bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal anti-idiotype antibody therapy of B-cell lymphoma: the addition of a short course of chemotherapy does not interfere with the antitumor effect nor prevent the emergence of idiotype-negative variant cells
Authors: Maloney DG , Brown S , Czerwinski DK , Liles TM , Hart SM , Miller RA , Levy R
Published: October 2019

The Ig idiotype of B-cell lymphoma can be used as a tumor-specific target. Prior trials with monoclonal anti-idiotype antibodies alone and combined with alpha-interferon have shown significant antitumor activity. In some patients, idiotype-negative tumors emerged after treatment. In this trial, patients with relapsed non-Hodgkin's lymphoma were treated with two identical courses of monoclonal anti-idiotype anti-body therapy. Concurrent with the second course, at a time when idiotype-negative cells were suspected to be proliferating, a pulse dose of chlorambucil was administered. Tumor biopsies obtained before the first and second courses of treatment and at relapse were analyzed for idiotype expression and proliferation. Thirteen patients received 24 courses of antibody with minimal toxicity. Eleven had tumor regression, with 1 complete remission, 8 partial remissions, and 2 minor remissions, with freedom from progression lasting a median of 7 months in responding patients. Idiotype-negative tumor cells appeared in some relapse specimens despite the use of chlorambucil. In retrospect, this was not surprising because there was no increase in the proliferative rate of these tumors at the time the drug was used. Anti-idiotype antibodies continue to demonstrate antitumor activity against B-cell lymphoma with minimal toxicity. The mechanism of the effect is presumed to involve both direct antiproliferative effects of the antibody on the tumor cells as well as indirect, more long-lasting effects on the host. The addition of a mild chemotherapeutic agent in the dose and schedule used here to the second cycle of antibody therapy did not interfere with the antitumor effect, nor did it decrease the emergence of idiotype-negative cells.
Granulocyte-macrophage colony-stimulating factor induces neutrophil adhesion to pulmonary vascular endothelium in vivo: role of beta 2 integrins
Authors: Yong KL , Rowles PM , Patterson KG , Linch DC
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) causes upregulation of neutrophil surface CD11b/CD18 expression, and enhances the adhesion of neutrophils to cultured human endothelial cells in vitro. Systemic administration of GM-CSF results in a rapid, transient decrease in circulating phagocyte numbers. Using a nonhuman primate model (Cynomolgus), we provide histologic evidence that this transient leukopenia is associated with the margination of neutrophils in the pulmonary microcirculation. In four animals receiving 2 to 15 micrograms/kg recombinant human GM-CSF (rhGM-CSF), light microscopic sections of lung contained 36 +/- 8, 17 +/- 7, 21 +/- 6, and 15 +/- 8 (mean +/- SD, n = 20) neutrophils within a graticule grid, as compared with two control animals receiving saline injections whose lung sections contained 2.1 +/- 1.6 and 3.1 +/- 2.1 (mean +/- SD, n = 20) neutrophils within the same grid. Scanning electron microscopy shows activated leukocytes adherent to pulmonary vascular endothelium, but no morphologic evidence of endothelial damage, and no migration of cells into the extravascular space. Margination is associated with an increase in surface expression of CD11b/CD18 on circulating phagocytes, which could contribute to the adhesion to capillary endothelial cells, but CD11b/CD18 levels remain elevated even when demargination is complete. In vitro, monoclonal antibodies (MoAbs) to CD18 and CD11b were able to inhibit neutrophil aggregation and adhesion to endothelium. FMLP-induced neutrophil aggregation was inhibited by 39.8% +/- 11.5% and 44.8% +/- 12.3%, respectively, by MoAbs to CD18 and CD11b (P less than .0005, n = 4 for both); a similar effect was demonstrated on TPA-induced aggregation. MoAb CD18 reduced the adhesion of unstimulated neutrophils to endothelium by 44% (P less than .01, n = 7), and inhibited the amount of GM-CSF-stimulated adhesion by 74% (P less than .001, n = 7), while MoAb to CD11b produced a reduction of unstimulated neutrophil adhesion by 30%, and of GM-CSF-stimulated adhesion by 40% (P less than .01, n = 5, for both). However, when administered in vivo, MoAb CD18 produced only a small, albeit significant, amelioration of GM-CSF-induced margination in vivo, while MoAb CD11b was without effect. These results show that GM-CSF-induced transient leukopenia is associated with enhanced neutrophil adherence to pulmonary vascular endothelium, but suggest that the beta 2 leukocyte integrins CD11/CD18 play only a minor role in this process.
Rapid detection of Spanish (delta beta)zero-thalassemia deletion by polymerase chain reaction
Authors: Vives-Corrons JL , Pujades MA , Miguel-Garcia A , Miguel-Sosa A , Cambiazzo S
Published: October 2019

