bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired Cotranslational Processing as a Mechanism for Type I Antithrombin Deficiency
Authors: Fitches Alison C. , Appleby Ruth , Lane David A. , De Stefano Valerio , Leone Giuseppe , Olds Robin J.
Published: October 2019

Most secretory proteins, including antithrombin (AT), are synthesized with a signal peptide, which is cleaved before the mature protein is exported from the cell. The signal peptide is important in the process whereby nascent protein is recognized as requiring subsequent modification within the endoplasmic reticulum (ER). We have identified a novel mutation, 2436T→C L(-10)P, which affects the central hydrophobic domain of the AT signal peptide, in a proband presenting with venous thrombotic disease and type I AT deficiency. We investigated the basis of the phenotype by examining expression in mammalian cells of a range of variant AT cDNAs with mutations affecting the –10 residue. Glycosylated AT was secreted from COS-7 cells transfected with wild-type AT, –10L deletion, -10V or -10M variants, but not variants with P, T, R, or G at -10. Cell-free expression of wild-type and variant AT cDNAs was then performed in the presence of canine pancreatic microsomes, as a substitute for ER. Variant AT proteins with P, T, R, or G at residue –10 did not undergo posttranslational glycosylation, and their susceptibility to trypsin digestion suggested they had not been translocated into microsomes. Our results suggest that the ability of AT signal peptide to direct the protein to ER for cotranslational processing events appears to be critically dependent on maintaining the hydrophobic nature of the region including residue –10. The investigations have defined impaired cotranslational processing as a hitherto unrecognized cause of hereditary AT deficiency.
Differential Leukocyte Recruitment From Whole Blood Via Endothelial Adhesion Molecules Under Shear Conditions
Authors: Reinhardt Paul H. , Kubes Paul
Published: October 2019

The objective of this study was to determine if vascular cell adhesion molecule (VCAM-1), E-selectin, and P-selectin could selectively recruit leukocyte subpopulations, and whether this was affected by shear force or adhesion molecule concentration. Cover slips coated with purified adhesion molecules were incorporated into laminar flow chambers. Whole human blood was perfused for 5 minutes over these cover slips at relative shear forces of 2 to 40 dynes/cm2. Chasing the whole blood with buffer permitted visualization of leukocyte-substratum interactions. Leukocytes were observed to roll on and adhere to VCAM-1 at shears between 2 and 15 dynes/cm2. As assessed by cover slip staining, the majority of these cells were lymphocytes, but eosinophils, monocytes, and, surprisingly, neutrophils were also recruited, events inhibitable by anti–4-integrin antibody (HP1/2). Neutrophils were effectively recruited onto the selectins, with interactions occurring at shears as high as 30 and 40 dynes/cm2 for E- and P-selectin respectively. Eosinophils had high affinity for P- but not E-selectin. Mononuclear cells did not have high affinity for either selectin, but interacted avidly with VCAM-1. Antibodies against P-selectin (G1) and E-selectin (ES-1) completely blocked interactions on these substrates. Reducing the concentration of adhesion molecules did not appreciably change recruitment patterns except for VCAM-1, where neutrophils were no longer recruited. The novel use of whole blood in flow chambers shows a partial selectivity of selectins and VCAM-1 for certain subpopulations of leukocytes under varying physiologic shear conditions.
Prevalence of Antibodies Against Proteins Derived From Leukemia Cells in Patients With Chronic Myeloid Leukemia
Authors: Ling Min , Wen Yue-Jin , Lim Seah H.
Published: October 2019

Although various studies supported the notion that leukemia cells in chronic myeloid leukemia (CML) may be recognized by the immune system, direct evidence showing the immunogenicity in vivo of proteins derived from the leukemia cells is lacking. In this study, we have constructed an expression cDNA library from the leukemia cells of a patient with CML and used the autologous serum to screen for high-titer IgG antibodies directed at the leukemia-derived proteins. We isolated eight distinct clones from the library, suggesting that multiple immune responses were elicited in the autologous host. Sequence analysis showed high degrees of homology to known gene sequences in six of the eight clones. Neither bcr-abl nor proteinase 3 sequences were isolated. Using Northern blot analysis, seven of the eight clones showed ubiquitous expression in normal bone marrow, leukemia cell lines, fresh leukemia cells, and normal tissues. However, clone no. 4 showed restricted mRNA expression, being only detected in some fresh leukemia cells, K562 cells, and normal testicular RNA. Using bacterial lysates in dot blot analysis, a panel of sera from normal individuals and patients with CML and other hematological malignancies were screened for high-titer antibodies against these eight clones. There were, among the CML patients, signficantly higher prevalence of antibodies against seven of the eight clones. They were observed even after omitting from the analysis patients with multiple myeloma whose associated immune paresis may impair immune responses to these proteins. Interestingly, antibodies against these proteins were also detected in a small number of normal individuals. Although the precise clinical significance of our findings remains to be determined, this study provides evidence in support of the potential immunogenicity of leukemia-derived proteins in the autologous host. It also provides basis for further investigations to characterize these proteins, especially clone no. 4, and determine their potential for immune targeting in CML.
Incidence of TEL/AML1 Fusion in Children With Relapsed Acute Lymphoblastic Leukemia
Authors: Loh Mignon L. , Silverman Lewis B. , Young Mary L. , Neuberg Donna , Golub Todd R. , Sallan Stephen E. , Gilliland D. Gary
Published: October 2019

