bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythropoiesis in Fanconi's anemia
Authors: Alter BP , Knobloch ME , Weinberg RS
Published: October 2019

Fanconi's anemia (FA) is an autosomal recessive condition in which greater than 90% of the homozygotes develop aplastic anemia. To determine the relation between erythroid progenitors and clinical status, blood and marrow mononuclear cells were cultured in methyl cellulose with erythropoietin, plus other hematopoietic growth factors, and growth in normal oxygen (20%) was compared with growth in low, physiologic oxygen (5%). Peripheral blood cultures were performed from 24 patients, and marrows from six. Patients were classified into six clinical groups. Group 1: Severe aplasia, transfused; one patient; no erythroid progenitors. Group 2: Severe, transfused, androgen unresponsive; one patient; no blood burst-forming units-erythroid (BFU- E). Group 3: Androgen responsive; eight patients, with decreased blood BFU-E. Group 4: Aplastic, about to start treatment; two patients; below normal numbers of colony-forming units-erythroid (CFU-E) and BFU-E. Group 5: Stable, with mild anemia, and/or thrombocytopenia, and/or macrocytosis; seven patients; with below normal numbers of blood BFU-E. Group 6: Hematologically normal; five patients; blood BFU-E low normal to normal. One marrow had normal numbers of CFU-E and BFU-E. Incubation in 5% oxygen doubled CFU-E and BFU-E only in the patients with close to normal or normal growth in 20% oxygen. Hemin and interleukin-3 increased growth slightly in those cultures where there was some growth with erythropoietin alone. Our data show that there is a correlation between current clinical status and in vitro erythropoiesis. Cultures of erythroid progenitors may also be useful predictors of hematologic prognosis in FA, although our follow-up period is too short to prove this hypothesis.
Frequent detection of Epstein-Barr virus DNA by the polymerase chain reaction in lymph node biopsies from patients with Hodgkin's disease without genomic evidence of B- or T-cell clonality
Authors: Knecht H , Odermatt BF , Bachmann E , Teixeira S , Sahli R , Hayoz D , Heitz P , Bachmann F
Published: October 2019

This study of 52 Swiss patients with Hodgkin's disease (HD), including 17 cases with a high content of Sternberg-Reed (SR) and Hodgkin (H) cells, was performed to determine the percentage of cases harboring Epstein-Barr virus (EBV) DNA and/or clonal rearrangements of Ig and T- cell antigen receptor (TcR) genes in diagnostic lymph node biopsies. Special attention was drawn to the heavily infiltrated cases to detect a possible relationship between clonality and EBV DNA identification. EBV DNA was detected by the polymerase chain reaction (PCR) using three different sets of specific primers. The viral origin of the amplification products was confirmed by hybridization with a radiolabeled internal probe or demonstration of a specific Sma I restriction site. Genomic rearrangement of Ig and TcR genes was studied by Southern blot analysis. EBV DNA was identified by PCR in 38 of 48 cases (79%). Clonal rearrangements were identified in only 4 of 52 cases (Ig genes) and were independent of the degree of infiltration by SR cells and the presence of EBV DNA. The absence of EBV DNA in three cases with numerous SR cells (only one of them showed clonal rearrangement) and the presence of only a few viral copies in four further cases with numerous SR cells (semiquantitative analysis of viral DNA by PCR was performed in 26 EBV-positive cases) suggests that this virus is modulating rather than an etiologic agent in a considerable proportion of HD cases.
Evidence of c-myc gene abnormalities in mediastinal large B-cell lymphoma of young adult age [see comments]
Authors: Scarpa A , Borgato L , Chilosi M , Capelli P , Menestrina F , Bonetti F , Zamboni G , Pizzolo G , Hirohashi S , Fiore-Donati L
Published: October 2019

Six cases of mediastinal large B-cell lymphoma (MLCL) with sclerosis were analyzed for the presence and patterns of c-myc and bcl-2 loci rearrangements, and for the presence of Epstein-Barr virus DNA sequences by Southern blot hybridization, c-myc gene alterations were found in three of six cases. Two cases showed the presence of mutations or small rearrangements at the 3′ end of the first exon. The c-myc gene abnormalities found in these two cases are similar to those observed in the translocation 8;14 of the endemic Burkitt's lymphomas or in its variants t(2;8) and t(8;22). A third case showed a major rearrangement of c-myc gene, with truncation within its first intron, similar to those observed in sporadic Burkitt's and in acquired immunodeficiency- associated lymphomas. None of the cases displayed bcl-2 gene rearrangements or contained viral sequences. Our data suggest a possible role for a translocation-mediated c-myc activation in the pathogenesis of MLCL. Conversely, bcl-2 gene and Epstein-Barr virus do not appear to be involved in the pathogenesis of these peculiar lymphomas. The association between c-myc structural modifications and MLCL also seems to be of relevance in light of the peculiar tendency of this tumor to involve unusual extranodal site (eg, kidney), reminiscent of the spreading attitude of Burkitt's limphomas.
Antibodies against human neutrophil LECAM-1 (LAM-1/Leu-8/DREG-56 antigen) and endothelial cell ELAM-1 inhibit a common CD18-independent adhesion pathway in vitro
Authors: Kishimoto TK , Warnock RA , Jutila MA , Butcher EC , Lane C , Anderson DC , Smith CW
Published: October 2019

