bluuebunny/crossref_metadata_2025 huggingface dataset:
Selection of benign primitive hematopoietic progenitors in chronic myelogenous leukemia on the basis of HLA-DR antigen expression
Authors: Verfaillie CM , Miller WJ , Boylan K , McGlave PB
Published: October 2019

Chronic myelogenous leukemia (CML) is a lethal malignancy of the human hematopoietic stem cell. Here we report that coexistent benign, primitive hematopoietic progenitors can be distinguished from their malignant counterparts in CML bone marrow by differences in cell surface antigen expression. Selection of bone marrow cells expressing the CD34 antigen but lacking the HLA-DR antigen results in recovery of small lymphocyte-like blasts, which initiate and sustain production of myeloid clonogenic progeny in vitro. Secondary clonogenic cells derived at week 1, 5, and 8 from long-term bone marrow cultures (LTBMCs) initiated with primitive progenitors, which lack HLA-DR antigens, exhibit neither the Philadelphia chromosome (Ph1) nor the corresponding bcr/abl mRNA characteristic of CML. In contrast, clonogenic cells recovered at week 1, 5, and 8 from LTBMCs initiated with the CML HLA- DR+ population contain Ph1 and express bcr/abl mRNA. This observation indicates that it may be possible to select a population of viable, exclusively benign hematopoietic stem cells from CML bone marrow capable of repopulating the hematopoietic compartment following autologous bone marrow transplantation.
Intensive chemotherapy with mitoxantrone and high-dose cytosine arabinoside followed by granulocyte-macrophage colony-stimulating factor in the treatment of patients with acute lymphocytic leukemia
Authors: Kantarjian HM , Estey EH , O'Brien S , Anaissie E , Beran M , Rios MB , Keating MJ , Gutterman J
Published: October 2019

Thirty-four adults with refractory acute lymphocytic leukemia received salvage therapy with mitoxantrone 5 mg/m2 intravenously over 1 hour daily for 5 days and cytosine arabinoside (ara-C) 3 g/m2 intravenously over 2 hours every 12 hours for six doses, followed by granulocyte- macrophage colony-stimulating factor (GM-CSF) 125 microgram/m2 intravenously over 4 hours daily until recovery of granulocytes above 2.0 x 10(3)/microL. Their outcome was compared with 29 prognostically similar historical control patients treated with the identical chemotherapy without GM-CSF. Overall, the complete response rates were similar in the treatment and control groups (13 of 34 [38%] v 11 of 29 [38%]). There was a trend for less remission induction mortality in the GM-CSF-treated patients (2 of 34 [6%] v 6 of 29 [21%]; P = .08), but, conversely, a higher rate of resistant disease (19 of 34 [56%] v 10 of 29 [34%]; P = .09). Recovery of granulocyte counts above 500/microL was significantly faster in the GM-CSF-treated group (25 days v 33 days; P less than .01), but there was no reduction in the incidence of febrile episodes (91% v 93%) or of documented infections (59% v 59%). Survival was prolonged in the GM-CSF-treated patients but was not of clinical relevance (31 v 20 weeks; P = .05). In summary, the addition of GM-CSF to intensive chemotherapy in refractory adult ALL was associated with a reduction in the remission induction mortality, probably secondary to a shorter duration of granulocytopenia, but not with an improvement in complete response rates.
Mutational activation of the N-ras oncogene assessed in primary clonogenic culture of acute myeloid leukemia (AML): implications for the role of N-ras mutation in AML pathogenesis
Authors: Bashey A , Gill R , Levi S , Farr CJ , Clutterbuck R , Millar JL , Pragnell IB , Marshall CJ
Published: October 2019

The number of steps involved in the pathogenesis of acute myeloid leukemia (AML) is unclear. The initiating event would be expected to exist in all leukemic cells, but subsequent events may be subclonal. If several genetic events occur, they may cooperate within the same cell or be alternatively acquired by different subclones. These possibilities cannot be adequately analyzed in DNA prepared directly from patient specimens. In this study, N-ras mutations demonstrable in DNA prepared from peripheral blood of 10 patients with AML were examined in primary in vitro colonies (AML-colony-forming units [CFU]) grown from these patients. Both colonies containing the mutant gene and colonies containing normal allele only were obtained from each patient. The proportion of colonies containing no mutant allele varied among patients (5% to 57%). A subset of mutation containing colonies appeared to have lost the normal allele in nine of 10 AML cases analyzed. In the four cases with two N-ras mutations, the two mutations were found to exist in different subclones. In these cases, macroscopic colonies (AML- MCFU) were also obtained using an assay system designed to select for earlier clonogenic cells than in the AML-CFU assay. The N12cys mutation in AML10 was found in the CFU, but not in the MCFU, and the N12asp mutation in AML43 was found in the MCFU, but not in the CFU. These results suggest that N-ras mutation is a postinitiation event in AML that contributes to the outgrowth of more malignant subclones. Where two mutations are found in a case of AML, they appear to have been acquired by separate subclones, which may show different degrees of differentiation.
On the Optical Properties of the Hemoglobin in Microdrepanocytic Disease
Authors: ASCENZI A. , SILVESTRONI E.
Published: October 2019

In analogy with sickle cell anemia of the Negro, white carriers of the sickle cell trait and patients with microdrepanocytic disease show sickling which is associated with the presence of type S hemoglobin. The sickled cells are birefringent and the reduced hemoglobin forms tactoids which reveal a striking resemblance to the sickled erythrocytes.