delta beta-Thalassemia and hereditary persistence of fetal hemoglobin (HPFH) are inherited disorders characterized by the persistent synthesis of fetal hemoglobin (HbF) during adult life. The Spanish type of delta beta-thalassemia is a mild thalassemic condition due to a large deletion starting at the Alu I repeat between the A gamma and delta-globin genes immediately 3′ to the RIH probe and extending 11 and 17 kb downstream of the 3′ endpoints of HPFH 1 and HPFH 2, respectively. Using probes from the Spanish (delta beta)zero- thalassemic DNA, the 3′ breakpoint region has been mapped to a point approximately 8.5 to 9.0 kb downstream from that of HPFH type 1 and, as we know the restriction sites 3′ to this breakpoint, the presence of the deletion can be identified with the polymerase chain reaction (PCR). In the present study, a PCR method using three specific oligonucleotides has been developed for the identification of the Spanish (delta beta)zero-thalassemia in 100 patients with delta beta- thalassemia (99 heterozygotes with mild anemia, decreased mean corpuscular volume, and 5% to 15% HbF, and one homozygote with 100% HbF and thalassemia intermedia phenotype). We conclude that the finding of the Spanish type of (delta beta)zero-thalassemia in all the patients studied here suggests Spain as the most probable origin of this thalassemic phenotype. Moreover, the amplification of the fragment encompassing the deletion junction and normal sequence is useful for the rapid molecular detection of Spanish (delta beta)zero-thalassemia.
Developmental switching of messenger RNA expression from the human alpha-globin cluster: fetal/adult pattern of theta-globin gene expression
Authors: Albitar M , Care A , Peschle C , Liebhaber SA
Published: October 2019

The alpha-globin gene cluster contains four functional globin genes, zeta, alpha 2, alpha 1, and theta. The developmental regulation of the embryonic zeta and fetal/adult alpha 2- and alpha 1-globin genes is well characterized at the level of protein synthesis. The developmental pattern of the theta-globin gene is not well characterized due to the inability to detect its encoded protein. Direct analysis of the globin switching at the steady-state messenger RNA (mRNA) level has been hampered by the difficulty in obtaining quantities of embryonic and early fetal mRNA sufficient for analysis. We analyzed the relative levels of the steady-state zeta-, alpha-, and theta-globin mRNAs in yolk sac in 5-, 6-, 7-, and 8-week postconception embryonic liver, and in cord and adult blood reticulocytes. We show that the switch in the alpha-globin gene cluster from the embryonic to fetal/adult pattern of expression begins at 5 to 6 weeks of gestation. Both the theta- and alpha-globin genes show similar patterns of developmental control that are reciprocal to zeta. alpha-globin RNA is barely detectable or undetectable at 5 weeks, and increases in the 6- to 8-week period, while theta-globin mRNA shows a parallel increase at 5 to 8 weeks postconception and is expressed in cord blood and adult reticulocytes. These data show that the theta-globin gene represents a fetal/adult gene, albeit expressed at a low level.
Promotion of murine marrow alloengraftment and hematopoietic recovery across the major histocompatibility barrier by administration of recombinant human interleukin-1 alpha [see comments]
Authors: Blazar BR , Widmer MB , Taylor PA , Vallera DA
Published: October 2019

Irradiated C57BL/6 (H-2b) recipients of T-cell-depleted (TCD) BALB/c (H- 2d) bone marrow (BM) and recombinant interleukin-1 alpha (IL-1 alpha) (1 microgram/d) had a significantly (P less than or equal to .006) higher 100-day actuarial survival rate, accelerated hematopoietic recovery, and higher levels of alloengraftment than a group of transplanted control mice treated identically, but given phosphate- buffered saline (PBS). To elucidate the mechanisms involved with IL-1 alpha-induced promotion of alloengraftment and hematopoietic recovery, we performed sequential splenic FACS studies on transplanted mice and secondary transfer studies in syngeneic mice given IL-1 alpha or PBS. Splenic phenotyping showed that recipients of IL-1 alpha had a higher proportion of donor granulocytes (52% v 19%) as compared with PBS controls as early as 7 days after bone marrow transplantation (BMT). On day 11 post-BMT, recipients of IL-1 alpha had a more than fourfold increase in splenocyte number, which included a higher percentage (90% v 59%) of donor cells, especially donor granulocytes (52% vs 32%), and a sevenfold increase in donor T cells as compared with controls. Host T- cell numbers were not affected. Taken together, these data suggest that IL-1 alpha stimulated bipotential (myeloid and lymphoid) donor cell engraftment. In a syngeneic BMT system, administration of IL-1 alpha resulted in a higher incidence of survival when recipients were transplanted with BM cells, indicating that IL-1 alpha administration probably either expanded or potentiated engraftment of a committed progenitor cell pool. Secondary transfer experiments using marrow from IL-1 alpha-treated mice showed that the number of day 12 colony-forming unit-spleen (CFU-S) cells was unaltered compared with untreated control mice, suggesting that more primitive, albeit committed, hematopoietic progenitor cells were not affected. We also examined the potential additive effects of IL-1 alpha and granulocyte-macrophage colony- stimulating factor (GM-CSF) administered in combination (for 14 days). Mice receiving a suboptimal amount of IL-1 alpha along with GM-CSF had significantly higher levels of donor alloengraftment (92%) with superior hematopoietic recovery, as compared with mice receiving either IL-1 alpha (57%) or GM-CSF (18%) alone.
Progress in understanding the pathogenesis of the anemia of chronic disease [see comments]
Authors: Means RT Jr , Krantz SB
Published: October 2019