Neutrophil adhesion to interleukin-1 (IL-1)-stimulated human umbilical vein endothelial cells (HUVEC) involves the CD18 family of leukocyte integrins (lymphocyte function-associated antigen-1 [LFA-1], Mac-1, and p150,95) and LECAM-1 (DREG-56/LEU-8/LAM-1 antigen) on neutrophils and intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1) on the endothelium. In this study, we compare CD18-independent adhesion pathways mediated by neutrophil LECAM- 1 and endothelial ELAM-1 and find that these two pathways overlap in a variety of assays: (1) anti-LECAM-1 and anti-ELAM-1 monoclonal antibody (MoAb) inhibit neutrophil binding to HUVEC, and the inhibitory effect is not additive; (2) anti-LECAM-1 MoAb, like anti-ELAM-1 MoAb, inhibits neutrophil binding to HUVEC stimulated for 3 hours with IL-1, but not to HUVEC stimulated for 8 hours, by which time ELAM-1 expression is downregulated; (3) anti-ELAM-1 MoAb has no effect on transendothelial migration, a CD18-dependent, LECAM-1-independent neutrophil function. Interestingly, anti-ELAM MoAb has a reduced but significant inhibitory effect on the adhesion of activated neutrophils that have shed their cell-surface LECAM-1. We also show that neutrophil binding to ELAM-1- transfected L cells is inhibited not only by anti-ELAM-1 but also by anti-LECAM-1 MoAb. These results suggest that LECAM-1 and ELAM-1 can operate in the same adhesion pathway, possibly as a receptor- counterreceptor pair. LECAM-1 and ELAM-1 are likely to interact with other ligands as well, perhaps through carbohydrate determinants that modify more than one glycoprotein.
Purification of human marrow progenitor cells and demonstration of the direct action of macrophage colony-stimulating factor on colony-forming unit-macrophage
Authors: Sato N , Sawada K , Kannonji M , Tarumi T , Sakai N , Ieko M , Sakurama S , Nakagawa S , Yasukouchi T , Krantz SB
Published: October 2019

To facilitate the investigation of the direct interaction between hematopoietic progenitors and colony-stimulating factors, we have developed a method to purify human marrow progenitor cells. Using density centrifugation, negative panning with concanavalin A coated plates, positive selection of CD34-positive cells with immunomagnetic microspheres, overnight adherence to a plastic dish, negative selection with a panel of monoclonal antibodies, and density centrifugation, human marrow progenitor cells were purified from 1.5% to 53.2%, a 42- fold purification, with a 4.8% yield. The purified cells consisted of 38% erythroid, 9% colony forming unit-granulocyte (CFU-G), 29% CFU- macrophage (CFU-M), 12% CFU-eosinophil/basophil (CFU-Eo/Ba), and 4% CFU- mix. The purified cells cultured in serum-free fibrin clots with recombinant human macrophage colony-stimulating factor (rM-CSF) for 14 days developed a pure population of CFU-M colonies. An appearance of CFU-M colonies was present after the addition of 1 U/mL of rM-CSF and the maximum stimulation was found at 100 U/mL. When the purified cells were cultured in serum-free medium with rM-CSF in a limiting dilution assay and the percentage of nonresponder wells for CFU-M colonies was plotted against cell concentration, serum-free cultures yielded a straight line through the origin, indicating that CFU-M development did not depend on accessory cells and that rM-CSF acted directly on the CFU- M.
Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil
Authors: Harrison DE , Lerner CP
Published: October 2019

To test whether primitive hematopoietic stem cells (PHSC) cycle rapidly during recovery from an initial 5-fluorouracil (5-FU) treatment, two doses of 5-FU were administered 1, 3, 5 or 8 days apart. Cells from treated marrow donors were mixed with untreated competitor marrow that would produce genetically distinguishable erythrocytes and lymphocytes, using hemoglobin (Hb) and glucosephosphate isomerase (GPI) transplantation markers. These cell mixtures were injected into lethally irradiated hosts. Functional abilities of donor marrow populations were assessed after 3, 6, and 12 months as percentages of donor type Hb and GPI in the host's circulating erythrocytes and lymphocytes, respectively. Bone marrow from mice treated with two doses of 5-FU 3 to 5 days apart was severely affected, producing circulating erythroid and lymphoid cells an average of only 25% of normal for doses 3 days apart, and 14% of normal for doses 5 days apart. Two doses of 5- FU administered 1 day or 8 days apart had much smaller effects, producing circulating cells 75% or 58% of normal. Thus, most PHSC are stimulated to proliferate rapidly 3 to 5 days after treatment with 5- FU, but far fewer PHSC proliferate as early as 1 day, or as late as 8 days, after the 5-FU treatment.
Proliferation and cytolytic function of anti-CD3 + interleukin-2 stimulated peripheral blood mononuclear cells following bone marrow transplantation
Authors: Katsanis E , Anderson PM , Filipovich AH , Hasz DE , Rich ML , Loeffler CM , Ochoa AC , Weisdorf DJ
Published: October 2019