The TEL/AML1 fusion associated with t(12;21)(p13;q22) is the most common gene rearrangement in childhood leukemia, occurring in approximately 25% of pediatric acute lymphoblastic leukemia (ALL), and is associated with a favorable prognosis. For example, a cohort of pediatric patients with ALL retrospectively analyzed for theTEL/AML1 fusion treated on Dana-Farber Cancer Institute (DFCI) ALL Consortium protocols between 1980 to 1991 demonstrated a 100% relapse-free survival in TEL/AML1-positive patients with a median of 8.3 years of follow-up. However, two recent studies analyzing pediatric patients with relapsed ALL have reported the same incidence of the TEL/AML1 rearrangement as in patients with newly diagnosed ALL, suggesting that TEL/AML1 positivity is not a favorable prognostic indicator. To clarify this apparent discrepancy, 48 pediatric patients treated on Dana-Farber Cancer Institute (DFCI) protocols with ALL at first or second relapse were tested forTEL/AML1 using reverse transcriptase-polymerase chain reaction (RT-PCR). The TEL/AML1 fusion was identified in only 1 of 32 analyzable relapsed ALL patients, in concordance with our previous reports of improved disease-free survival in TEL/AML1-positive patients. The low frequency of TEL/AML1-positive patients at relapse is significantly different than that reported in other studies. Although there are several potential explanations for the observed differences in TEL/AML1-positive patients at relapse, it is plausible that relapse-free survival in TEL/AML1-positive patients may be changed with different therapeutic approaches. Taken together, these results support the need for prospective analysis of prognosis in TEL/AML1-positive patients.
An Activating Mutation in the Kit Receptor Abolishes the Stroma Requirement for Growth of ELM Erythroleukemia Cells, But Does Not Prevent Their Differentiation in Response to Erythropoietin
Authors: Leslie Nick R. , O’Prey Jim , Bartholomew Chris , Harrison Paul R.
Published: October 2019

We have previously shown that murine ELM erythroleukemia cells can only be grown in vitro in the presence of a stromal feeder layer, or alternatively stem cell factor (SCF), without which they differentiate. When grown in the presence of SCF, ELM cells can still differentiate in response to erythropoietin (Epo), but growth on stroma prevents this. We previously isolated a stroma-independent ELM variant, ELM-I-1, that is also defective in Epo-induced differentiation. We show here that this variant has an activating mutation in the Kit receptor, converting aspartic acid 814 to histidine. Expression of the mutant receptor in stroma-dependent ELM-D cells causes growth factor-independent proliferation and also gives the cells a selective advantage, in terms of proliferation rate and clonegenicity, compared with ELM-D cells grown in optimal amounts of SCF. Expression of the mutant receptor in ELM-D cells also prevents spontaneous differentiation, but not differentiation induced by Epo. Analysis of mitogenic signaling pathways in these cells shows that the mutant receptor induces constitutive activation of p42/p44 mitogen-activated protein kinases. It also selectively inhibits the expression of p66Shc but not the p46/p52 Shc isoforms (as did treatment of ELM cells with SCF), which is of interest, because p66Shc is known to play an inhibitory role in growth factor signaling.
Presence of a p53 Gene Deletion in Patients With Multiple Myeloma Predicts for Short Survival After Conventional-Dose Chemotherapy
Authors: Drach Johannes , Ackermann Jutta , Fritz Elke , Krömer Elisabeth , Schuster Rudolf , Gisslinger Heinz , DeSantis Maria , Zojer Niklas , Fiegl Michael , Roka Sebastian , Schuster Judith , Heinz Renate , Ludwig Heinz , Huber Heinz
Published: October 2019

In multiple myeloma (MM), previous studies showed that mutations of the p53 gene are rare events in patients with newly diagnosed disease, but it is not known whether deletions of p53 are of any significance in MM. To address this question, we used interphase fluorescence in situ hybridization (FISH) with a DNA probe specific for the p53 locus at 17p13 and investigated bone marrow plasma cells from 72 patients with MM (59 patients = 81.9% before therapy). By FISH, deletions of p53, which were found to be predominantly monoallelic, were detected in 32.8% and 54.5% of patients with newly diagnosed and relapsed MM, respectively. Karyotypes from six of the patients with a p53 deletion by FISH showed a structural abnormality of 17p in only one of them. Additional FISH studies including a distal-17p probe (specific for theD17S34 locus) provided evidence for an interstitial deletion on 17p resulting in loss of p53 hybridization signals in myeloma cells. Among all 59 patients with newly diagnosed MM, presence of a p53 deletion was associated with stage III (P = .054), but not with other laboratory and clinical parameters. Patients with a p53 deletion had significantly shorter survival time compared with those without a deletion, both from the time of diagnosis (median 13.9v 38.7 months; P < .0001) and from the time of initiation of induction treatment consisting of conventional dose chemotherapy (median 15.9 months v median not reached at 38 months; P < .0002). On stepwise multivariate regression analysis, presence of a p53 deletion was the most significant independent parameter predicting for shortened survival (P = .002). We conclude that a p53 gene deletion, which can be identified by interphase FISH in almost a third of patients with newly diagnosed MM, is a novel prognostic factor predicting for short survival of MM patients treated with conventional-dose chemotherapy.