Improved understanding of the inflammatory response and the identification and characterization of the specific cytokines involved, as well as improved understanding of erythropoiesis, and the availability of recombinant human growth factors such as EPO, have greatly enhanced our appreciation of the pathogenesis of ACD by allowing development of a number of informative models for studying this syndrome. It appears that a variety of cytokines are involved in all aspects of the pathogenesis of ACD, from the inhibition of erythroid progenitors and EPO production to impairment of iron release. A schematic of the contributions of some of these cytokines to the development of ACD is shown in Fig 6. The exact biochemical mechanisms by which these effects occur is still to be determined. The progress outlined in this report has allowed us to develop a more precise understanding of the pathogenesis of this common and important clinical syndrome. In 1983, Hansen subtitled a review of ACD “A Bag of Unsolved Questions.” Although this description is still accurate, our understanding of ACD has now developed to the point where we can offer a more defined subtitle: “A Bag of Cytokines.”
Enhanced marrow recovery by short preincubation of marrow allografts with human recombinant interleukin-3 and granulocyte-macrophage colony- stimulating factor
Authors: Naparstek E , Hardan Y , Ben-Shahar M , Nagler A , Or R , Mumcuoglu M , Weiss L , Samuel S , Slavin S
Published: October 2019

We studied an alternative method of using hematopoietic growth factors (HGFs) to enhance hematopoietic recovery in patients undergoing bone marrow transplantation (BMT), by short in vitro preincubation. Twenty consecutive patients with leukemia received T-cell-depleted allografts using Campath-1G. Two thirds of the marrow was infused on the scheduled day of transplant and one third of the marrow following preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on day 4. Engraftment parameters and duration of hospitalization were compared by actuarial analysis to those of 40 historical controls. Patients receiving the incubated boost had significantly faster platelet recovery (P = .017) and shorter hospitalization period (P = .001) when compared with the control subjects. Platelet count reached greater than 25 x 10(9)/L on day 17 (median) in the study group and on day 23 in the controls. The median duration of hospitalization was 20 and 36 days, respectively. In the early posttransplantation follow-up, two of four patients in the study group died as a result of graft rejection, while all 13 deaths in the control group resulted from complications associated with marrow suppression. We suggest that pretransplant in vitro activation of bone marrow cells with IL-3 and GM-CSF may prove to be an efficient method for enhancing marrow recovery after BMT.
Acute lymphoblastic leukemia in patients aged 60 years and over: a population-based study of incidence and outcome
Authors: Taylor PR , Reid MM , Bown N , Hamilton PJ , Proctor SJ
Published: October 2019

Data on all patients aged 60 or older in whom a diagnosis of acute lymphoblastic leukemia (ALL) was made in the Northern Health Region of the United Kingdom, over a period of 8 1/2 years, were studied to determine the incidence and range of biological subtypes, and the outcome of a variety of therapeutic approaches. Forty-nine cases were diagnosed (31% of all adult ALL), giving an incidence of 0.9 per 100,000 age-specific population (ASP). Immunophenotyping was performed in 41 patients. Eighteen had common, two T-ALL, 10 null, and 7 B-ALL. Four were biphenotypic with both lymphoid and myeloid markers. One patient refused treatment and four were considered too frail for chemotherapy. Twenty-two were treated palliatively. Aggressive “curative” treatment was planned for 22, but three died before it started. The actuarial survival for the whole group was 4%. The median survival of the palliatively treated group was 1 month, compared with 3 months for those given aggressive treatment. Only two patients (aged 74 and 67) survived more than 2 years in complete remission (CR), both having been treated aggressively. We conclude that the prognosis for those over 60 years of age is extremely poor, but that for a small number of patients a useful prolongation of good quality life may be achieved. Age should not, by itself, be a bar to treatment intended to induce remission. Patients and their relatives should be made aware of the overall poor prognosis and advised of the therapeutic goals when decisions regarding management are being made.
Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt
Authors: Erickson P , Gao J , Chang KS , Look T , Whisenant E , Raimondi S , Lasher R , Trujillo J , Rowley J , Drabkin H
Published: October 2019