We evaluated the proliferation, cytolytic function, and phenotypic characteristics of anti-CD3 plus interleukin-2 (IL-2) stimulated peripheral blood mononuclear cells (PBMCs) from 44 patients with leukemia or non-Hodgkin's lymphoma (NHL) treated with multiagent chemotherapy or following bone marrow transplantation (BMT). BMT patients had decreased cell growth with only a 1.35 +/- 0.25 (autologous BMT for acute lymphoblastic leukemia [ALL]), 1.24 +/- 0.25 (autologous BMT for NHL), and 0.8 +/- 0.1 (allogeneic BMT for leukemia) mean fold increase by day 5 of culture compared with controls (4.0 +/- 0.4), P less than .001. Anti-CD3 + IL-2 activated cells from patients with ALL and NHL who had received autologous BMT and cells from patients with leukemia who underwent allogeneic BMT were more effective in lysing the natural killer (NK) sensitive target, K562, and the NK- resistant target, Daudi, compared with controls. In contrast, cytolysis of K562 and Daudi by cultured PBMCs from patients with ALL and NHL receiving multi-agent chemotherapy was similar to that of controls. Cultures from BMT recipients had a significant increase in CD16+ (autologous ALL 5.7 +/- 1.5%, P less than .01; autologous NHL 12.4 +/- 3.5%, P less than .001; allogeneic 14.3 +/- 2.9%, P less than .001) and CD56+ cells (autologous ALL 27.6 +/- 12.0%, P less than .01; autologous NHL 39.3 +/- 9.5%, P less than .001; allogeneic 42.7 +/- 7.4%, P less than .001) compared with controls (CD16+ 2.5 +/- 0.4%; CD56+ 6.9 +/- 0.9%). Stimulation of PBMCs with anti-CD3 + IL-2 is effective in generating cells with high cytolytic function post-BMT.
Effect of age on second messenger generation in neutrophils
Authors: Lipschitz DA , Udupa KB , Indelicato SR , Das M
Published: October 2019

Neutrophils from healthy elderly donors generate significantly less diacylglycerol (DAG) and inositol triphosphate (IP3) than neutrophils from young donors, following stimulation by the chemotactic peptide, formyl-methionyl-leucylphenylalanine (FMLP). The defect in signal transduction occurred at a point proximal to the generation of IP3 and DAG, since the reduction in FMLP-induced superoxide generation was corrected if the intervening signal transduction steps were bypassed, either by priming with a substimulatory dose (1.62 nmol/L) of phorbol myristate acetate (PMA), by ionophore elevation of cytosolic calcium, or by using a stimulatory dose of PMA (1.62 mumol/L). FMLP receptor number and affinity were unaffected by aging. On FMLP activation, neutrophils from old, as compared with young, volunteers showed significantly greater and more long-lasting decreases in the concentrations of phosphatidylinositol (PI), phosphatidylinositol 4- monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). This indicates a reduction with age in the metabolically active precursor pools responsible for the generation of IP3 and DAG. In contrast, aging had little effect on the production of phosphatidic acid (PA), which has recently been suggested to serve as a major activator of the NADPH oxidase. This may explain why the decrease in IP3 and DAG production was not accompanied by a comparable decrement in superoxide generation, which was only 17% lower in the old than in young donor neutrophils. Thus, aging is associated with reductions in the concentration of critically important phosphoinositides, resulting in diminution in the ability to produce key second messengers. Although the aged neutrophil is largely able to compensate for the decrements in signal transduction, its reserve capacity is compromised, making it particularly vulnerable to external insults that also impair function.
Human herpesvirus-6 infection in bone marrow transplantation
Authors: Yoshikawa T , Suga S , Asano Y , Nakashima T , Yazaki T , Sobue R , Hirano M , Fukuda M , Kojima S , Matsuyama T
Published: October 2019

Twenty-five pediatric patients who received bone marrow transplantation (BMT) were studied prospectively to determine the relationship between BMT and human herpesvirus-6 (HHV-6) infection by the virus isolation from peripheral blood and/or bone marrow and by determining neutralizing antibodies to HHV-6 during the 2 months following BMT. All of the 25 donors and the recipients were immune to HHV-6 at the time of BMT and the virus was not isolated from them. HHV-6 was isolated from peripheral blood and/or bone marrow mononuclear cells in ten (40%) of the 25 recipients between day 14 and day 22 of BMT, but not from any other day. Two additional recipients showed a significant increase in the antibody titer. Thus, infection with HHV-6 was confirmed in 12 (48%) of the 25 recipients. Four of the 12 developed skin rashes; three of these four had a febrile episode when the virus was isolated, whereas none of the remaining 13 developed the skin rash. These results suggest a frequent infection with HHV-6 only a few weeks after BMT and a close association between the infection with the virus and the development of skin rashes.
Identification of the c-kit ligand: end of the road for understanding aplastic anemia in steel mutant mice?
Authors: Pantel K , Boertman J , Nakeff A
Published: October 2019

We here report the initiation of hematopoietic recovery in congenitally hypoplastic S1/S1d mice by the cytotoxic ablation of cells bearing the natural killer (NK) phenotype (NK 1.1+). The most striking finding was the early several-fold increase in the cycling fraction of stem and progenitor cells (with the exception of progenitors committed to megakaryocytopoiesis) in the anti-NK 1.1+ antibody-treated group. This increase resulted in an early, complete restoration of total marrow cellularity to the normal (+/+) littermate level. Our data suggest that NK 1.1+ cells exert functions critical to the negative control of hematopoietic cell proliferation in S1/S1d mice.
Synergistic interactions between interleukin-11 and interleukin-4 in support of proliferation of primitive hematopoietic progenitors of mice
Authors: Musashi M , Clark SC , Sudo T , Urdal DL , Ogawa M
Published: October 2019

