bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical importance of extraordinary integration patterns of human T- cell lymphotropic virus type I proviral DNA in adult T-cell leukemia/lymphoma [see comments]
Authors: Shimamoto Y , Suga K , Shibata K , Matsuzaki M , Yano H , Yamaguchi M
Published: October 2019

The proviral DNA of human T-cell lymphotrophic virus type I (HTLV-I) is known to be integrated monoclonally in the malignant cells of adult T- cell leukemia/lymphoma (ATL), which is a peripheral T-cell malignancy caused by this virus. We studied the relationship between the integration patterns of HTLV-I and clinical characteristics in 89 patients with ATL. The proviral DNA of HTLV-I was examined by the standard Southern blot analysis using the endonucleases EcoRI and Pst I. One clear band of greater than 9 kb was detected in most of the patients (83 case) when cellular DNA was digested with EcoRI. On the other hand, extraordinary integration patterns of HTLV-I proviral DNA were detected in 6 patients; 3 of them showed two bands, while the other 3 showed one band smaller than 9 kb. When cellular DNA was digested with PstI, the band patterns of these patients were quite different from those of typical patients. The patients with the extraordinary integration patterns had clinical characteristics dissimilar to those of the other 83 patients with the ordinary integration pattern. The patients with two bands by EcoRI digestion always had severe hypoxemia with extremely high levels of serum lactate dehydrogenase at first presentation and showed peculiar organ infiltrations, such as retina and muscle, which were less frequent in the other ordinary 83 patients. They all died within 8 months after the onset. In contrast, the patients with one smaller band by EcoRI digestion always had small and mature T lymphocytes with bilobulated nuclei without lymphadenopathy and showed a favorable clinical course, which was uncommon in the ordinary cases. They were alive 20 to 38 months after diagnosis. Rearranged bands of the T-cell receptor gene were detected in all patients with unusual integration. These findings indicate that the integration patterns of HTLV-I proviral DNA have a clinical implication and may be one of the explanations for heterogeneity in the behavior of this disease.
High-dose therapy for refractory multiple myeloma: improved prognosis with better supportive care and double transplants
Authors: Vesole DH , Barlogie B , Jagannath S , Cheson B , Tricot G , Alexanian R , Crowley J
Published: October 2019

One hundred and thirty-five patients with advanced and refractory myeloma received one of three high-dose therapy regimens: melphalan at doses of 90 to 100 mg/m2 (MEL 100; 47 patients) without autotransplant; total body irradiation (TBI; 850 cGy) with either melphalan 140 mg/m2 or thiotepa 750 mg/m2 and autologous bone marrow transplant (ABMT) (< or = 30% plasma cells; 21 patients); melphalan 200 mg/m2 (MEL 200) supported by both peripheral blood stem cells (PBSC) and ABMT plus GM- CSF intended as a double-transplant program (67 patients; 42 have completed and 3 are still awaiting a second autotransplant; 5 additional patients received an allograft for their second transplant). Mortality within 2 months of therapy was 20% to 25% with MEL 100 and TBI regimens, but less than 1% with MEL 200, mainly because severe neutropenia (< 500/microL) was shortened to less than 1 week due to infusion of PBSC and use of growth factor therapy. Low beta 2- microglobulin (beta 2M) levels < or = 2.5 mg/L and MEL 200 therapy were identified as the two most important independent favorable variables associated with prolonged event-free survival (EFS) and overall survival (OS). On the basis of these two parameters, three risk groups were defined: 29 good-risk patients with low beta 2M receiving MEL 200 had the best outcome, with median durations of EFS of 37 months and projected OS of > or = 43 months; 54 intermediate-risk patients displaying one of the two favorable parameters had EFS and OS durations of 16 and 36 months, respectively; and 52 poor-risk patients with high beta 2M not receiving MEL 200 had a dismal prognosis, with EFS of 3 months and OS of 5 months (all P > .0001). Further analysis that excluded treatment as a variable identified high beta 2M and resistant relapse as the two major adverse prognostic factors, one of which was present in 80% of the 135 patients. Among these 108 high-risk patients, prognosis was improved markedly with MEL 200 because of both better supportive care (PBSC and hematopoietic growth factors) and more intensive therapy using the double-transplant approach. This study supports the concept that safer and potentially more-effective therapies can be developed in the setting of advanced and resistant disease.
Effect of low-dose interleukin-2 on disease relapse after T-cell- depleted allogeneic bone marrow transplantation
Authors: Soiffer RJ , Murray C , Gonin R , Ritz J
Published: October 2019