© 1998 by The American Society of Hematology.
Infusion of Cytotoxic T Cells for the Prevention and Treatment of Epstein-Barr Virus–Induced Lymphoma in Allogeneic Transplant Recipients
Authors: Rooney Cliona M. , Smith Colton A. , Ng Catherine Y.C. , Loftin Susan K. , Sixbey John W. , Gan Yanjun , Srivastava Deo-Kumar , Bowman Laura C. , Krance Robert A. , Brenner Malcolm K. , Heslop Helen E.
Published: October 2019

Epstein-Barr virus (EBV) causes potentially lethal immunoblastic lymphoma in up to 25% of children receiving bone marrow transplants from unrelated or HLA-mismatched donors. Because this complication appears to stem from a deficiency of EBV-specific cytotoxic T cells, we assessed the safety and efficacy of donor-derived polyclonal (CD4+ and CD8+) T-cell lines as immunoprophylaxis and treatment for EBV-related lymphoma. Thirty-nine patients considered to be at high risk for EBV-induced lymphoma each received 2 to 4 intravenous infusions of donor-derived EBV-specific T lymphocytes, after they had received T-cell–depleted bone marrow from HLA-matched unrelated donors (n = 33) or mismatched family members (n = 6). The immunologic effects of this therapy were monitored during and after the infusions. Infused cells were identified by detection of the neo marker gene. EBV-specific T cells bearing theneo marker were identified in all but 1 of the patients. Serial analysis of DNA detected the marker gene for as long as 18 weeks in unmanipulated peripheral blood mononuclear cells and for as long as 38 months in regenerated lines of EBV-specific cytotoxic T cells. Six patients (15.5%) had greatly increased amounts of EBV-DNA on study entry (>2,000 genome copies/106 mononuclear cells), indicating uncontrolled EBV replication, a complication that has had a high correlation with subsequent development of overt lymphoma. All of these patients showed 2 to 4 log decreases in viral DNA levels within 2 to 3 weeks after infusion and none developed lymphoma, confirming the antiviral activity of the donor-derived cells. There were no toxic effects that could be attributed to prophylactic T-cell therapy. Two additional patients who did not receive prophylaxis and developed overt immunoblastic lymphoma responded fully to T-cell infusion. Polyclonal donor-derived T-cell lines specific for EBV proteins can thus be used safely to prevent EBV-related immunoblastic lymphoma after allogeneic marrow transplantation and may also be effective in the treatment of established disease.

© 1998 by The American Society of Hematology.
Phosphorothioate Oligonucleotides Inhibit the Intrinsic Tenase Complex
Authors: Sheehan John P. , Lan Hao-Chang
Published: October 2019

Systemic administration of ISIS 2302, a 20-mer antisense phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both monkey and human studies. The anticoagulant effects of ISIS 2302 were investigated with both in vitro coagulation assays in human plasma and purified enzyme systems. At high oligonucleotide plasma concentrations (>100 μg/mL), prolongation of the prothrombin and thrombin times was observed. In a thrombin time assay using purified components, high concentrations of ISIS 2302 inhibited thrombin clotting activity both by stimulating inhibition by heparin cofactor II and directly competing with fibrinogen for binding to anion binding exosite I. In contrast, low concentrations of ISIS 2302 (<100 μg/mL) showed a selective, linear prolongation of the activated partial thromboplastin time (PTT). The rate limiting effect of 50 μg/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was identified by sequential modification of the clotting assay. Delaying addition of oligonucleotide until after contact activation failed to correct prolongation of the PTT. The calcium-dependent steps of the intrinsic pathway were individually assessed by adding sufficient activated coagulation factor to correct the PTT in plasma deficient in that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor X–deficient plasma with or without oligonucleotide present. ISIS 2302 (50 μg/mL) did not prolong a modified Russel viper venom time, suggesting no significant inhibition of prothrombinase. Thus, 50 μg/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic tenase activity. ISIS 2302 showed partial inhibition of intrinsic tenase activity (to approximately 35% of control) at clinically relevant oligonucleotide concentrations in a chromogenic assay. This activity was oligonucleotide sequence–independent but required the phosphorothioate backbone, suggesting that inhibition of intrinsic tenase is a general property of this class of oligonucleotides. These results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.