Interleukin-11 (IL-11) is a newly identified lymphohematopoietic cytokine originally derived from the primate bone marrow stromal cell line, PU-34. Separately, we reported that IL-11 augments IL-3-dependent proliferation of primitive murine hematopoietic progenitors in culture. We have now examined the synergistic interactions between IL-11 and IL- 4 in support of colony formation from marrow cells of mice treated 2 days before with 150 mg/kg 5-fluorouracil. Neither recombinant human IL- 11 nor murine IL-4 alone was effective in the support of colony formation. When the two factors were combined, there was major enhancement of colony formation, including that of multilineage colony- forming cells. Serial observations (mapping studies) of development of multipotential blast cell colonies indicated that the synergy between IL-11 and IL-4 is due in part to shortening of the dormant period of the stem cells, an effect very similar to that of IL-6 and granulocyte colony-stimulating factor. The combination of IL-11 and IL-4 may be useful in the stimulation of dormant hematopoietic stem cells in vivo.
Effects of human interleukin-6 on megakaryocyte development and thrombocytopoiesis in primates
Authors: Stahl CP , Zucker-Franklin D , Evatt BL , Winton EF
Published: October 2019

Recombinant human interleukin-6 (IL-6) has previously been shown to increase platelet counts in mice and primates. To elucidate the mechanisms underlying this phenomenon, serial analyses were performed on megakaryocytes obtained from rhesus monkeys treated for 8 days with 30 micrograms/kg/d of recombinant human IL-6. Platelet counts increased to a maximum of 7.8 x 10(5)/microL with biphasic peaks on days 7 and 12 without significant changes in platelet volumes. Large increases in DNA content were seen by two-color flow cytometry and digital image analysis. Ploidy distribution underwent a significant shift between study days 3 and 11 (P less than .0001) with large increases in the frequency of 64N and 128N megakaryocytes. The modal ploidy increased from the normal 16N to 64N. Megakaryocyte size, as measured by area, was increased 2- to 2.7-fold. On day 3, multiple megakaryocytes were seen in endomitosis, along with an abundance of young cells with wide, organelle-free peripheral zones. The giant megakaryocytes seen on days 5 to 7 exhibited marked membrane hyperplasia that occupied much of the cell. Emperipolesis occurred frequently, as did megakaryocyte cell death. No giant platelets were seen. We conclude that IL-6 significantly alters the process of megakaryocyte maturation and thrombocytopoiesis, and that these effects, at least in the doses of IL- 6 administered, should not be equated with the physiologic mechanisms operative during accelerated platelet production.
Decreased CD45RA T cells in B-cell chronic lymphatic leukemia patients: correlation with disease stage
Authors: Peller S , Kaufman S
Published: October 2019

T-cell subsets CD4, CD8 and suppressor-inducers (CD45RA) were determined in 20 patients with B-cell chronic lymphatic leukemia (B- CLL). The proportion of CD4 and CD45RA was decreased when compared with T cells from normal subjects. CD8 was markedly increased. The activity of concanavalin A-induced suppressor cells was not significantly different from that of normal controls and was negatively correlated to the percentage of CD4 of B-CLL patients. The selective loss of CD45RA cells was more prominent in patients in advanced Rai stages of the disease (III to IV) than in early stages (0 to II). Six patients of the advanced stages group suffered from autoimmune hemolytic anemia, whereas no patient in the early stages of disease showed an autoimmune phenomenon. Our results may indicate a mechanism of autoimmunity in B- CLL similar to that of patients with autoimmune diseases.
Enhanced repopulation of murine hematopoietic organs in sublethally irradiated mice after treatment with ciprofloxacin
Authors: Kletter Y , Riklis I , Shalit I , Fabian I
Published: October 2019

We analyzed the effect of ciprofloxacin, fleroxacin, and ceftazidime on production of colony-stimulating factors (CSF) by cultured murine spleen cells in the presence of pokeweed mitogen (PWM). Ciprofloxacin at concentrations of 5 to 10 micrograms/mL in concert with PWM stimulated CSF production by cultured spleen cells. A 3.5-fold increase in the number of CFU-C was observed in the presence of ciprofloxacin- PWM spleen conditioned medium (SCM) as compared with control cultures exposed to PWM-SCM only. Antimurine GM-CSF and antimurine interleukin-3 (IL-3) antibodies inhibited colony formation stimulated by PWM-SCM or ciprofloxacin-PWM-SCM. Fleroxacin and ceftazidime at concentrations of 1 to 100 micrograms/mL and ciprofloxacin at high concentration (greater than 10 micrograms/mL) either did not affect CSF production by spleen cells or had an inhibitory effect. In vivo treatment of sublethally irradiated (650 rad) mice with ciprofloxacin (15 mg/kg per dose three times daily for 5 days) resulted in an increased number of myeloid progenitors in the spleen and bone marrow (BM) of treated mice. In contrast, treatment with ceftazidime did not affect progenitor cell numbers. On days 4 and 8 postirradiation ciprofloxacin-treated mice had a 2.3- and 3.8-fold increase, respectively, in the number of CFU-C in the BM. The number of CFU-C in the spleen did not increase on day 4 postirradiation, but on day 8, the number increased 1.7-fold. On day 4 postirradiation, sublethally irradiated mice treated with ciprofloxacin had a higher WBC count, RBC count, and hemoglobin level as compared with ceftazidime- and saline-treated mice. Twenty-four days postirradiation, 45% of saline-treated mice (20 of 44), and 35% of ceftazidime-treated mice (8 of 23) died, as compared with 13% (5 of 38) of ciprofloxacin-treated mice (P less than .05). These studies indicate that ciprofloxacin may have an immune-enhancing effect on the hematopoietic system in neutropenic mice.
Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule
Authors: Okada S , Nakauchi H , Nagayoshi K , Nishikawa S , Nishikawa S , Miura Y , Suda T
Published: October 2019