We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5′ sequences of AML1 to 3′ sequences of ETO, oriented telomere to centromere on the der(8) chromosome.
Retinoic acid-resistant HL-60R cells harbor a point mutation in the retinoic acid receptor ligand-binding domain that confers dominant negative activity
Authors: Robertson KA , Emami B , Collins SJ
Published: October 2019

Retinoic acid (RA) induces granulocytic differentiation of acute promyelocytic leukemia (APL) cells and is a useful therapeutic agent for patients with this disease. In the HL-60 promyelocytic leukemia cell line, this RA-induced granulocytic differentiation appears to be directly mediated through the RA receptor (RAR-alpha). We have previously identified a mutant subclone of HL-60 (designated HL-60R) that exhibits relative resistance to RA and that harbors RA receptors with markedly reduced affinity for RA. In the present study, we have now identified the genetic basis for this aberrant RA receptor activity. DNA sequencing of polymerase chain reaction-amplified cDNA products corresponding to the RAR-alpha ligand-binding domain shows a point mutation in RAR-alpha codon 411 in this mutant HL-60R subclone. This specific C-->T mutation generates a termination codon resulting in the truncation of 52 amino acids at the COOH terminal end of RAR-alpha. In cotransfection studies, expression vectors harboring this mutated RAR-alpha exhibit dominant negative activity with respect to the trans- activating function of the normal RAR-alpha. Although our observations are limited to HL-60 cells, similar RA receptor mutations might play an important role in the acquisition of RA resistance in RA-treated APL patients.
Thrombotic microangiopathies in the 1980s: clinical features, response to treatment, and the impact of the human immunodeficiency virus epidemic
Authors: Thompson CE , Damon LE , Ries CA , Linker CA
Published: October 2019

We reviewed the medical records of 44 adults with 50 consecutive episodes of thrombotic thrombocytopenia purpura (TTP) or hemolytic uremic syndrome (HUS) seen at the University of California, San Francisco affiliated hospitals during the past decade. Patients were treated according to a uniform plan in which initial therapy included daily large volume plasmapheresis using fresh frozen plasma. Patients not responding completely to initial therapy were treated with a salvage regimen including splenectomy, dextran, and corticosteroids. At the time of diagnosis, the lactate dehydrogenase (LDH) was elevated in 98% of cases, with a median value of 1,208 U/L. Other clinical features were present inconsistently, and only 34% of “TTP” episodes involved the classic pentad of hemolytic anemia, thrombocytopenia, neurologic disorders, noninfectious fever, and renal impairment. Primary treatment with plasma exchange produced complete remission in 56% (27 of 48) of the episodes. Previously splenectomized patients uniformly responded to plasma therapy (12 of 12). In patients not responding completely to primary therapy, salvage splenectomy produced complete responses in 81% (13 of 16) of the cases. The pattern of clinical response to therapy was consistent, with initial resolution of neurologic dysfunction (median, 3 days) followed by normalization of LDH levels (5 days) and platelet count (7 days). Normalization of renal function occurred significantly later (15 days). Although short-term responses to plasma therapy in human immunodeficiency virus (HIV)-seropositive patients did not differ from other patients, no HIV-positive patient survived more than 2 years from diagnosis of thrombotic microangiopathy (TMA). We conclude that the diagnosis of TMA requires a high degree of clinical suspicion and that the diagnostic criteria should consist of microangiopathic hemolytic anemia, thrombocytopenia, and an elevated LDH. Initial therapy with plasma exchange leads to disease control in the majority of cases, but an optimal treatment strategy requires the use of alternative methods if initial remission is transient or not achieved. Salvage therapy with splenectomy, steroids, and dextran is highly effective in this setting.
Dissociation of human cytokine receptor expression and signal transduction
Authors: Linnekin D , Park LS , Farrar WL
Published: October 2019

We have examined the relationship between granulocyte-macrophage colony- stimulating factor (GM-CSF) and interleukin-3 (IL-3) receptor expression and signal transduction in populations of HL-60 cells differing in proliferative capacity to these cytokines. GM-CSF or IL-3 stimulation of HL-60 cells pretreated with either dimethyl sulfoxide (DMSO) or retinoic acid results in increases in proliferative response as well as both tyrosine and serine phosphorylation. In contrast, neither GM-CSF or IL-3 stimulation of parental HL-60 cells (those not treated with DMSO or retinoic acid) produced any changes in either proliferation or protein phosphorylation. Thus, although parental HL-60 cells expressed both GM-CSF and IL-3 receptors, treatment with either DMSO or retinoic acid was necessary to confer the capacity for signal transduction as assessed by both a biologic and biochemical response. Pretreatment of cells with genistein, a protein tyrosine kinase inhibitor, resulted in inhibition of GM-CSF-induced protein tyrosine phosphorylation as well as proliferation. These data show a strong correlation between cytokine-induced increases in protein phosphorylation and subsequent biologic responses. Further, this work demonstrates that cytokine receptor expression and signal transduction can be disassociated and suggests the potential for independent regulation of these two components of signal transduction.
Enhanced hematopoiesis in vivo and in vitro by splenic stromal cells derived from the mouse with recombinant granulocyte colony-stimulating factor
Authors: Fukushima N , Nishina H , Koishihara Y , Ohkawa H
Published: October 2019