The mean retardation of each sickled red cell is 7.5 ± 0.15 mµ and the birefringence is approximately 0.0037.
White Thrombo-embolism and Vascular Fragility in the Hamster Cheek Pouch after Anticoagulants
Authors: FULTON GEORGE P. , AKERS ROBERT P. , LUTZ BRENTON R.
Published: October 2019

1. Heparin, dicumarol, Tromexan and phenylindanedione did not prevent, but actually enhanced, the formation of platelet plugs at the site of hemorrhages produced at the tip of a stimulating microelectrode in contact with the walls of small blood vessels in the hamster cheek pouch. Furthermore, these anticoagulants per se produced an increased adhesiveness of platelets and leukocytes to the endothelium of small venules. In particular, heparin produced platelet embolism immediately after administration. The circulating emboli varied in size from that of leukocytes to cylindrical masses which filled the lumina of vessels measuring up to 75 µ in diameter. Platelet embolism was not found during the first five days of administration of dicumarol, but appeared subsequently.

2. The significance of increased platelet agglutinability during the hypocoagulable state induced by anticoagulants and the need for a critical distinction between agglutinability of the formed elements and coagulation involving fibrin are discussed.

3. An increase in the fragility of the walls of venules followed the administration of heparin, dicumarol, Tromexan and phenylindanedione. A brief series of relatively weak faradic shocks from a micro-electrode produced a hemorrhage. Shocks of much greater strength applied to venules of normal untreated hamsters produced no visible effect. This method is proposed as a new semiquantitative procedure for investigation of vascular fragility in accessible membrane preparations. It may possibly distinguish between fragility in the sense of rhexis or breaking of the wall and petechial formation such as that produced by the application of negative pressure and perhaps erroneously referred to in the literature as "fragility."

4. Petechial hemorrhages appeared at the points of junction of the postcapillary venules after anticoagulant administration, but not on the capillaries. Spontaneous ruptures were not found in capillaries. No evidence was found for an increase in capillary fragility. Consequently, the term "vascular fragility" is proposed to replace "capillary" fragility.
The Adrenalin (Epinephrin) Test as Applied to Hematologic Disorders
Authors: CHATTERJEA JYOTI B. , DAMESHEK WILLIAM , STEFANINI MARIO
Published: October 2019

1.The epinephrin test was performed in 12 normal subjects and 63 patients with various hematologic disorders to evaluate its role in the diagnosis of hypersplenic syndromes and aplastic anemias. Attention was directed solely to the early phases of the reaction, that is to the pre-adrenocortical stimulatory effect, which has been claimed to reflect the functional activity of the spleen and bone marrow.

2. The epinephrin response in normal subjects was characterized by transitory pancytosis. Leukocytosis and thrombocytosis were conspicuous features whereas erythrocytosis and reticulocytosis were not regularly seen. Leukocytosis was due to an increase of mature forms of neutrophils, lymphocytes, monocytes and eosinophils. The Arneth count did not show any significant variation after epinephrin. There was no correlation in different cases between the degree of hemopoietic response and the degree of rise of blood pressure, although in each case the time of maximum cellular response usually ran parallel to the time of maximum rise of blood pressure.

3. The response in splenectomized individuals was essentially similar to or slightly greater than that seen in normal subjects. In splenomegalic conditions there was no correlation between the degree of splenic contraction and the degree of hemopoietic response.

4. Results of epinephrin injection into the splenic artery showed that the increase of cellular elements was in general pancellular in type, and not confined to any particular cell deficient in the peripheral blood; it was obviously independent of the nature and degree of peripheral cytopenia.

5. The splenic contraction following epinephrin may possibly be due to a marked constriction of the splenic artery and in its intrasplenic branches with the resultant passive deflation of the organ.

6. In each case there was a close correlation between the epinephrin response and the functional activity of the bone marrow.

7. The epinephrin test per se is not diagnostic of any clinical condition. In the hypersplenic syndromes interpretation of negative tests may actually be misleading. At most, the test provides indirect corroborative evidence regarding the functional status of the blood forming tissues and to some extent of the "reservoir" of blood cells present not only in the bone marrow, but in various tissues and organs throughout the body. The exact degree of splenic participation in the epinephrin response is not known, but our studies indicate that it is minimal.
Some Aspects of the Metabolism of Antimegaloblastic Substances in Man
Authors: GIRDWOOD RONALD H.
Published: October 2019

1. When the sera of pernicious anemia patients or controls were heated at 100 C. for 30 minutes, they developed the ability to support the growth of L. leichmannii by virtue of some substance other than vitamin B12. It seemed likely, however, that following the administration of the vitamin, such heating also liberated free B12 in the serum from a combined form.

2. The L. leichmannii assay did not appear to be satisfactory for showing possible difference in the levels of vitamin B12 in the urines or sera of pernicious anemia patients and controls.

3. Parenterally administered vitamin B12 did not cause any measurable rise in the serum level of folic acid or citrovorum factor in pernicious anemia patients or controls.