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK- 2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr- 1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c- kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.
Duplication of a methionine within the glycoprotein Ib binding domain of von Willebrand factor detected by denaturing gradient gel electrophoresis in a patient with type IIB von Willebrand disease
Authors: Ribba AS , Lavergne JM , Bahnak BR , Derlon A , Pietu G , Meyer D
Published: October 2019

von Willebrand disease (vWD) type IIB is characterized by an increased reactivity of von Willebrand factor (vWF) with platelets and a lack of large multimers. Exon 28 of the vWF gene encodes for functional domains involved in the binding of vWF to GPIb, and it is presumed that the defects in type IIB vWD lie within or adjacent to these functional domains. We screened overlapping DNA fragments generated by the polymerase chain reaction (PCR) that spanned the 1,379 bp of exon 28 of a type IIB vWD patient using denaturing gradient gel electrophoresis (DGGE). To increase the power of DGGE to detect base changes, we used the PCR to attach a G + C-rich sequence. In the type IIB patient, a DNA fragment at the 5′ end of exon 28 demonstrated homoduplex and heteroduplex complexes after DGGE, a pattern characteristic of heterozygous genes after melting and reannealing during the PCR. Sequencing of the cloned insert from the patient showed a duplication of an ATG in one gene coding for a Met at amino acids 540 to 541 in the mature vWF subunit. This duplication leads to three consecutive methionines in the patient's sequence. The duplicated Met resides within a disulfide bond loop proposed to be important in the function of the GPIb binding domain of vWF. The patient's nephew, who also has type IIB vWD, showed the same duplicated codon, linking the defect to the abnormal phenotype in this family. These nucleotide changes were not found in 100 chromosomes analyzed either by DGGE or hybridization with an allele specific oligonucleotide containing the duplicated ATG codon. In addition, the same oligonucleotide hybridized only to DNA from type IIB vWD individuals and not to DNA from normal members of the family. Therefore, we conclude that this duplicated Met modifies the GPIb binding domain of vWF and causes type IIB vWD in this family.
Platelet activation by immobilized monoclonal antibody: evidence for a CD9 proximal signal
Authors: Griffith L , Slupsky J , Seehafer J , Boshkov L , Shaw AR
Published: October 2019

Anti-CD9 monoclonal antibodies (MoAbs) are reported to activate human platelets through stimulation of the Fc gamma II receptor. We show here that nonstimulatory F(ab')2 fragments of the anti-CD9 MoAb 50H.19 induce dense-granule release and dose-dependent platelet aggregation when attached to polystyrene latex beads. Cross-linking F(ab')2 fragments of MoAb 50H.19 by F(ab')2 fragments of goat anti-mouse IgG does not result in platelet aggregation unless the second antibody is bound to latex beads, indicating that immobilization, and not cross- linking of the stimulus, is critical to the initiation of the CD9 signal. In contrast, F(ab')2 fragments of the second antibody readily induce the aggregation of platelets treated with the anti-Fc gamma II receptor MoAb IV.3. Immobilization of MoAb per se is insufficient to induce an activation signal because intact and F(ab')2 fragments of nonstimulatory MoAb directed to glycoprotein Ib and HLA class I do not become stimulatory when attached to beads. CD9-induced activation requires cytoskeletal rearrangement because it is inhibited by cytochalasin B. Aggregation is blocked by inhibitors of the thromboxane pathway, indicating that CD9 activates phospholipase C indirectly through prior activation of phospholipase A2.\
Prognostic importance of flow cytometric immunophenotyping of 540 consecutive patients with B-cell chronic lymphocytic leukemia
Authors: Geisler CH , Larsen JK , Hansen NE , Hansen MM , Christensen BE , Lund B , Nielsen H , Plesner T , Thorling K , Andersen E
Published: October 2019

Blood mononuclear cells from 540 newly diagnosed, unselected patients with B-cell chronic lymphocytic leukemia (CLL) were examined by immunofluorescence flow cytometry for a panel of surface membrane markers, including IgM and IgD, the monoclonal antibodies anti-CD3, -5, -20, -21, -22, -FMC7, and, for the final 125 patients, anti-CD23. There were 503 CD5+ and 37 CD5- cases. In the CD5+ cases, the cells typically expressed IgM, IgD, CD20, CD21, CD22, and CD23. In univariate analysis, age, clinical stage, IgM-fluorescence intensity, CD23, and FMC7 had significant prognostic importance, with high IgM-fluorescence intensity, high FMC7, and low CD23 expression being associated with a short survival. There was no significant difference in survival between 351 cases expressing IgMD and 55 cases expressing IgM without IgD, or between kappa and lambda light chain monoclonal cases. CD20, CD21, and CD22 had no prognostic importance. In Cox multiple regression analyses, age, CD23, IgM-fluorescence intensity, and clinical stage (International Workshop System) had independent prognostic importance. Thus, besides clinical variables, CD23 and IgM intensity might be useful prognostic markers in the management of CD5+, B-cell CLL. The survival of CD5- patients was on the borderline of being significantly shorter than that of CD5+ patients. The majority of the CD5- cases were FMC7+, CD23-, had strong IgM fluorescence, and had splenomegaly.
S100-positive, T-cell chronic lymphoproliferative disease: an aggressive disorder of an uncommon T-cell subset
Authors: Hanson CA , Bockenstedt PL , Schnitzer B , Fox DA , Kueck B , Braun DK
Published: October 2019

