophages. The high numbers of peritoneal macrophages presumably result from an influx of blood monocytes into the peritoneum, and/or from local proliferation of peritoneal macrophages. Once in the peritoneal cavity, monocytes differentiate into tissue macrophages. Mononuclear phagocyte proliferation and differentiation are influenced by different cytokines, including macrophage colony-stimulating factor (M-CSF). The purpose of this study was to determine the relationship of M-CSF levels in human PF and plasma to the macrophage content, and to the patient diagnoses. Mean concentrations of PF M-CSF were higher than plasma levels (2.44 +/- 0.13 v 0.95 +/- 0.06 ng/mL, respectively). The mean concentrations of plasma M-CSF did not differ in samples from women of different diagnostic groups (normal, peritoneal adhesions, endometriosis, inactive pelvic inflammatory disease, uterine fibroids, and idiopathic infertility), but the PF concentration was slightly higher in normal women. The absolute (total) amount of PF M-CSF in normal women was lower than in those of the other diagnostic groups. The total amount of PF M-CSF in all women correlated closely with the total number of peritoneal macrophages. The tubal patency status (open versus closed) did not influence the plasma and PF concentrations of M-CSF, nor the PF absolute amount of M-CSF. The PF M-CSF may have come from peritoneal macrophages, fibroblasts, mesothelial cells, or endothelial cells. PF M- CSF may play important roles in the proliferation and/or the differentiation of peritoneal mononuclear phagocytes.
An insertional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin nice (beta 220/216)
Authors: Tse WT , Gallagher PG , Pothier B , Costa FF , Scarpa A , Delaunay J , Forget BG
Published: October 2019

Spectrin Nice (beta 220/216) is a spectrin variant associated with a shortened beta chain found in a patient with elliptocytosis. The shortened beta chain (beta' chain) appeared as an additional band of approximately 216 Kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was defective in its ability to be phosphorylated. There were increased amounts of spectrin dimers in crude spectrin extracts from the propositus and the association constant of spectrin dimer self-association was decreased. There was an associated increase of the alpha I 74-Kd fragment from the alpha chain after partial trypic digestion of spectrin. To identify the underlying molecular defect, we analyzed cDNA for beta spectrin obtained by polymerase chain reaction amplification of reverse-transcribed reticulocyte messenger RNA from peripheral blood of the propositus. DNA sequencing of individual as well as pooled subclones showed that two extra bases (GA) are inserted in codon no. 2046 in one allele of the beta-spectrin gene. The insertion results in a frameshift mutation and generates an aberrant C- terminus truncated by about 4 Kd, consistent with the estimated size of the beta' chain observed. By allele-specific oligonucleotide hybridization, the insertion was shown to be present in the propositus and absent in his parents, confirming a previous proposal that it is a de novo mutation. The determination of the location of the mutation in spectrin Nice points to specific regions of the beta-spectrin chain where phosphorylation may occur. A model is proposed to describe the interaction between the alpha- and beta-spectrin chains and to explain the effects of the mutation found in spectrin Nice on the trypsin digestion pattern of its associated alpha chain.
Adoptive transfer of anti-cytomegalovirus effect of interleukin-2- activated bone marrow: potential application in transplantation
Authors: Agah R , Charak BS , Chen V , Mazumder A
Published: October 2019

This work is a continuation of our studies that showed that interleukin- 2 (IL-2)-activated murine bone marrow (ABM) cells have potent cytotoxic potential against murine cytomegalovirus (MCMV)-infected targets in vitro, without loss of reconstitutive ability in vivo. Our data show that ABM cells lyse the MCMV-infected cells in vitro, at both acute and chronic stages of infection; this lysis is specific for the MCMV- infected cells. ABM cells supplemented with IL-2 therapy virtually eradicated the viral infection and prolonged the survival of MCMV- infected Balb/c mice, whether or not they were immunocompromised by irradiation (P less than .001 in both situations). Efficacy of ABM cells alone or IL-2 alone was less than the combination of ABM cells and IL-2. The efficacy of combination treatment with ABM cells and IL-2 in improving the survival of MCMV-infected mice was comparable, whether used in a preventive or a therapeutic setting. Therapy with ABM plus IL- 2 also prevented the reactivation of chronic MCMV infection after irradiation. Preliminary findings indicate that Thy-1+ and asialo GM1+ cells limited the MCMV proliferation by approximately 30% and 80%, respectively, while BM macrophages limited the proliferation of MCMV by 100%. These results suggest that BM transplantation (BMT) with ABM cells followed by IL-2 therapy may constitute a novel strategy to improve the host resistance against cytomegalovirus infection after BMT.
Detection of lymphomatous bone marrow involvement with magnetic resonance imaging [see comments]
Authors: Hoane BR , Shields AF , Porter BA , Shulman HM
Published: October 2019

We reviewed magnetic resonance (MR) staging examinations of 98 patients with malignant lymphoma who failed other therapy and were under evaluation for bone marrow transplantation. MR scan results were compared with blind posterior iliac crest aspirations and biopsies. Images of vertebral, pelvic, and femoral marrow were obtained using a standard T1-weighted, short repetition time (TR), short time to echo (TE) (TR700/TE22), spin-echo (T1-SE) method in 92 patients and short TI inversion recovery (STIR) technique (TR1,500/TE36/TI100) in all. On standard T1-SE sequence, normal marrow is bright due to the predominance of marrow fat, and tumor is dark. With STIR images, water containing tumor has a very high signal intensity in a dark (fat suppressed) background. Thirteen patients had positive MR scans and marrow biopsies, whereas 49 had negative MR scans and biopsies. Of 36 discordant MR/histology results, 10 had positive biopsies and negative MR exams; eight of these had microscopic infiltration (less than or equal to 5%) with tumor. MR detected marrow tumor either in the crests or elsewhere in 25 of 75 (33%) patients with negative study biopsies. We could confirm marrow involvement in 15 of these 25 (60%) by clinical methods. Therefore, up to one third of the patients evaluated with routine biopsies may have occult marrow tumor detectable by MR exam. In patients with negative marrow biopsies, especially those with Hodgkin's disease or intermediate to high-grade non-Hodgkin's lymphomas, MR scans found focal lesions distant from the crests. Biopsy better detected lower grade microscopic involvement. We conclude that optimal marrow staging of lymphoma patients incorporates both biopsy and MR imaging.