4. The synthetic folic acid conjugates pteroyldiglutamic acid and pteroyltriglutamic acid appeared in the sera and urines of pernicious anemia patients as pteroylglutamic acid or some related substance with folic acid activity for S. faecalis.

5. Orally administered citrovorum factor appeared to be largely converted by the gastric juice to folic acid if free hydrochloric acid was present. When administered parenterally, however, citrovorum factor was excreted in the urine largely unchanged.
Preservation of Leukocytes
Authors: TULLIS JAMES L.
Published: October 2019

A method for the separation and preservation of leukocytes is presented and discussed. The significance of different methods for measurement of cell viability are delineated.
Hemolytic Disease of the Newborn Due to Anti-A
Authors: CRAWFORD HAL , CUTBUSH MARIE , MOLLISON P. L.
Published: October 2019

Eleven cases of hemolytic disease of the newborn are described in which the only blood group antibody in the mother's serum, incompatible with the infant's cells, was anti-A. The direct antiglobulin (Coombs) test on the infant's red cells was weakly positive in 7 cases and negative in 4 cases. In every case the mother's serum displayed immune characteristics, in particular the ability to lyse A cells.

Osmotic fragility was increased in 10 out of 11 cases. This finding is contrasted with those in a series of cases of hemolytic disease of the newborn due to anti-Rh.
Production of Nucleophagocytosis by Rabbit Antileukocytic Serum
Authors: ZIMMERMAN HYMAN J. , WALSH JOHN R. , HELLER PAUL
Published: October 2019

Dried buffy coat layer of the blood from a patient with granulocytic leukemia was injected into a rabbit. Serum from this rabbit, when mixed with the buffy coat layer of the blood from the same patient, resulted in the production of nucleophagocytosis. The serum of the rabbit also showed the ability to produce marked conglutination and destruction of the neutrophilic polymorphonuclear leukocytes.
Bone Marrow Changes in Patients with Chronic Leukemia Treated by Splenic X-irradiation
Authors: GUNZ F. W.
Published: October 2019

1. Following therapeutic x-irradiation of the spleen in patients suffering from chronic myeloid leukemia alterations in the composition of the bone marrow have been noted in a high proportion of cases. These consisted of a decrease in the number of immature myeloid cells or an increase in that of the immature erythroid cells or both.

2. Eighty minutes after local irradiation of the leukemic spleen with therapeutic doses of x-rays there was found a diminution in the number of mitoses among myeloid cells in the bone marrow.
Acquired Hemolytic Anemia
Authors: DACIE J. V.
Published: October 2019

Eighteen patients with acquired hemolytic anemia were studied with particular reference to the direct and indirect antiglobulin (Coombs) reaction. The abnormal antibodies present were of the warm type in eleven patients and of the cold type in seven patients.

With warm antibodies a well marked zone of inhibition was usually observed when sensitized red cells were suspended in high concentrations of antiglobulin serum, and the reaction was readily inhibited by the previous addition of very small amounts of human γ globulin to the antiglobulin serum. Acidification of the serum to pH 6.5 caused in most cases a moderate increase in the intensity of sensitization in the indirect antiglobulin test. Previous heating of the serum at 56 C. for 30 minutes did not prevent sensitization.

With cold antibodies, on the other hand, no inhibition of the antiglobulin reaction took place in the highest concentration of antiglobulin serum used, and the reaction was not inhibited by the previous addition of small amounts of human γ globulin to the antiglobulin serum. Acidification of the serum to pH 6.5 caused, in most cases, a considerable increase in the intensity of the reaction. Previous heating of the serum at 56 C. for 30 minutes completely prevented sensitization.

The separation of the abnormal antibodies of acquired hemolytic anemia into warm and cold types cuts across clinical classification. Warm antibodies were found in the majority of idiopathic cases; cold antibodies were present in some idiopathic cases and in hemolytic anemia following virus pneumonia. Splenectomy was followed by sustained remissions in some of the idiopathic cases of both the warm antibody and cold antibody varieties.
Secondary Hemochromatosis
Authors: AUFDERHEIDE ARTHUR C. , HORNS HOWARD L. , GOLDISH ROBERT J.
Published: October 2019

1. Secondary hemochromatosis has been sharply separated from simple hemosiderosis by defining the former as "a condition acquired as a consequence of anemia, blood transfusions, or both, and characterized by increased hepatic and total body iron content and unequivocal portal cirrhosis of the liver."

2. Previously reported cases are critically reviewed in the light of this definition.

3. Two new cases of secondary (exogenous) hemochromatosis are reported.

4. Anemia is postulated as the basic etiologic factor in secondary hemochromatosis by causing increased iron absorption; iron introduced in the form of blood transfusions probably only accelerates a process already in progress.