Splenic stromal cells (CF-1 cells) were established from a mouse administered recombinant human granulocyte colony-stimulating factor (rG-CSF) to clarify the mechanism of splenic extramedullary hematopoiesis induced by the factor. The cells were negative for alkaline phosphatase, factor VIII-related antigen, mac I, and phagocytosis. They were positive for acid phosphatase, collagen type I, collagen type III, and fibronectin. CF-1 cells were not converted to adipocytes in a confluent culture with 10(-6) mol/L hydrocortisone. [35S]rG-CSF bound to CF-1 cells specifically in the growth phase but not in the resting phase. The CF-1 cells had greater colony-stimulating activities than the normal splenic stromal cells. When CF-1 cells were added to bone marrow cells in the spleen colony-forming cells (CFU-S) assay, the number of colonies in the spleen increased between 1.4 and 1.8 times the control without these stromal cells. On the other hand, the normal splenic stromal cells had no effect on increasing the number of CFU-S colonies. Therefore, these data suggest that a factor- dependent hematopoietic microenvironment is generated in the spleen by rG-CSF, and the stromal cells that have the hematopoietic potency become dominant in splenic extramedullary hematopoiesis induced by rG- CSF.
BALB/3T3 fibroblast-conditioned medium attracts cultured mast cells derived from W/W but not from mi/mi mutant mice, both of which are deficient in mast cells
Authors: Jippo-Kanemoto T , Adachi S , Ebi Y , Matsuda H , Kasugai T , Nishikawa S , Kitamura Y
Published: October 2019

Proliferation of murine mast cells is induced by both T-cell-derived and fibroblast-derived growth factors. Because the most potent T-cell- derived mast cell growth factor, interleukin-3, promotes the migration of mast cells, we investigated whether fibroblast-derived growth factors had the chemoattractive activity as well. Conditioned medium (CM) of BALB/3T3 fibroblasts induced the migration of cultured mast cells (CMC) derived from normal (+/+) mice. BALB/3T3-CM contained the mast cell growth factor (MGF)/stem cell factor (SCF)/kit ligand (KL), which is the ligand for the receptor encoded by the W (c-kit) gene. CMC derived from the spleen of W/W mice lack the extracellular domain of the W (c-kit) receptor, and W/W CMC did not proliferate in response to BALB/3T3-CM. However, W/W CMC did migrate normally toward BALB/3T3-CM and, moreover, the antibody to the extracellular domain of the W (c- kit) receptor did not inhibit the chemoattractive activity of +/+ CMC toward BALB/3T3-CM. These results indicated that MGF/SCF/KL itself did not represent the major chemoattractive activity. On the other hand, BALB/3T3-CM induced neither proliferation nor migration of CMC derived from mi/mi mice. Both W/W and mi/mi mice are deficient in mast cells, but the present results suggest that the mechanism of the abnormality is different between W/W and mi/mi mice.
Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids A alpha 461-610 (Lys 461 AAA-->stop TAA)
Authors: Koopman J , Haverkate F , Grimbergen J , Egbring R , Lord ST
Published: October 2019

In the A alpha-chain gene coding for an abnormal fibrinogen (fibrinogen Marburg) we identified a single base substitution (A-->T) that changes the codon A alpha 461 AAA (Lys) to TAA (Stop). The propositus was found to be homozygous for the mutation, whereas the father and five siblings were heterozygous, and three other siblings contained only the normal sequence. The stop codon at position 461 results in the deletion of the carboxyl-terminal segment A alpha 461–610. Purified fibrinogen Marburg contained an A alpha-chain with a relative molecular weight of approximately 47,000. The FpA release by thrombin was not affected by this deletion, whereas the fibrin polymerization was strongly decreased. The binding of endothelial cells to immobilized fibrinogen Marburg was almost completely abolished compared with normal fibrinogen. Fibrinogen Marburg contained a substantial amount of albumin linked to the fibrinogen molecule by disulfide bonds, and these fibrinogen-albumin complexes were also present in plasma. The plasma fibrinogen concentration of the propositus was measured by three different methods: a functional method (< 0.25 mg/mL), an immunologic method using polyclonal antibodies (0.6 mg/mL), and an immunologic method based on two monoclonal antibodies specific for the amino- terminus and carboxyl-terminus of the A alpha-chain (< 0.05 mg/mL). Using the two immunologic methods, it appeared that only 10% to 15% of the plasma fibrinogen of the heterozygous siblings was abnormal.
Factor Xa interacts with two sites on monocytes with different functional activities
Authors: Worfolk LA , Robinson RA , Tracy PB
Published: October 2019