© 1998 by The American Society of Hematology.
Retinoic Acid Inhibits CD40 + Interleukin-4–Mediated IgE Production In Vitro
Authors: Worm M. , Krah J.M. , Manz R.A. , Henz B.M.
Published: October 2019

To elucidate the role of retinoic acid (RA) in anti-CD40 + interleukin-4 (IL-4)–mediated B-cell activation, the effect of 10−12 to 10−6 mol/L RA was studied in anti-CD40 (1 μg/mL) + IL-4 (5 ng/mL)-mediated proliferation and Ig synthesis by human peripheral blood mononuclear cells (PBMC) and B cells in healthy donors. Anti-CD40 + IL-4–mediated proliferation of PBMC and B cells was inhibited by RA in a dose-dependent manner, with maximal inhibition of 62% ± 5% in PBMC and 55% ± 4.4% in B cells by all-trans RA, and 58% ± 6.7% and 51% ± 4.7%, respectively by 13-cis RA. IgE synthesis was even more markedly inhibited by RA starting at concentrations of >10−14mol/L for B cells and >10−10 mol/L for PBMC. Maximal inhibition of IgE production for B cells was at 10−8mol/L for all-trans RA (94% ± 1.8%) and 96% ± 3.2% for 13-cis RA. Low concentrations of RA inhibiting IgE synthesis (10−10 mol/L) affected neither B-cell proliferation nor the production of IgA, IgG, and IgM. Elucidation of the mechanism involved in this inhibition of IgE production shows that epsilon germline transcription is decreased by RA, whereas production of interferon-γ (IFN-γ) was not enhanced in the presence of RA. To differentiate whether the RA effect was mediated by RA receptors , β, and γ, the expression of the retinoic acid receptors (RAR) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The data show that unstimulated human peripheral B cells express mRNA of the RA receptor , β, and γ. Using retinoids with different receptor binding specificity (CD336, CD437, CD2019, CD367), dose-dependent inhibition of IgE synthesis was shown by all four derivates, but was most marked by an RA binding the  receptor with high specificity. Taken together, this study shows that RA inhibits IgE production of anti-CD40 + IL-4–stimulated B cells in vitro.

© 1998 by The American Society of Hematology.
Efficacy and Costs of Granulocyte Colony-Stimulating Factor in Allogeneic T-Cell Depleted Bone Marrow Transplantation
Authors: Lee Stephanie J. , Weller Edie , Alyea Edwin P. , Ritz Jerome , Soiffer Robert J.
Published: October 2019

Hematopoietic growth factors have shown clinical benefits in patients undergoing chemotherapy and stem cell transplantation, but few studies have been performed to assess whether the benefits are worth the costs. We reviewed 196 patients undergoing T-cell depleted related donor bone marrow transplantation (BMT) between 1990 and 1996 to assess the effect of growth factor use on time to engraftment and costs of hospitalization. Beginning in 1994, based on encouraging results in autologous transplantation, patients (n = 81) were treated with granulocyte colony-stimulating factor (G-CSF) starting at day +1 after marrow infusion until engraftment. Between January 1, 1990 and January 1, 1994, patients (n = 115) did not receive growth factor. CD6 depletion of donor marrow was the only form of prophylaxis against graft-versus-host disease (GVHD). Despite receiving a lower stem cell dose (P = .004), the group receiving G-CSF had a decreased time to engraftment (20 days v 12 days, P < .0001) and time from marrow infusion to discharge (23 days v 17 days,P < .0001). In multivariate modeling, the use of G-CSF was the most significant factor predicting time to engraftment and discharge. Incidence of grades II-IV GVHD, early mortality, percentage of patients who engrafted, and relapse rates did not differ between the groups. Inpatient charges during the first 50 days after marrow infusion (including readmissions) were available on 110 patients and were converted to costs using departmental ratios of costs of charges. Median costs were significantly lower in the group receiving G-CSF ($80,600 v $84,000, P = .0373). Thus, use of G-CSF in this setting allows earlier hospital discharge with lower costs.

© 1998 by The American Society of Hematology.
Expression of Macrophage Inflammatory Protein-1 Receptors in Human CD34+ Hematopoietic Cells and Their Modulation by Tumor Necrosis Factor- and Interferon-γ
Authors: Dürig Jan , de Wynter Erika A. , Kasper Christoph , Cross Michael A. , Chang James , Testa Nydia G. , Heyworth Clare M.
Published: October 2019

Macrophage inflammatory protein-1 (MIP-1) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1 may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1 receptors on CD34+ cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1 receptors on CD34+cells was analyzed by two-color flow cytometry using a biotinylated MIP-1 molecule. The mean percentage of LP CD34+ cells expressing the MIP-1 receptors was 67.7 ± 7.2% (mean ± SEM; n = 22) as compared with 89.9 ± 2.6% (n = 10) and 74.69 ± 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1 receptor subtypes on LP CD34+ cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34+ cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34+ cells for 24 to 36 hours in the presence of tumor necrosis factor- (TNF-) and interferon-γ (IFN-γ) resulted in a significant increase in MIP-1 receptor expression. TNF- induced MIP-1 receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1 receptor expression on hematopoietic cells.