5. Prolonged futile oral iron therapy may be harmful.

6. A plea is made for a strict concept of secondary hemochromatosis as well as for thorough documentation of future reports.
Deletion of chromosome 20q in myelodysplasia can occur in a multipotent precursor of both myeloid cells and B cells
Authors: White NJ , Nacheva E , Asimakopoulos FA , Bloxham D , Paul B , Green AR
Published: October 2019

Deletions of the long arm of chromosome 20 are associated with several myeloid malignancies and, in particular, with myeloproliferative disorders and myelodysplastic syndromes (MDS). Together with deletions of chromosome 5q and chromosome 7q, chromosome 20q deletions have previously been thought to be restricted to myeloid cells in patients with MDS. We report here that deletion of chromosome 20q in MDS can arise in a multipotent precursor of both myeloid cells and B cells. Clonal Epstein-Barr virus (EBV)-transformed cell lines, both with and without a 20q deletion, have been isolated from a patient with MDS. Moreover, these cell lines have been shown to provide a useful physical mapping tool and have been used to confirm the interstitial nature of the 20q deletion. Microsatellite polymerase chain reaction (PCR) and PCR analysis of PGK gene methylation have been used to study highly purified populations of peripheral blood cells. The 20q deletion was detectable by microsatellite PCR in peripheral blood granulocytes and monocytes but not in B cells or T cells. Clonality of the different lineages followed the same pattern as the 20q deletion. This represents the first report in which a chromosome abnormality associated with MDS has been immortalized in an EBV-transformed lymphoblastoid cell line. Furthermore, our data show that patients with apparent myeloid restriction of a chromosome deletion may in fact have a disease arising in a multipotent cell with both myeloid and lymphoid potential.
Detection of 14q32 translocations in B-cell malignancies by in situ hybridization with yeast artificial chromosome clones containing the human IgH gene locus
Authors: Taniwaki M , Matsuda F , Jauch A , Nishida K , Takashima T , Tagawa S , Sugiyama H , Misawa S , Abe T , Kashima K
Published: October 2019

Partner sites of 14q32 translocations found in B-cell malignancies were detected by fluorescence in situ hybridization (FISH) using yeast artificial chromosome (YAC) clones, Y20 and Y6, containing the human Ig heavy chain (IgH) gene locus. Y20 spans a 160-kb upstream and 40-kb downstream region of the JH segments on chromosome band 14q32.33. Y6 is 300-kb upstream of Y20, and spans a further 320-kb telomeric region. The human DNA sequences amplified by Alu polymerase chain reaction of the YAC clones were used as probes for FISH to study six patients with non-Hodgkin's lymphoma (NHL), one patient with acute lymphoblastic leukemia, and one cell line FR4 established from a plasmacytoma. Three telomeric YAC clones each specific for 3q, 8q, and 18q were also used to further characterize 14q32 translocations. The IgH YACs were successfully applied to detect cytogenetically invisible subtelomeric translocation of the IgH gene locus to each partner site in t(14;18), t(8;14), and t(14;19), and to identify t(3;14) (q27;q32.33) in three patients with 14q32 translocation of unknown origin. Furthermore, complex translocations involving more than three chromosomes were detected in an NHL patient with t(8;14), and t(3;12), and in the FR4 with der(14)t(8;14), der(8)dic(1;8), and del(1)(q21). The technique would be a useful tool in elucidating the mechanisms of a 14q32 translocation in B-cell malignancies.
Serum procollagen type III is an early and sensitive marker for veno- occlusive disease of the liver in children undergoing bone marrow transplantation
Authors: Heikinheimo M , Halila R , Fasth A
Published: October 2019

Veno-occlusive disease of the liver (VOD) is a life-threatening complication occurring in patients undergoing bone marrow transplantation (BMT). Although clinical signs and laboratory parameters such as elevation of serum bilirubin often suggest this condition, it would be useful to identify early biochemical markers for VOD. Fibrous alterations in the hepatic venules and small lobular veins occur during development of VOD; these changes are accompanied by the deposition of types I and III collagen in the liver tissue. Since the N- terminal propeptide of type III procollagen (PIIINP) is a sensitive marker of liver and lung fibrosis, we undertook a study to evaluate the usefulness of measurements of serum PIIINP in children with VOD. Seven of the 28 children who underwent BMT, both allogenic and autologous, developed VOD. All seven had an increase of more than 100 ng/mL in the serum PIIINP level, whereas only one of the remaining 21 children not affected by VOD had an increment of PIIINP more than 100 ng/mL (P = .0001). The levels of serum PIIINP were higher in the VOD group during the follow-up period of up to 91 days after BMT. The elevation of PIIINP also occurred at a stage of the disease usually preceding any other laboratory or clinical signs of VOD. Serum concentration of PIIINP thus seems to be of value as an early marker for VOD in children undergoing BMT.
Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML
Authors: Deisseroth AB , Zu Z , Claxton D , Hanania EG , Fu S , Ellerson D , Goldberg L , Thomas M , Janicek K , Anderson WF
Published: October 2019