Studies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IX alpha through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (alpha-HFV1). At the monocyte surface, the factor Va/Xa- catalyzed activation of prothrombin was maximally inhibited with 0.25 mumol/L alpha-HFV1, whereas 1.0 mumol/L alpha-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did not cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.
Maturation of the hemostatic system during childhood
Authors: Andrew M , Vegh P , Johnston M , Bowker J , Ofosu F , Mitchell L
Published: October 2019

The hemostatic system is assumed to be similar in children and adults and reference ranges established for adults are commonly used to evaluate children suspected of having congenital or acquired hemostatic problems. However, we know that the hemostatic system is not fully mature by 6 months of age and comprehensive studies of healthy older children have not been published. Therefore, we conducted a prospective cohort study of the hemostatic system in healthy children having minor, elective day surgery. After obtaining informed consent, a 3-mL blood sample was obtained at the time routine preoperative blood work was drawn. The plasma was fractioned and stored at -70 degrees C for batch assaying. We measured the concentration of 33 components of the hemostatic system (functional and immunologic assays) and the bleeding time (automated pediatric device) in 246 children aged 1 to 16 inclusive (a minimum of four subjects at each age). Eleven components of hemostasis (fibrinogen, prekallikrein, high-molecular weight kininogen, factors VIII and XIII, antithrombin III [ATIII], heparin cofactor II [HCII], alpha 1-antitrypsin [alpha 1AT], protein S, plasminogen, alpha 2-antiplasmin [alpha 2AP]) had mean values and ranges of normal that were similar to adults. Mean values of seven coagulants (II, V, VII, IX, X, XI, XII) were significantly lower than adult values and varied with age. Values for three inhibitors, alpha 2- macroglobulin (alpha 2M), protein C, and protein C1-inhibitor (C1-Inh) also differed from adults. Alpha 2M and C1-Inh inhibitor levels were elevated throughout childhood, whereas protein C levels were low, with a lower limit of normal of 0.40 U/mL until the age of 11. Finally, the upper limit of normal for the bleeding time was longer in children during the first 10 years of life, but decreased to adult values in the teenage years. In summary, there are important physiologic differences in the hemostatic system in children compared with adults. The decreased levels of several critical coagulants and increased levels of alpha 2M may contribute in part to the lower risk of thrombotic events in childhood. Age-matched controls should be used for evaluation of the hemostatic system in children with suspected congenital or acquired defects.
Thrombocytosis in patients with tumors producing colony-stimulating factor
Authors: Suzuki A , Takahashi T , Nakamura K , Tsuyuoka R , Okuno Y , Enomoto T , Fukumoto M , Imura H
Published: October 2019

We investigated the cause of thrombocytosis in 14 patients with tumors producing colony-stimulating factor (CSF). Of the 14 patients, 10 had tumors producing granulocyte-CSF (G-CSF) and 4 had tumors producing granulocyte-macrophage--CSF (GM-CSF). Thrombocytosis of greater than 400 x 10(9)/L was noted in 8 of 10 patients with G-CSF-producing tumors and all 4 patients with GM-CSF-producing tumors. Median peak platelet counts were, respectively, 511 x 10(9)/L (range, 384 to 694 x 10(9)/L) and 579 x 10(9)/L (range, 526 to 910 x 10(9)/L) in patients with tumors producing G-CSF and GM-CSF. In most patients, thrombocytosis declined towards the terminal stage. High interleukin-1 (IL-1) and IL-6 levels were found in addition to CSFs in the plasma or culture supernatants of tumor cells obtained from most patients. In patients with GM-CSF- producing tumors, these specimens had megakaryocyte-CSF (Meg-CSF) activity, which was abolished by anti-GM-CSF antibody. These specimens also had megakaryocyte potentiating (Meg-Pot) activity attributable to both GM-CSF and IL-6. In patients with G-CSF-producing tumors, only Meg- Pot activity due to IL-6 was detected. These results indicate that the thrombocytosis in GM-CSF-producing tumors was caused by both the Meg- CSF activity of GM-CSF and the Meg-Pot activity of IL-6 plus GM-CSF, while that in G-CSF-producing tumors was due to the Meg-Pot activity of IL-6.
Identification and expression of a common missense mutation (L302P) in the acid sphingomyelinase gene of Ashkenazi Jewish type A Niemann-Pick disease patients
Authors: Levran O , Desnick RJ , Schuchman EH
Published: October 2019