S100-positive T lymphocytes account for less than 3% of peripheral blood T cells. Rare cases of S100-positive T-cell lymphoma have been previously described. We report four such cases of S100-positive T-cell chronic lymphoproliferative disease. In all cases, hepatosplenomegaly was observed, without prominent lymphadenopathy. Central nervous system (CNS) involvement by the leukemic cells was suggested in three cases by physical symptoms and confirmed in two cases by cerebrospinal fluid studies. Despite treatment, three patients died at 3, 6, and 8 months after diagnosis. Although there was a leukemic presentation, only minimal bone marrow infiltration was evident. Splenectomy showed red pulp infiltration. Liver and lymph node biopsies showed sinusoidal leukemic involvement. In all cases, the leukemic cells expressed mature T-cell- and natural killer cell-associated antigens. Cytoplasmic S100 was detected in the leukemic cells in the blood, spleen, liver, and lymph node. Southern blot studies in two cases showed T-beta, T-gamma, and T-delta gene rearrangements. RNA Northern blots showed T-alpha and T-beta chain transcripts with no T-gamma or T-delta RNA identified. Southern blot analysis showed no hybridization to probes specific for Epstein-Barr virus, cytomegalovirus, human immunodeficiency virus-1, or human T-cell lymphotropic virus type-1. These findings show that S100- positive T-cell chronic lymphoproliferative disorder is an aggressive, extramedullary-based disease frequently associated with CNS involvement and characterized by short survivals.
Radiolabeled anti-CD45 monoclonal antibodies target lymphohematopoietic tissue in the macaque
Authors: Matthews DC , Appelbaum FR , Eary JF , Hui TE , Fisher DR , Martin PJ , Durack LD , Nelp WB , Press OW , Badger CC
Published: October 2019

Despite bone marrow transplantation, many patients with advanced leukemia subsequently relapse. If an additional increment of radiation could be delivered to lymphohematopoietic tissues with relative specificity, the relapse rate may decrease without a marked increase in toxicity. We have examined the biodistribution of two 131I-labeled monoclonal antibodies reactive with the CD45 antigen in Macaca nemestrina. Three animals received 0.5 mg/kg BC8, an IgG1 of low avidity (6 x 10(7) L/mol). Three received 0.5 mg/kg AC8, an IgG2a of moderate avidity (5 x 10(8) L/mol), and two received 4.5 mg/kg AC8. Estimates of radiation absorbed dose demonstrated that these antibodies could deliver up to five times more radiation to lymph nodes, and up to 2.6 times more to bone marrow, than to lung or liver. The higher avidity AC8 antibody at 0.5 mg/kg was cleared more rapidly from blood and resulted in lower antibody uptake in lymph nodes than did BC8 at 0.5 mg/kg. Increasing the dose of AC8 to 4.5 mg/kg resulted in slower blood clearance and higher lymph node uptake. These studies suggest that radiolabeled anti-CD45 antibodies can deliver radiation with relative specificity to lymphohematopoietic tissues. This approach, in combination with marrow transplantation, may improve treatment of hematologic malignancies.
The impact of a very high purity factor VIII concentrate on the immune system of human immunodeficiency virus-infected hemophiliacs: a randomized, prospective, two-year comparison with an intermediate purity concentrate [see comments]
Authors: de Biasi R , Rocino A , Miraglia E , Mastrullo L , Quirino AA
Published: October 2019

Pathophysiologic considerations as well as non-comparative clinical results suggest that very high purity concentrates may slow immunologic deterioration in human immunodeficiency virus (HIV)-infected hemophiliacs. In an attempt to evaluate this hypothesis, we prospectively compared CD4 cell counts, skin testing responses, and changes of the clinical status in 20 asymptomatic HIV-positive hemophiliacs, randomly assigned to continue the treatment with an intermediate purity concentrate or to receive a very high purity product, purified by immunoaffinity chromatography with monoclonal antibodies. In the group switched to the very high purity concentrate there was no significant change of the CD4 cell counts over the 96-week follow-up period, whereas in the group continued on the intermediate purity concentrate, a highly significant decline was detected (P less than .013). Furthermore, in the very high purity group, four of six anergic patients at entry acquired reactivity to skin testing. The results of this study clearly support the use of very high purity concentrates for the replacement therapy of HIV-infected hemophiliacs.
T-cell depletion of HLA-identical transplants in leukemia
Authors: Marmont AM , Horowitz MM , Gale RP , Sobocinski K , Ash RC , van Bekkum DW , Champlin RE , Dicke KA , Goldman JM , Good RA
Published: October 2019