© 1998 by The American Society of Hematology.
Defining an Antigenic Epitope on Platelet Factor 4 Associated With Heparin-Induced Thrombocytopenia
Authors: Ziporen L. , Li Z.Q. , Park K.S. , Sabnekar P. , Liu W.Y. , Arepally G. , Shoenfeld Y. , Kieber-Emmons T. , Cines D.B. , Poncz M.
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy. Antibodies to platelet factor 4 (PF4)/heparin complexes have been implicated in the pathogenesis of this disorder, but the antigenic epitope(s) on the protein have not been defined. To address this issue, we studied the binding of HIT antibodies to a series of recombinant proteins containing either point mutations in PF4 or chimeras containing various domains of PF4 and the related protein, neutrophil activating peptide-2 (NAP-2). Serum samples from 50 patients with a positive 14C-serotonin release assay (14C-SRA) and a clinical diagnosis of HIT and 20 normal controls were studied. HIT antibodies reacted strongly with wild-type (WT) PF4/heparin complexes, but reacted little, if at all, with NAP-2/heparin complexes (optical density [OD]405 = 2.5 and 0.2, respectively). Alanine substitutions at three of the four lysine residues implicated in heparin binding, K62, K65, and K66, had little effect on recognition by HIT antibodies (OD405 = 2.2, 2.8, and 2.0, respectively), whereas an alanine substitution at position K61 led to reduced, but still significant binding (OD405 = 1.0). Similar studies involving chimeras between PF4 and NAP-2 localized a major antigenic site to the region between the third and fourth cysteine residues for more than half of the sera tested. This site appears to involve a series of amino acids immediately after the third cysteine residue beginning with P37. Thus our studies suggest that whereas the C-terminal lysine residues of PF4 are important for heparin binding, they do not comprise a critical antigenic site for most HIT antibodies. Rather, we propose that maintaining a region near the third cysteine residue of PF4, distal from the proposed heparin-binding domain, is required to form the epitope recognized by many HIT antibodies.

© 1998 by The American Society of Hematology.
Formation of the Human Fibrinogen Subclass Fib420: Disulfide Bonds and Glycosylation in Its Unique (EChain) Domains
Authors: Fu Yiping , Zhang Jian-Zhong , Redman Colvin M. , Grieninger Gerd
Published: October 2019

COS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel E chain and those of the more abundant subclass whose  chains lack E’s globular C-terminus. That region, referred to as the EC domain, is closely related to the ends of β and γ chains of fibrinogen (βC and γC). Transfection of COS cells with E, β, and γ cDNAs alone results in secretion of the symmetrical molecule (Eβγ)2, also known as Fib420. Cotransfection with cDNA for the shorter  chain yielded secretion of both (βγ)2 and (Eβγ)2 but no mixed molecules of the structure E(βγ)2. Exploiting the COS cells’ fidelity with regard to Fib420 production, identification was made of the highly conserved Asn667 as the sole site of N-linked glycosylation in the E chain. No evidence from Cys → Ser replacements was found for interchain disulfide bridges involving the four cysteines of the EC domain. However, for fibrinogen secretion, the E, β, and γ subunits do exhibit different requirements for integrity of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dissimilar structural roles in the process of fibrinogen assembly.

© 1998 by The American Society of Hematology.
Identification of Mutations and Polymorphisms in the Factor XI Genes of an African American Family by Dideoxyfingerprinting
Authors: Martincic Danko , Zimmerman Sherri A. , Ware Russell E. , Sun Mao-Fu , Whitlock James A. , Gailani David
Published: October 2019

Congenital deficiency of factor XI is a rare condition associated with a mild to moderate bleeding diathesis that is most commonly found in persons of Jewish ancestry. The disorder has been reported sporadically in a number of other ethnic groups, but rarely in the black population. We report on the genetic analysis of the factor XI genes of two African American patients: a 9-year-old boy (the propositus) with mild factor XI deficiency and his mother. Both individuals have lifelong histories of excessive bleeding. Dideoxyfingerprinting, a technique combining components of single-strand conformational polymorphism analysis and dideoxy-chain termination sequencing, was used in the analysis. Both patients were found to be heterozygous for a mutation changing serine 248 to glutamine, whereas the propositus was heterozygous for an additional mutation on the paternal allele changing glutamine 226 to arginine. Both mutations reside in the third apple domain of the factor XI heavy chain, an area that has been shown to contain binding sites for factor IX, platelets, and glycosaminoglycans. Previously reported mutations in the factor XI gene seem to cause deficiency primarily by reducing protein expression. Because both alleles in the propositus contain amino acid substitutions, the significant amount of circulating factor XI in his plasma must be comprised entirely of abnormal molecules. Factor XI circulates as a homodimer, and the presence of mutations in both alleles of the factor XI gene suggests that his bleeding disorder is caused in part by the effect of the two abnormal gene products forming dimers in different combinations. Three neutral (not associated with amino acid changes) DNA polymorphisms were also identified in the two subjects: a C to T change at nucleotide 472 in exon 5, A to G at nucleotide 844 in exon 8, and T to C at nucleotide 1234 in exon 11. Analysis of a random sample of normal volunteers showed that these polymorphisms are relatively common, with allele frequencies of 7.4%, 19%, and 18%, respectively. This suggests that there is considerable genetic heterogeneity in the factor XI gene.