Types A and B Niemann-Pick disease (NPD) result from the deficient activity of acid sphingomyelinase (ASM; E.C. 3.1.4.12) and the resultant lysosomal accumulation of sphingomyelin. Type A disease is a fatal, neurodegenerative disorder of infancy, whereas type B disease has no neurologic manifestations and is characterized primarily by reticuloendothelial involvement and survival into adulthood. Both disorders occur more frequently among individuals of Ashkenazi Jewish ancestry than in the general population. Recently, a missense mutation in the ASM gene (designated R496L) was detected in more than 30% of the ASM alleles from Ashkenazi Jewish type A NPD patients. We report a second, common mutation that resulted from a T to C transition at nucleotide 905 and predicted a leucine to proline substitution at ASM codon 302 (designated L302P). Notably, the L302P mutation occurred in 23.5% (8 of 34) of the Ashkenazi Jewish type A NPD alleles studied. In contrast, it was not found in any of the ASM alleles from non-Jewish type A patients, in 36 alleles from type B patients, or in 100 ASM alleles from normal Ashkenazi Jewish individuals. To confirm the authenticities of the L302P and R496L mutations, each nucleotide change was separately introduced into the full-length ASM cDNA by site- directed mutagenesis and transiently expressed in COS-1 cells. Neither mutation expressed ASM catalytic activity, consistent with the type A phenotype of homoallelic patients. The identification of the L302P mutation should further facilitate molecular carrier detection for NPD in the Ashkenazi Jewish population, particularly because the L302P mutation can be easily detected using the restriction enzyme, AlwNl.
Fetal compensation of the hemolytic anemia in mice homozygous for the normoblastosis (nb) mutation
Authors: Peters LL , Birkenmeier CS , Barker JE
Published: October 2019

The mouse autosomal recessive mutation nb causes a deficiency of erythroid ankyrin and generates a life-threatening hemolytic anemia in adult mice; however, at birth, nb/nb mice appear to be robust and show no pallor. In our study, the time of disease onset was sought by comparison of nb/nb and +/? mice both in utero and postnatally. Erythroid ankyrin messenger RNA (mRNA) is expressed in fetal erythroid progenitors from normal mice, but is reduced to 10% of normal levels in mutant fetuses. Despite the deficiency of erythroid ankyrin mRNA, 16 and 18 day nb/nb fetuses have normal levels of red blood cells (RBCs) and the RBCs are morphologically normal by scanning electron microscopy. The earliest signs of any clinical anomaly are an increase in the number of circulating reticulocytes and the deposition of minor amounts of iron just before birth in the 18 day fetal nb/nb liver, suggesting that RBCs are being destroyed. Within 24 hours after birth, nb/nb neonates have a slight but significant decrease of their RBC counts. During the next 5 days, the nb/nb RBC counts decrease markedly, the reticulocyte counts assume the mutant adult levels of 60%, the erythrocytes become microcytic and fragmented, and iron deposits accumulate in the liver. The rapid onset of clinical disease postnatally, coupled with our findings that the erythroid ankyrin gene is transcribed in fetal erythroid cell precursors from normal mice, suggest that mechanisms exist in the nb/nb fetus to compensate for the erythroid ankyrin deficiency.
Evidence that the antigens of the Yt blood group system are located on human erythrocyte acetylcholinesterase
Authors: Spring FA , Gardner B , Anstee DJ
Published: October 2019

The Yt blood group system comprises two antigens, Yta and Ytb. Human anti-Yta and human anti-Ytb immune precipitate a component of the same apparent molecular weight as acetylcholinesterase from radioiodinated erythrocytes of appropriate Yt phenotype. Immune precipitates obtained with anti-Yta and anti-Ytb contained acetylcholinesterase activity. In contrast, immune precipitates obtained with human anti-Gya and murine monoclonal anti-CD55, which identify other glycosylphosphatidylinositol- linked erythrocyte surface proteins, did not have acetylcholinesterase activity. Quantitative binding assays using murine monoclonal antiacetylcholinesterase antibodies (AE-1 and AE-2) gave 3,000 to 5,000 binding sites/cell for IgG and 7,000 to 10,000 sites/cell for Fab fragments. Endo F digestion of immune precipitates obtained with AE-1 and anti-Yta indicated that approximately 10% of the enzyme comprises N- glycans. These results indicate that the Yt antigens define an inherited polymorphism on erythrocyte acetylcholinesterase and that the recent assignment of the Yt blood group locus to the long arm of chromosome 7 (Zelinski et al, Genomics 11:165, 1991) provisionally identifies the position of the acetylcholinesterase gene.
Monoclonal antibody against the common beta subunit (beta c) of the human interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony- stimulating factor receptors shows upregulation of beta c by IL-1 and tumor necrosis factor-alpha
Authors: Watanabe Y , Kitamura T , Hayashida K , Miyajima A
Published: October 2019