We analyzed the effects of T-cell depletion on the outcome of HLA- identical sibling bone marrow transplants for leukemia by comparing 731 T-cell-depleted transplants with 2,480 non-T-cell-depleted transplants. T-cell depletion decreased acute graft-versus-host disease (GVHD) (relative risk [RR] 0.45; P less than .0001) and chronic (GVHD) (RR 0.56; P less than .0001). However, it increased graft failure (RR 9.29; P less than .0001). Leukemia relapse also was increased. In first remission acute leukemia or chronic phase chronic myelogenous leukemia, leukemia relapse was 2.75 times more likely after T-cell-depleted transplants (P less than .0001). T-cell depletion increased the risk of treatment failure (RR 1.35; P less than .0003) and decreased leukemia- free survival. We also studied controllable variables associated with outcome of T-cell-depleted transplants. The unique findings were that among recipients of T-cell-depleted transplants for early leukemia, radiation doses greater than or equal to 11 Gy (RR 0.54; P less than .01), dose rates greater than 14 cGy/min (RR 0.56; P less than .002), and additional posttransplant immune suppression with cyclosporine alone (RR 0.53; P less than .0006) or cyclosporine plus methotrexate (RR 0.36; P less than .01) were associated with fewer treatment failures. Use of monoclonal antibodies rather than physical techniques for T-cell depletion (RR 2.01; P less than .03) and fractionated radiation (RR 1.69; P less than .05) were associated with increased treatment failure and lower leukemia-free survival. These data may be useful in designing strategies to improve results of T-cell-depleted transplants.
Absence of the human retinoblastoma gene product in the megakaryoblastic crisis of chronic myelogenous leukemia
Authors: Towatari M , Adachi K , Kato H , Saito H
Published: October 2019

The human retinoblastoma gene (RB) product, which is involved in the control of cell cycle and tumor suppression, is constitutively expressed as a nuclear phosphoprotein in normal human cells. We examined leukemic cells from 22 patients with blast crisis of chronic myelogenous leukemia (CML) for alterations of the RB expression. Western blotting and flow cytometry with anti-RB-protein antibodies showed that all of five cases with megakaryoblastic crisis lacked the expression of the RB-encoded protein, whereas none of 17 cases with the other phenotypes such as myeloblastic or lymphoblastic crisis showed any abnormality. These findings suggest that megakaryoblastic transformation of CML might be lineage-specifically associated with loss of the RB protein.
High-dose recombinant interleukin-2 and acute myeloid leukemias in relapse
Authors: Maraninchi D , Blaise D , Viens P , Brandely M , Olive D , Lopez M , Sainty D , Marit G , Stoppa AM , Reiffers J
Published: October 2019

Interleukin-2 (IL-2) is able to induce the regression of metastatic cancers when administered in vivo. IL-2-activated natural killer cells and lymphocytes show, in vitro, activities against leukemic cells. To assess if in vitro observations could have significant clinical relevance, we evaluated the in vivo activity of high-dose recombinant IL-2 (6 to 8 x 10(6) IU/m2/8H intravenous bolus for 5 days) in 10 patients with acute myeloid leukemias (AML) in relapse after chemotherapy (n = 7) or autologous bone marrow transplantation (n = 3). Two patients achieved a complete remission and one had a minimal improvement in his marrow blast cells. Response was observed after one cycle of IL-2 in the two patients achieving a complete remission. These two patients relapsed at 3 and 4 months. These results showing clinical activity of high-dose recombinant IL-2 in AML invite further evaluation of this new form of immunotherapy in other clinical situations, like an adjuvant setting for selected groups of high-risk patients.
Minimal residual disease in patients with chronic myelogenous leukemia undergoing long-term treatment with recombinant interferon alpha-2b alone or in combination with interferon gamma
Authors: Opalka B , Wandl UB , Becher R , Kloke O , Nagel-Hiemke M , Moritz T , Beer U , Seeber S , Niederle N
Published: October 2019

Interferon (IFN) therapy has become widely used for the treatment of chronic myelogenous leukemia. Hematologic remissions can be induced in about 60% of patients. Moreover, in a small number of patients loss of the Philadelphia (Ph) chromosome and of the BCR-ABL rearrangement is observed. We have used genomic Southern blotting as well as a two-step polymerase chain reaction (PCR) method to score for BCR-ABL messenger RNA (mRNA) in patients with hematologic remission induced by treatment with IFN alpha-2b alone or in combination with IFN gamma. Concomitantly, cytogenetic analysis was performed. In 11 of 115 patients reported here, a complete loss of rearranged BCR fragments was observed in Southern blots of peripheral blood (PB) and/or bone marrow (BM) cell samples. Malignant marker bands disappeared first in the PB. In six patients, this genotype remained stable, whereas in five patients, low-intensity, rearranged bands reappeared despite continuation of treatment. The reappearance of the malignant marker was not accompanied by a clinical relapse. Ph-negative metaphases were observed in PB cells of four patients and in the PB and BM cells of two of these patients. In the samples of the other patients, residual Ph- positive cells were detected. By two-step PCR, residual BCR-ABL rearranged transcripts were found in samples of 10 patients.
Bidirectional effects of transforming growth factor beta (TGF-beta) on colony-stimulating factor-induced human myelopoiesis in vitro: differential effects of distinct TGF-beta isoforms
Authors: Jacobsen SE , Keller JR , Ruscetti FW , Kondaiah P , Roberts AB , Falk LA
Published: October 2019