© 1998 by The American Society of Hematology.
Selective Ablation of Acute Myeloid Leukemia Using Antibody-Targeted Chemotherapy: A Phase I Study of an Anti-CD33 Calicheamicin Immunoconjugate
Authors: Sievers E.L. , Appelbaum F.R. , Spielberger R.T. , Forman S.J. , Flowers D. , Smith F.O. , Shannon-Dorcy K. , Berger M.S. , Bernstein I.D.
Published: October 2019

Leukemic blast cells express the CD33 antigen in most patients with acute myeloid leukemia (AML), but this antigen is not expressed by hematopoietic stem cells. We conducted a study to determine whether normal hematopoiesis could be restored in patients with AML by selective ablation of cells expressing the CD33 antigen. In a dose escalation study, 40 patients with relapsed or refractory CD33+ AML were treated with an immunoconjugate (CMA-676) consisting of humanized anti-CD33 antibody linked to the potent antitumor antibiotic calicheamicin. The capacity of leukemic cells to efflux 3,3’-diethyloxacarbocyanine iodide (DiOC2) was used to estimate pretreatment functional drug resistance. Leukemia was eliminated from the blood and marrow of 8 (20%) of the 40 patients; blood counts returned to normal in three (8%) patients. A high rate of clinical response was observed in leukemias characterized by low dye efflux in vitro. Infusions of CMA-676 were generally well tolerated, and a postinfusion syndrome of fever and chills was the most common toxic effect. Two patients who were treated at the highest dose level (9 mg/m2) were neutropenic >5 weeks after the last dose of CMA-676. These results show that an immunoconjugate targeted to CD33 can selectively ablate malignant hematopoiesis in some patients with AML.
Sickle Cell Anemia as a Possible State of Enhanced Anti-Apoptotic Tone: Survival Effect of Vascular Endothelial Growth Factor on Circulating and Unanchored Endothelial Cells
Authors: Solovey Anna , Gui Lizhen , Ramakrishnan Sundaram , Steinberg Martin H. , Hebbel Robert P.
Published: October 2019

The biologic processes of apoptosis and angiogenesis are linked in endothelial biology because some endothelial cell growth factors also exert anti-apoptotic effects. We studied whether apoptosis is occurring in circulating endothelial cells (CEC) that have lost the survival signals derived from anchorage to extracellular matrix. Consistent with this expectation, 64% ± 16% of CEC from normal donors showed evidence of apoptosis (by morphology and TdT-mediated dUTP nick end labeling [TUNEL] assay). However, only 30% ± 15% (P < .001 v normal) of CEC from donors with sickle cell anemia were apoptotic. Vascular endothelial growth factor (VEGF) levels were significantly (P = .001) higher in plasma of sickle donors (120.1 ± 81.4 pg/mL) than that of normal donors (37.6 ± 34.6 pg/mL), and there was an inverse correlation between VEGF and CEC apoptosis (r = .612,P = .001). Consistent with stimulation by VEGF, CEC from sickle donors exhibited increased expression of vβ3. In vitro experiments showed that VEGF inhibits apoptosis for cultured endothelial cells that are kept unanchored and not allowed to re-establish attachment to extracellular matrix, thus demonstrating that VEGF provides survival signals independent of its ability to promote matrix reattachment. These data suggest the hypothesis that sickle cell anemia is a state of enhanced anti-apoptotic tone for endothelial cells. If true, this has implications for disease pathobiology, particularly the development of neovascularizing retinopathy.
A Large Proportion of Patients With a Diagnosis of Essential Thrombocythemia Do Not Have a Clonal Disorder and May Be at Lower Risk of Thrombotic Complications
Authors: Harrison Claire N. , Gale Rosemary E. , Machin Samuel J. , Linch David C.
Published: October 2019

Essential thrombocythemia (ET) is traditionally considered to be a clonal disorder. No specific karyotypic abnormalities have been described, but the demonstration of clonality using X-chromosome inactivation patterns (XCIPs) has been used to differentiate ET from a non-clonal reactive thrombocytosis. However, these assays may be difficult to interpret, and contradictory results have been reported. We have studied 46 females with a diagnosis of ET according to the Polycythemia Vera Study Group (PVSG) criteria. XCIP results in 23 patients (50%) were uninterpretable due to either constitutive or possible acquired age-related skewing. Monoclonal myelopoiesis could be definitively shown in only 10 patients. Thirteen patients had polyclonal myelopoiesis, and in 8, it was possible to exclude clonal restriction to the megakaryocytic lineage. Furthermore, there was no evidence of clonal progenitors in purified CD34+CD33− and CD34+CD33+ subpopulations from bone marrow of 2 of these 13 patients. There was no difference between patients with monoclonal and polyclonal myelopoiesis with respect to age or platelet count at diagnosis, duration of follow-up, incidence of hepatosplenomegaly, or hemorrhagic complications. However, polyclonal patients were less likely to have experienced thrombotic events (P = .039). These results suggest that ET is a heterogeneous disorder, and the clinical significance of clonality status warrants investigation in a larger study.
Human Growth Factor–Enhanced Regeneration of Transplantable Human Hematopoietic Stem Cells in Nonobese Diabetic/Severe Combined Immunodeficient Mice
Authors: Cashman Johanne D. , Eaves Connie J.
Published: October 2019