High-affinity receptors for human granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are composed of two distinct subunits, alpha and beta. Each receptor has its own ligand-specific alpha subunit, and the three receptors share the common beta subunit, beta c. Using a transfectant of NIH3T3 cells expressing the high-affinity human GM-CSF receptor, monoclonal antibodies (MoAbs) against beta c were generated. These MoAbs specifically bound to cells bearing beta c and immunoprecipitated the beta c protein of 120 Kd. Using these MoAbs, expression of beta c was examined. It is known that IL-1 augments the proliferative response of a human factor-dependent hematopoietic cell line TF-1 to either GM-CSF, IL-3, or IL-5, and that it upregulates the high-affinity receptors for GM-CSF, IL-3, and IL-5. Antibody binding and immunoprecipitation demonstrated that IL-1 increased cell surface expression of beta c. This enhancement by IL-1 was accompanied by an increased level of beta c mRNA. In addition, we found that tumor necrosis factor-alpha (TNF- alpha) also increased the expression of beta c, although it did not augment the proliferative response of TF-1 to GM-CSF, IL-3, and IL-5.
Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture
Authors: Valent P , Spanblochl E , Sperr WR , Sillaber C , Zsebo KM , Agis H , Strobl H , Geissler K , Bettelheim P , Lechner K
Published: October 2019

In the murine system, a number of cytokines (including interleukin-3 [IL-3], IL-4, and stem cell factor [SCF]) promote the growth of mast cells (MCs). However, so far little is known about factors controlling differentiation of human MCs. Recent data suggest that human MCs express receptors (R) for SCF. The aim of the present study was to investigate whether recombinant human (rh) SCF induces differentiation of human MCs from their precursor cells. For this purpose, bone marrow (BM; normal donors, n = 6) and peripheral blood (PB; normal donors, n = 11) mononuclear cells (MNC) were cultured in the presence of rhSCF, rhIL-3, rhIL-4, rhIL-9, recombinant human macrophage colony-stimulating factor (rhM-CSF), or control medium in long-term (8 weeks) suspension cultures. After 4 weeks, up to 5% of the MNC (BM and PB) cultured in the presence of rhSCF, but not in the presence of other cytokines, were found to exhibit the characteristics of MCs. These MCs expressed the YB5.B8-reactive domain of the SCF R as well as IgE R, as determined by combined toluidine blue/immunofluorescence staining. Myeloid antigens, likewise expressed on human basophils (ie, CD11b, CDw65, and Bsp-1), could not be detected on these cells. Furthermore, rhSCF, but not rhIL- 3, rhIL-4, rhIL-9, or rhM-CSF, induced dose- and time-dependent increases in the formation of cellular tryptase (an MC-specific enzyme) (rhSCF [100 ng/mL], 1,308 +/- 679 ng/mL v control medium, 18 +/- 6 ng/mL tryptase on day 35 of PB cell cultures), as well as an increase in cellular histamine. After 6 to 8 weeks, when other mature hematopoietic cells decreased, MCs still could be detected in culture, with up to 40% of all cells being MCs. To test whether rhSCF also activates tissue MCs, we performed histamine release experiments (dispersed tissue; lung, n = 3; uterus, n = 3). SCF was found to enhance (by up to 3.4-fold) the capacity of the MCs to release histamine upon cross-linkage of IgE R with anti-IgE. Together, these observations suggest that rhSCF induces in vitro differentiation of human MCs from their BM and PB precursor cells in long-term culture and upregulates MC releasability.
Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles
Authors: Kelton JG , Warkentin TE , Hayward CP , Murphy WG , Moore JC
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.
Characterization of platelet-releasable forms of beta-amyloid precursor proteins: the effect of thrombin
Authors: Smith RP , Broze GJ Jr
Published: October 2019

Activated platelets release a potent inhibitor of factor XIa previously identified as a Kunitz proteinase inhibitor domain-containing form of the beta-amyloid precursor proteins (beta APP). Two carboxy-terminal truncated forms of the beta APP, beta APP-751 and beta APP-770, are shown to be the predominant isoforms secreted by platelets. The release of beta APP from platelets is responsible for the higher concentration of beta APP in serum compared with plasma, and thrombin dose-response data show that release of beta APP is most consistent with alpha granule localization within the platelet. Thrombin induces a limited and specific proteolysis of platelet-secreted beta APP, resulting in loss of a carboxy-terminal fragment. This phenomena is dependent on both thrombin concentration and duration of incubation and is inhibited by the thrombin-specific inhibitor hirudin, characteristics that can be duplicated in a mixture of purified recombinant beta APP-751 and thrombin. A similar effect of thrombin on full-length transmembrane forms of beta APP would result in a membrane-bound remnant containing the intact beta-amyloid protein.