Relapse after autologous bone marrow transplantation for chronic myelogenous leukemia (CML) can be due either to the persistence of leukemia cells in systemic tissues following preparative therapy, or due to the persistence of leukemia cells in the autologous marrow used to restore marrow function after intensive therapy. To help distinguish between these two possible causes of relapse, we used safety-modified retroviruses, which contain the bacterial resistance gene NEO, to mark autologous marrow cells that had been collected from patients early in the phase of hematopoietic recovery after in vivo chemotherapy. The cells were then subjected to ex vivo CD34 selection following collection and 30% of the bone marrow were exposed to a safety-modified virus. This marrow was infused after delivery of systemic therapy, which consisted of total body irradiation (1,020 cGy), cyclophosphamide (120 mg/kg), and VP-16 (750 mg/m2). RT PCR assays specific for the bacterial NEO mRNA, which was coded for by the virus, and the bcr-abl mRNA showed that in two evaluable CML patients transplanted with marked cells, sufficient numbers of leukemia cells remained in the infused marrow to contribute to systemic relapse. In addition, both normal and leukemic cells positive for the retroviral transgenome persisted in the systemic circulation of the patients for at least 280 days posttransplant showing that the infused marrow was responsible for the return of hematopoiesis following the preparative therapy. This observation shows that it is possible to use a replication-incompetent safety-modified retrovirus in order to introduce DNA sequences into the hematopoietic cells of patients undergoing autologous bone marrow transplantation. Moreover, this data suggested that additional fractionation procedures will be necessary to reduce the probability of relapse after bone marrow transplantation in at least the advanced stages of the disease in CML patients undergoing autologous bone marrow transplantation procedures.
Platelet and Megakaryocyte Dense Granules Contain Glycoproteins Ib and IIb-IIIa
Authors: Youssefian Tayebeh , Massé Jean-Marc , Rendu Francine , Guichard Josette , Cramer Elisabeth M.
Published: October 2019

Platelets contain two main types of secretory organelles, the dense granules and the α-granules. P-selectin, a specific receptor for leukocytes that is present in the α-granule membrane, has also been demonstrated to be associated with the dense granule limiting membrane, showing that a relationship exists between these two types of secretory granules. We have previously shown that the plasma membrane receptors glycoproteins (Gp) IIb-IIIa and Ib are also present in the α-granule membrane. To document further the composition of the dense granule membrane, we have used immunoelectron microscopy in the present work to determine if the dense granule membrane also contains these glycoproteins. First, the cytochemical method of Richards and Da Prada (J Histochem Cytochem 25:1322, 1977), which specifically enhances dense body electron density, was combined with immunogold-labeled anti–Gp IIb-IIIa or anti–Gp Ib antibody. A consistent and reproducible labeling for Gp IIb-IIIa, but less for Gp Ib, was found in the membrane of platelet dense granules. Subsequently, double immunogold labeling was performed on frozen thin sections of resting platelets using antibodies directed against the dense body components granulophysin or P-selectin, followed by anti–Gp IIb-IIIa or anti–Gp Ib. Consistent labeling for Gp IIb-IIIa and weaker labeling for Gp Ib were detected in dense bodies. The possibility that the granulophysin-positive structures could be lysosomes was excluded by the presence of P-selectin. Immunogold labeling of isolated dense granule fractions confirmed these results. Identical findings were made on human cultured megakaryocytes using double immunolabeling. In conclusion, this study demonstrates the presence of Gp IIb-IIIa and Gp Ib on the dense granule membrane. This observation provides additionnal evidence of similarities between the α-granule and dense granule membranes and raises the possibility of a dual mechanism responsible for the formation of dense granules similar to that of α-granules, ie, endogenous synthesis as well as endocytosis from the plasma membrane.
Metabolism of Thrombopoietin (TPO) In Vivo: Determination of the Binding Dynamics for TPO in Mice
Authors: Stefanich Eric , Senn Tauri , Widmer Ramon , Fratino Christine , Keller Gilbert-André , Fielder Paul J.
Published: October 2019

Previous in vivo studies have established that plasma thrombopoietin (TPO) levels are regulated by binding to c-Mpl on platelets and that, in vitro, platelets bind and degrade TPO. To determine if the in vivo metabolism of TPO was specific and saturable, we injected normal CD-1 mice IV with trace amounts of 125I-rmTPO with or without a saturating concentration of rmTPO. The amount of radioactivity present in the spleen, blood cell fraction, platelet fraction, tibia/fibula, and femur was significantly greater in the mice receiving 125I-rmTPO alone. Conversely, the amount of radioactivity present in the plasma was significantly greater in the mice receiving both 125I-rmTPO and rmTPO, thus suggesting the uptake of rmTPO by the spleen, platelets, and bone marrow in vivo was saturable. Platelet and spleen homogenates from animals receiving 125I-rmTPO alone showed a degradation pattern of 125I-rmTPO similar to that observed in vitro using mouse platelet rich plasma. To determine the in vivo binding dynamics for rmTPO, mice were injected with 125I-rmTPO alone or with increasing concentrations of rmTPO; spleen and blood cell-associated radioactivity was determined at 2 hours postinjection. A 4-parameter curve fit of the data indicated that the “in vivo binding affinity” for rmTPO was approximately 6.4 μg/kg. These data indicate that after a dose of approximately 6.4 μg/kg, 50% of all c-Mpl receptors will be saturated with rmTPO. Electron microscopy indicated that radioactivity was present bound to and within megakaryocytes and platelets in both sternum and spleen and platelets in circulation. Together these data demonstrate that in vivo, 125I-rmTPO is mainly metabolized by platelets and to a small extent by cells of the megakaryocyte lineage, via a specific and saturable mechanism.
Detection of Trisomy 12 and Rb-Deletion in CD34+ Cells of Patients With B-Cell Chronic Lymphocytic Leukemia
Authors: Gahn B. , Schäfer C. , Neef J. , Troff C. , Feuring-Buske M. , Hiddemann W. , Wörmann B.
Published: October 2019