Self-renewal is considered to be the essential defining property of a stem cell. Retroviral marking, in vitro amplification, and serial transplantation of human cells that can sustain long-term lymphomyelopoiesis in vivo have provided evidence that human hematopoietic stem cell self-renewal occurs both in vitro and in vivo. To investigate whether this process can be manipulated by cytokines, we administered two different combinations of human growth factors to sublethally irradiated nonobese diabetic/severe combined immunodeficient (SCID) mice transplanted with 107 light-density human cord blood cells and then performed secondary transplants to compare the number of transplantable human lymphomyeloid reconstituting cells present 4 to 6 weeks post-transplant. A 2-week course of Steel factor + interleukin (IL)-3 + granulocyte-macrophage colony-stimulating factor + erythropoietin (3 times per week just before sacrifice) specifically and significantly enhanced the numbers of transplantable human lymphomyeloid stem cells detectable in the primary mice (by a factor of 10). Steel factor + Flt3-ligand + IL-6 (using either the same schedule or administered daily until sacrifice 4 weeks post-transplant) gave a threefold enhancement of this population. These effects were obtained at a time when the regenerating human progenitor populations in such primary mice are known to be maximally cycling even in the absence of growth factor administration suggesting that the underlying mechanism may reflect an ability of these growth factors to alter the probability of differentiation of stem cells stimulated to proliferate in vivo.
Downregulation of c-kit (Stem Cell Factor Receptor) in Transformed Hematopoietic Precursor Cells by Stroma Cells
Authors: Heberlein Christoph , Friel Jutta , Laker Christine , von Laer Dorothee , Bergholz Ulla , Bögel Martina , Ashman Leonie K. , Klingler Karl , Ostertag Wolfram
Published: October 2019

We show a dramatic downregulation of the stem cell factor (SCF) receptor in different hematopoietic cell lines by murine stroma. Growth of the human erythroid/macrophage progenitor cell line TF-1 is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, TF-1 cells clone and proliferate equally well on stroma. Independent stroma-dependent TF-1 clones (TF-1S) were generated on MS-5 stroma. Growth of TF-1S and TF-1 cells on stroma still requires interaction between c-kit (SCF receptor) and its ligand SCF, because antibodies against c-kit inhibit growth to less than 2%. Surprisingly, c-kit receptor expression (RNA and protein) was downregulated by 2 to 3 orders of magnitude in TF-1S and TF-1 cells grown on stroma. This stroma-dependent regulation of the kit receptor in TF-1 was also observed on exposure to kit ligand-negative stroma, thus indicating the need for heterologous receptor ligand interaction. Removal of stroma induced upregulation by 2 to 4 orders of magnitude. Downregulation and upregulation of c-kit expression could also be shown for the megakaryocytic progenitor cell line M-07e and was comparable to that of TF-1, indicating that stroma-dependent regulation of c-kit is a general mechanism. Downregulation may be an economic way to compensate for the increased sensitivity of the c-kit/ligand interaction on stroma. The stroma-dependent c-kit regulation most likely occurs at the transcriptional level, because mechanisms, such as splicing, attenuation, differential promoter usage, or mRNA stability, could be excluded.
Fibrin-Dependent Platelet Procoagulant Activity Requires GPIb Receptors and von Willebrand Factor
Authors: Béguin S. , Kumar R. , Keularts I. , Seligsohn U. , Coller B.S. , Hemker H.C.
Published: October 2019

Thrombin generation in platelet-rich plasma (PRP) involves complex interactions between platelets and coagulation proteins. We previously reported that the addition of fibrin to PRP enhances tissue-factor initiated thrombin generation by ≈ 40%, and the current studies were designed to assess the mechanism(s) underlying thrombin generation in the absence and presence of fibrin. Blocking platelet GPIIb/IIIa + vβ3 receptors with a monoclonal antibody (MoAb) inhibited basal thrombin generation, but did not affect the enhancement produced by fibrin. In contrast, blocking GPIb with any of three different MoAbs had no effect on basal thrombin generation, but essentially eliminated fibrin enhancement of thrombin generation. When thrombin generation was tested in PRP deficient in von Willebrand factor (vWF), both basal and fibrin-enhanced thrombin generation were markedly reduced, and the addition of factor VIII did not normalize thrombin generation. Botrocetin, which induces the binding of vWF to GPIb, enhanced thrombin generation. In all studies, the ability of PRP to support thrombin generation correlated with the production of platelet-derived microparticles and serum platelet-derived procoagulant activity. Thus, two separate mechanisms, both of which depend on vWF, appear to contribute to platelet-derived procoagulant activity: one is independent of fibrin and relies primarily on GPIIb/IIIa, but with a minor contribution from vβ3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the mechanisms of the abnormalities in serum prothrombin times reported in Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD), and the increased risk of thrombosis associated with elevated vWF levels.
Definition of Dendritic Cell Subpopulations Present in the Spleen, Peyer’s Patches, Lymph Nodes, and Skin of the Mouse
Authors: Anjuère Fabienne , Martı́n Pilar , Ferrero Isabel , Fraga Marta López , del Hoyo Gloria Martı́nez , Wright Natalia , Ardavı́n Carlos
Published: October 2019