Transforming growth factor-beta (TGF-beta) has potent antiproliferative effects on human hematopoietic progenitor cells. We report here that TGF-beta 1 and -beta 2 also exert bimodal dose-dependent stimulation of granulocyte-macrophage colony-stimulating factor (CSF) and granulocyte- CSF-induced day 7 granulocyte-macrophage colony-forming units. This increase in colony formation was restricted to low doses (0.01 to 1.0 ng/mL) of TGF-beta 1 and was due to increased granulopoiesis, showing that TGF-beta can affect the differentiation as well as the proliferation of hematopoietic progenitors. Furthermore, TGF-beta 3 was found to be a more potent inhibitor of hematopoietic progenitor cells than TGF-beta 1 and -beta 2. In contrast to the bidirectional proliferative effects of TGF-beta 1 and -beta 2, the effects of TGF- beta 3 on human hematopoiesis were only inhibitory, showing for the first time that TGF-beta isoforms differ not only in potencies but also with regard to the nature of the response they elicit.
The binding domain of von Willebrand factor to sulfatides is distinct from those interacting with glycoprotein Ib, heparin, and collagen and resides between amino acid residues Leu 512 and Lys 673
Authors: Christophe O , Obert B , Meyer D , Girma JP
Published: October 2019

A series of proteolytic fragments of human von Willebrand Factor (vWF) was purified to characterize the functional site that supports its interaction with sulfatides. SpIII, an N-terminal homodimer generated by V-8 protease (amino acids [AA] 1 to 1365), bound to sulfatides in a dose-dependent and saturable way. SpIII also totally inhibited the binding of vWF to sulfatides and SpIII binding was completely abolished by vWF. In contrast, SpII, the complementary C-terminal homodimer (AA 1366 to 2050), did not exhibit any binding affinity for sulfatides. Four purified fragments overlapping the sequence of SpIII were also tested for their ability to interact with sulfatides. An N-terminal monomeric 34-Kd fragment (P34, AA 1 to 272) generated by plasmin, a central monomer (SpI, AA 911 to 1365) produced by digestion with V-8 protease, and a tetrameric fragment III-T2 (comprising a pair of the two sequences AA 273 to 511 and AA 674 to 728) produced by secondary digestion of SpIII with trypsin did not interact with sulfatides. In contrast, a monomeric 39/34-Kd fragment produced by dispase (AA 480 to 718) bound specifically and with a high affinity to sulfatides and totally displaced vWF or SpIII binding. Conversely, binding of the 39/34-Kd species was totally abolished by vWF or SpIII. Thus, a functional site responsible for sulfatide binding was localized between AA 480 and 718 and comparison of the binding properties of the 39/34-Kd and III-T2 fragments indicated that the sequence 512 to 673 is necessary for the binding to sulfatides. Further mapping of this new functional domain of vWF, based on experiments of competitive inhibition of binding by either heparin or monoclonal antibodies directed toward vWF, showed that the site interacting with sulfatides is distinct from those involved in binding to platelet glycoprotein Ib, collagen, or heparin. This finding was confirmed by experiments using synthetic peptides which also indicated that the sequence comprising AA 569 to 584 is part of the sulfatide-binding domain or influences its activity.
Identification of risk factors for bleeding during treatment of acute venous thromboembolism with heparin or low molecular weight heparin
Authors: Nieuwenhuis HK , Albada J , Banga JD , Sixma JJ
Published: October 2019

In a prospective double-blind trial, we treated 194 patients with acute venous thromboembolism with heparin or low molecular weight heparin (LMWH; Fragmin). To evaluate the most important prognostic factors for bleeding, the presenting clinical features of the patients, the patients' anticoagulant responses, and the doses of the drugs were analyzed using univariate and multivariate regression analyses. No significant differences in clinical risk factors associated with bleeding were observed between heparin and LMWH. The univariate analyses ranked the parameters in the following order of importance: World Health Organization (WHO) performance status, history of bleeding tendency, cardiopulmonary resuscitation, recent trauma or surgery, leukocyte counts, platelet counts, duration of symptoms, and body surface area. Patients with WHO grade 4 had an eightfold increase in risk of bleeding as compared with WHO grade 1. Assessment of the individual contribution of each variable using multivariate regression analysis showed that the WHO performance status was the most important independent factor predicting major bleeding. A history of a bleeding tendency, recent trauma or surgery, and body surface area were also independent risk factors. The risk of bleeding was influenced by two factors related to the treatment, the patient's anticoagulant response as measured with the anti-Xa assay and the dose of the drug expressed as U/24 h/m2. An increased risk of bleeding was only observed at mean anti-Xa levels greater than 0.8 U/mL for both drugs. Significantly more major bleedings occurred in patients treated with high doses of the drugs, an observation that was independent of the concomitant anti-Xa levels. It should be considered whether choosing an appropriate initial dose adapted to the patient's body surface area and clinical risk factors can improve the efficacy to safety ratio of heparin treatment.
Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens
Authors: Silberstein LE , Jefferies LC , Goldman J , Friedman D , Moore JS , Nowell PC , Roelcke D , Pruzanski W , Roudier J , Silverman GJ
Published: October 2019

To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B- cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline- encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti- I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.