B-cell chronic lymphocytic leukemia (B-CLL) is a slowly progressive disease characterized by the clonal expansion of CD5+/CD23+ B lymphocytes. The malignant transformation is assumed to occur at the level of mature B lymphocytes. We asked whether CD34+ progenitor cells are involved in the malignant process in B-CLL. Furthermore, we investigated the possibility of aberrant CD34 expression by the malignant B-cell clone. Bone marrow and peripheral blood samples from 75 patients with B-CLL were tested for the presence of trisomy 12 and deletion of the retinoblastoma gene (Rb) by fluorescence in situ hybridization. CD34+ subpopulations were isolated by fluorescence-activated cell sorting and analyzed for the presence of the informative genetic marker. Bone marrow and peripheral blood samples of 10 B-CLL patients were analyzed for coexpression of CD34/CD5/CD20. Trisomy 12 was detected in 15 of 75 (20%) and Rb-deletion was detected in 6 of 30 patients (20%). In 7 patients with trisomy 12, hematopoietic progenitor cells were sorted, with the sort purity being between 85% and 99.8%. The genetic marker was detected in the CD34+/CD38+ cells as well as in the CD34+/38− subsets in 3 patients. Progenitor cells were also sorted in 2 patients with Rb-deletion. In 1 patient, Rb-deletion was present in 10% of CD34+/38+ cells. In the other patient, Rb-deletion was neither detected in the CD34+/38+ nor in the CD34+/CD38− subsets. In all 10 patients investigated for coexpression of CD34/CD5/CD20, we could not find a subpopulation coexpressing these markers. We conclude that trisomy 12 and Rb-deletion are present in a considerable subset of patients with B-CLL. In part of these patients, the genetic marker was detected at the level of CD34+ stem cells. CD34 expression is not related to an aberrant phenotype of the malignant B-cell clone. These results suggest that the malignant transformation in B-CLL may involve early hematopoietic stem cells and place a note of caution on future strategies using autologous stem cell transplantation.
Hematopoietic Potential and Retroviral Transduction of CD34+Thy-1+ Peripheral Blood Stem Cells From Asymptomatic Human Immunodeficiency Virus Type-1–Infected Individuals Mobilized With Granulocyte Colony-Stimulating Factor
Authors: Junker Uwe , Moon James J. , Kalfoglou Creton S. , Sniecinski Irena , Forman Stephen J. , Zaia John A. , Kaneshima Hideto , Böhnlein Ernst
Published: October 2019

The potential of hematopoietic stem cells (HSCs) from human immunodeficiency virus type-1 (HIV-1)–infected individuals, eg, self-renewal and multilineage differentiative capacity, might be perturbed due to the underlying disease. In this study, we assessed the HSC activity in the CD34+Thy-1+ cell population of peripheral blood stem cells (PBSCs) of three asymptomatic HIV-1–infected individuals after granulocyte colony-stimulating factor (G-CSF; 10 μg/kg/d) mobilization. On day 4 of G-CSF treatment, 0.8% to 1% of the total blood mononuclear cells were CD34+. Leukapheresis followed by a two-step cell isolation process yielded a CD34+Thy-1+ cell population of high purity (76% to 92% CD34+Thy-1+ cells). This cell population showed no evidence of HIV-1–containing cells based on a semiquantitative HIV-1 DNA polymerase chain reaction. Furthermore, the purified cells showed normal hematopoietic potential in in vitro clonogenic assays. Successful gene transfer into committed progenitor cells (colony-forming units-cells) and more primitive stem/progenitor cells (long-term culture colony-forming cells) could be shown after amphotropic retroviral transduction. These data provide evidence that the CD34+Thy-1+ stem cell compartment can be mobilized and enriched in early stage HIV-1–infected patients. Furthermore, successful transduction of this cell population as a prerequisite for stem cell-based clinical gene therapy protocols was demonstrated.
In Vitro Maintenance of Highly Purified, Transplantable Hematopoietic Stem Cells
Authors: Moore Kateri A. , Ema Hideo , Lemischka Ihor R.
Published: October 2019

The cellular and molecular mechanisms that regulate the most primitive hematopoietic stem cell are not well understood. We have undertaken a systematic dissection of the complex hematopoietic microenvironment to define some of these mechanisms. An extensive panel of immortalized stromal cell lines from murine fetal liver were established and characterized. Collectively, these cell lines display extensive heterogeneity in their in vitro hematopoietic supportive capacity. In the current studies, we describe a long-term in vitro culture system using a single stromal cell clone (AFT024) that qualitatively and quantitatively supports transplantable stem cell activity present in highly purified populations. We show multilineage reconstitution in mice that received the equivalent of as few as 100 purified bone marrow and fetal liver stem cells cultured for 4 to 7 weeks on AFT024. The cultured stem cells meet all functional criteria currently ascribed to the most primitive stem cell population. The levels of stem cell activity present after 5 weeks of coculture with AFT024 far exceed those present in short-term cytokine-supported cultures. In addition, maintenance of input levels of transplantable stem cell activity is accompanied by expansion of other classes of stem/progenitor cells. This suggests that the stem/progenitor cell population is actively proliferating in culture and that the AFT024 cell line provides a milieu that stimulates progenitor cell proliferation while maintaining in vivo repopulating activity.
Role of Glycoprotein V in the Formation of the Platelet High-Affinity Thrombin-Binding Site
Authors: Dong Jing-Fei , Sae-Tung Garunee , López José A.
Published: October 2019

The glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ibα, GP Ibβ, GP IX, and GP V. The site within the complex that binds thrombin has been localized to a 45-kD region at the amino terminus of GP Ibα, which also contains the site through which the complex interacts with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be efficiently expressed on the surface of transfected cells. We examined the ability of L cells expressing the GP Ib-IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells. Because it is one of the few substrates for thrombin on the platelet surface, GP V has also been implicated as possibly participating in thrombin's actions on the platelet. To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of thrombin to L2H cells versus cells that express the entire GP Ib-IX-V complex (L2H/V cells). Surface expression of GP Ibα was equivalent in these two stable cell lines. Thrombin binding to L2H/V cells was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L. No binding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently expressing GP V was compared with its binding to sham-transfected L2H cells. Again, only cells transiently expressing GP V bound thrombin specifically. As with the platelet polypeptide, thrombin cleaved GP V from the surface of L2H/V cells. To test whether GP V cleavage was required for enhancing thrombin binding to the complex, we tested the binding of enzymatically inactive D-phenylalanyl-Lprolyl-L-arginine chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating that GP V cleavage is not a prerequisite for the formation of the high-affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the high-affinity thrombin receptor on the platelet surface has complex allosteric requirements.
The Missense Mutation Arg593 → Cys Is Related to Antibody Formation in a Patient With Mild Hemophilia A
Authors: Fijnvandraat Karin , Turenhout Ellen A.M. , van den Brink Edward N. , ten Cate Jan W. , van Mourik Jan A. , Peters Marjolein , Voorberg Jan
Published: October 2019

The development of inhibitory antibodies to factor VIII in patients affected by a mild form of hemophilia A (factor VIII < 0.05 IU/mL) is considered a rare event. In this study, we evaluated the relationship between genotype and anti-factor VIII antibody formation in a patient with mild hemophilia A. Mutation analysis showed that a missense mutation in the factor VIII gene leading to replacement of Arg593 by Cys in the A2 domain of factor VIII was associated with hemophilia A in this patient. The anti-factor VIII antibodies present in the patient's plasma were characterized using metabolically labeled factor VIII fragments expressed in insect cells. The anti-factor VIII antibodies, composed of subclasses IgG2 and IgG4, reacted with both the fragment corresponding to the factor VIII heavy chain and the A2 domain. The Arg593 → Cys substitution was introduced into the cDNA encoding the A2 domain of factor VIII and the resulting construct was expressed in insect cells. Strikingly, the metabolically labeled A2 domain carrying the Arg593 → Cys mutation was not recognized by the anti-factor VIII antibodies present in the plasma of the patient. These data indicate that the anti-factor VIII antibodies are exclusively directed against exogenous factor VIII. This strongly suggests that the Arg593 → Cys substitution results in recognition of wild-type factor VIII as nonself and is thereby related to the formation of anti-factor VIII antibodies after factor VIII replacement therapy in this particular patient.
Reassessment of Protein Tyrosine Phosphorylation in Thrombasthenic Platelets: Evidence That Phosphorylation of Cortactin and a 64-kD Protein Is Dependent on Thrombin Activation and Integrin αIIbβ3
Authors: Rosa Jean-Philippe , Artçanuthurry Valérie , Grelac Françoise , Maclouf Jacques , Caen Jacques P. , Lévy-Toledano Sylviane
Published: October 2019

Tyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin αIIbβ3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack αIIbβ3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% αIIbβ3, functional), and variant type (50% αIIbβ3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional αIIbβ3 in initiating protein tyrosine phosphorylation. We noted that in addition to the characteristic absence of the 100 to 105 kD doublet, a 77 to 80 kD doublet and to a lesser extent a 64-kD band, exhibited low phosphorylation kinetics, but with normal initial phosphorylation rates (up to 60 seconds). Similar results were obtained by inhibition of thrombin aggregation of control platelets by αIIbβ3 antagonists (the RGDS peptide or the monoclonal antibody 10E5), or in the absence of stirring (fibrinogen binding, but no aggregation). These results suggest that tyrosine phosphorylation of the 77 to 80 kD doublet, identified by immunoprecipitation as the cytoskeletal protein cortactin, and the 64 kD band are dependent both on thrombin activation during early steps and on the late steps of αIIbβ3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.
The Conversion of Fibrinogen to Fibrin: Recombinant Fibrinogen Typifies Plasma Fibrinogen
Authors: Gorkun Oleg V. , Veklich Yuri I. , Weisel John W. , Lord Susan T.
Published: October 2019