Dendritic cells (DC) are highly efficient antigen-presenting cells (APC) that have an essential function in the development of immune responses against microbial pathogens and tumors. Although during the past few years our understanding of DC biology has remarkably increased, a precise characterization of the different DC subpopulations remains to be achieved with regard to their phenotype and lineage relationships. In this report, we have extensively studied the DC subpopulations present in the thymus, spleen, Peyer’s patches, lymph nodes (LN) and skin of the mouse. Thymus DC and 60% spleen DC have a lymphoid DC phenotype, ie, CD8+DEC-205high Mac-1low, whereas 40% spleen DC have a myeloid DC phenotype, ie, CD8−DEC-205low Mac-1high. Both CD8+and CD8− DC are leukocyte function-associated antigen-1 (LFA-1)high and highly adherent. Within Peyer’s patches the majority of DC correspond to the CD8+DEC-205high Mac-1lowlymphoid category. In the LN, together with CD8+ and CD8− DC, an additional nonadherent CD8intLFA-1int subpopulation with lymphoid DC characteristics is described. Finally, in the skin both epidermal Langerhans cells (LC) and dermal DC are CD8−DEC-205high Mac-1high , and do not express LFA-1. Interestingly, LC migration experiments indicate that LC underwent the upregulation of CD8 and LFA-1 upon migration to the LN, supporting the hypothesis that LC belong to the CD8+ lymphoid lineage.
Caspases Mediate Tumor Necrosis Factor-–Induced Neutrophil Apoptosis and Downregulation of Reactive Oxygen Production
Authors: Yamashita Kouhei , Takahashi Atsushi , Kobayashi Susumu , Hirata Hirokazu , Mesner Peter W. , Kaufmann Scott H. , Yonehara Shin , Yamamoto Kokichi , Uchiyama Takashi , Sasada Masataka
Published: October 2019

Tumor necrosis factor- (TNF-) exerts two separate effects on neutrophils, stimulating effector functions while simultaneously inducing apoptosis. We examined here the involvement of caspases in neutrophil apoptosis and the effect of TNF-–induced apoptosis on reactive oxygen production. Immunoblotting and affinity labeling showed activation of caspase-8, caspase-3, and a caspase with a large subunit of 18 kD (T18) in TNF-–treated neutrophils. Active caspase-6 and -7 were not detectable in this cell type. Caspase-8 activated caspase-3 and T18 in neutrophil cytoplasmic extracts. zVAD-fmk blocked neutrophil apoptosis, in parallel with the inhibition of caspase activation. TNF-–induced caspase activation was accompanied by a decrease in the ability of neutrophils to release superoxide anion. Conversely, TNF- treatment in the presence of zVAD-fmk caused a prolonged augmentation of superoxide release. Granulocyte-macrophage colony-stimulating factor inhibited TNF-–induced caspase activation and apoptosis, while reversing the diminution in superoxide release. These observations not only suggest that a caspase cascade mediates apoptotic events and downregulates oxygen radical production in TNF-–treated neutrophils, but also raise the possibility that suppression of caspase activation with enhanced proinflammatory actions of TNF- may underlie the pathogenesis of inflammatory diseases.
Long-Term Survival (10 Years or More) in 30 Patients With Primary Amyloidosis
Authors: Kyle Robert A. , Gertz Morie A. , Greipp Philip R. , Witzig Thomas E. , Lust John A. , Lacy Martha Q. , Therneau Terry M.
Published: October 2019

The median survival in primary systemic (AL) amyloidosis is less than 18 months. No published series of patients with AL amyloidosis have reported survival of more than 10 years. The records of all Mayo Clinic patients with a diagnosis of AL amyloidosis between January 1, 1966 and March 1, 1987 were reviewed. Patients with secondary amyloidosis, familial amyloidosis, senile systemic amyloidosis, and localized amyloidosis were excluded. During the 21 years of the study, 841 patients with AL amyloidosis were seen. Of these, 29 were excluded because the diagnosis was made at autopsy, and 2 others were excluded because no follow-up data were available. Actuarial survival for the 810 patients was 51% at 1 year, 16% at 5 years, and 4.7% at 10 years. Thirty patients survived for 10 years or more after the histologic diagnosis of AL amyloidosis; all received alkylating-agent therapy. In 14 patients, the monoclonal protein disappeared from the serum or urine. Of 10 patients with nephrotic syndrome, 4 had an objective response. Congestive heart failure, older age, creatinine value of 2 mg/dL or more, bone marrow plasma cell value of 20% or more, platelet count of 500 × 109/L or less, and the presence of peripheral neuropathy were underrepresented in the 10-year survivors and are unfavorable prognostic features. Five percent of patients with AL amyloidosis survived for 10 years or more.
Apoptotic Role of Fas/Fas Ligand System in the Regulation of Erythropoiesis
Authors: De Maria R. , Testa U. , Luchetti L. , Zeuner A. , Stassi G. , Pelosi E. , Riccioni R. , Felli N. , Samoggia P. , Peschle C.
Published: October 2019

The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.