Plasma fibrinogen is a mixture of multiple molecular forms arising mainly through alternative mRNA processing and subsequent posttranslational modification. Recombinant fibrinogen is synthesized without alternative mRNA processing in a cultured cell system that may generate novel posttranslational modifications. Thus, to show that recombinant fibrinogen can serve as a functional model for plasma fibrinogen, we have examined the conversion of fibrinogen to fibrin, comparing the recombinant with the plasma protein. We examined the kinetics of (1) thrombin-catalyzed fibrinopeptide release, (2) thrombin-catalyzed polymerization of fibrinogen, (3) the polymerization of fibrin monomers, and (4) FXIIIa-catalyzed cross-link formation. We saw small differences in polymerization, suggesting that the ordered assembly of protofibrils and fibers was not identical. In all other analyses, we found that plasma fibrinogen and recombinant fibrinogen were remarkably similar. Using electron microscopy, we examined the structures of individual fibrinogen molecules and fibrin clots. Individual fibrinogen molecules were predominantly three nodule structures for both recombinant and plasma proteins. Both samples also displayed four nodule structures, but fewer four nodule structures were found with recombinant fibrinogen. Fibrin clot structures were essentially indistinguishable. We concluded that recombinant fibrinogen can serve as a accurate model for plasma fibrinogen.
Chimeric CLL-1 Antibody Fusion Proteins Containing Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2 With Specificity for B-Cell Malignancies Exhibit Enhanced Effector Functions While Retaining Tumor Targeting Properties
Authors: Hornick Jason L. , Khawli Leslie A. , Hu Peisheng , Lynch Maureen , Anderson Peter M. , Epstein Alan L.
Published: October 2019

Although monoclonal antibody (MoAb) therapy of the human malignant lymphomas has shown success in clinical trials, its full potential for the treatment of hematologic malignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman B-cell MoAb (chCLL-1) constructed using the variable domains cloned from the murine Lym-2 (muLym-2) hybridoma, fusion proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)-2 (chCLL-1/IL–2) were generated and evaluated for in vitro cytotoxicity and in vivo tumor targeting. The glutamine synthetase gene amplification system was employed for high level expression of the recombinant fusion proteins. Antigenic specificity was confirmed by a competition radioimmunoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony formation assay, and the bioactivity of chCLL-1/IL–2 was confirmed by supporting the growth of an IL-2–dependent T-cell line. Antibody-dependent cellular cytotoxicity against ARH-77 target cells demonstrated that both fusion proteins mediate enhanced tumor cell lysis by human mononuclear cells. Finally, biodistribution and imaging studies in nude mice bearing ARH-77 xenografts indicated that the fusion proteins specifically target the tumors. These in vitro and in vivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2 have potential as immunotherapeutic reagents for the treatment of B-cell malignancies.
GABP Cooperates With c-Myb and C/EBP to Activate the Neutrophil Elastase Promoter
Authors: Nuchprayoon Issarang , Simkevich Carl P. , Luo Menglin , Friedman Alan D. , Rosmarin Alan G.
Published: October 2019

Neutrophil elastase (NE) is a serine protease that is transcriptionally regulated during early myeloid differentiation. The murine NE (mNE) promoter contains functionally important c-Myb, C/EBP, and ets binding sites. Deletion of the ets site reduced promoter activity by 90%. Although the ets transcription factor, PU.1, bound to this ets site, it only modestly activated the mNE promoter. Here, we show that a second transcription factor from myeloid cells — GABP — binds to the mNE ets site but strongly activates the mNE promoter. GABP is a heteromeric transcription factor complex that consists of GABPα, an ets factor, and GABPβ, a Notch-related protein. GABPα bound to the mNE ets site and, in turn, recruited GABPβ to form a transcriptionally active complex. GABPα and PU.1 competed with each other for binding to the mNE ets site. GABP increased the activity of the mNE promoter sevenfold in U937 myeloid cells. GABP cooperated with c-Myb and C/EBPα to activate the mNE promoter more than 85-fold in otherwise nonpermissive, nonhematopoietic NIH 3T3 cells. Thus, GABP binds to the crucial mNE promoter ets site and powerfully activates its expression alone and in cooperation with the transcription factors c-Myb and C/EBP.
Pertussis Toxin Shows Distinct Early Signalling Events in Platelet-Activating Factor–, Leukotriene B4–, and C5a-Induced Eosinophil Homotypic Aggregation In Vitro and Recruitment In Vivo
Authors: Teixeira Mauro M. , Giembycz Mark A. , Lindsay Mark A. , Hellewell Paul G.
Published: October 2019

The present study was performed to investigate the early signalling events responsible for eosinophil activation in response to platelet-activating factor (PAF ), C5a, and leukotriene B4 (LTB4 ). We evaluated the effect of pertussis toxin (PTX) on eosinophil aggregation in vitro and cutaneous eosinophil recruitment in vivo. Further studies using the protein kinase inhibitors Ro 31-8220 and staurosporine were performed in vitro to assess in more detail the early signalling events induced by these three mediators. Our results show that C5a and LTB4 signal predominantly or exclusively through a PTX-sensitive G protein that is negatively modulated by protein kinase C, possibly at the level of phospholipase C-β. In contrast, PAF activates eosinophils independent of Gi by a mechanism that is abolished by Ro 31-8220, a selective protein kinase C inhibitor. In addition, these results show for the first time that a receptor-operated event on the eosinophil is essential for chemoattractant-induced eosinophil recruitment in vivo.