T-cell depletion of donor bone marrow has been associated with an increased risk of disease relapse after allogeneic bone marrow transplantation (BMT). Recombinant interleukin-2 (IL-2), which is capable of increasing the antileukemic activity of peripheral blood lymphocytes obtained from patients who have undergone BMT, has been proposed as a potentially useful agent to reduce the risk of relapse post-BMT. We have previously shown that IL-2 administered to patients at very low doses after BMT is both clinically tolerable and immunologically active. We now report on the clinical outcome of 29 patients treated with low-dose IL-2 after CD6-depleted allogenic BMT for hematologic malignancy. IL-2 was administered by continuous infusion for up to 3 months beginning at a median of 67 days post-BMT. Eligibility requirements for IL-2 therapy included demonstration of stable engraftment and absence of acute grade 2–4 graft-versus-host disease (GVHD). Low-dose IL-2 was well tolerated by the majority of patients, with only 4 of 29 subjects withdrawn early. Acute GVHD developed in only one individual. After 12 weeks of treatment, the mean number of circulating natural killer cells in patients increased 10- fold without any significant change in T-cell number. Of the 25 patients who received > or = 1 month of IL-2, only 6 have relapsed. Relapse rate and disease-free survival (DFS) were determined in the 25 patients who completed at least 4 weeks of IL-2 treatment and compared with historical controls transplanted at our institution for the same conditions and treated with an identical ablative regimen and method of T-cell depletion. Only control patients who had survived disease free for 100 days post-BMT were included in this analysis. Cox's proportional hazards regression model suggested that, compared with control patients without a history of GVHD, patients treated with IL-2 had a lower risk of disease relapse (hazard ratio 0.34; range, 0.14 to 0.82) and superior DFS (hazard ratio 0.39; range 0.18 to 0.87). A randomized controlled trial of IL-2 immunotherapy after T-cell-depleted BMT should now be undertaken.
Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families
Authors: Koeleman BP , Reitsma PH , Allaart CF , Bertina RM
Published: October 2019

Heterozygous protein C deficiency is associated with an increased risk for thrombosis. This association is restricted to a minority of protein C-deficient families, which have been defined as clinically dominant protein C-deficient. In contrast, in the clinically recessive protein C- deficient families, only the homozygous family members are (severely) affected. One possible explanation for this difference in thrombotic risk between families may be the presence of a second hereditary risk factor. A good candidate for this second risk factor is the recently identified resistance to activated protein C (APC). APC resistance, which is associated with a mutation in the FV gene (FV Leiden), is a common and strong risk factor for thrombosis. We show here that the prevalence of the FV Leiden mutation is high among symptomatic protein C-deficient probands (19%). In 6 clinically dominant protein C- deficient families, the segregation of the FV Leiden mutation and the protein C gene mutation was studied. A thrombotic episode had been experienced by 73% of the family members having both the protein C gene mutation and the FV Leiden mutation. In contrast, respectively, 31% and 13% of the family members having either the protein C gene mutation or the FV Leiden mutation had experienced a thrombotic episode. Moreover, the result of a two locus linkage analysis support the assumption that the FV gene and the protein C gene are the two trait loci responsible for the thrombophilia. These results indicate that carriers of both gene defects have an increased risk for thrombosis compared with related carriers of the single defect.
Progression of the myeloid differentiation program is dominant to transforming growth factor-beta 1-induced apoptosis in M1 myeloid leukemic cells
Authors: Selvakumaran M , Reed JC , Liebermann D , Hoffman B
Published: October 2019

Hematopoiesis is a profound example of cell homeostasis that is regulated throughout life. This process requires the participation of many factors, including positive and negative regulators of growth and differentiation, that determine survival, growth stimulation, differentiation, functional activation, and programmed cell death. Understanding the effects of multiple stimuli on specific cells at the molecular and cellular level is crucial towards understanding how the population of blood cells maintains a homeostatic state. Two appropriate stimuli for analysis, both of which are found in bone marrow, are differentiation-inducing cytokines, which induce terminal differentiation associated with growth arrest, ultimately culminating in programmed cell death, and transforming growth factor-beta 1 (TGF- beta 1), which induces rapid growth arrest and apoptosis of hematopoietic cells. Previously, we have shown, using M1 myeloblastic leukemic cells as a model system, that differentiation-inducing cytokines induce terminal differentiation associated with growth arrest and, only after 5 to 7 days, apoptosis, whereas TGF-beta 1 induces rapid growth arrest and apoptosis. In this report, we show that M1 myeloid leukemic cells treated concomitantly with the differentiation inducer interleukin-6 and TGF-beta 1 undergo terminal differentiation, in which modulators of the MyD118 gene product, previously shown to be a positive regulator of TGF-beta 1-induced apoptosis, are implicated to play a role in protecting the cells from TGF-beta 1-induced apoptosis. Furthermore, using M1 cell variants blocked at different stages after induction of differentiation, including M1myb and M1myc, as well as conditionally blocked M1mycer, it has been shown that the dominance of interleukin-6 to TGF-beta 1-induced apoptosis is dependent on the progression of the differentiation program. Further studies with M1 and the genetically engineered M1 cell variants will be instrumental towards molecularly dissecting the interaction of hematopoietic differentiation with a variety of apoptotic pathways.
Aggregated IgE mimic interleukin-3-induced histamine synthesis by murine hematopoietic progenitors
Authors: Salachas F , Schneider E , Lemoine FM , Lebel B , Daeron M , Navarro S , Ziltener H , Dy M
Published: October 2019

Similar to interleukin-3 (IL-3), IgE acts on murine bone marrow cells by inducing histamine production. This effect does not result from degranulation of histamine-containing cells, but from histamine synthesis, as assessed by the following findings. (1) The histamine content of freshly isolated bone marrow cells is too low to account for the increase in extracellular histamine levels. (2) Neither IL-3 nor IgE induced histamine production in the presence of the specific inhibitor of histidine decarboxylase (HDC), the histamine-forming enzyme. (3) Both the enzymatic activity and the mRNA expression of HDC were enhanced in response to IL-3 or IgE. Artificial aggregation or formation of IgE immune complexes augmented ther effect on histamine synthesis, indicating that the aggregated form is responsible for this biologic activity. Yet, it is apparently not mediated by Fc epsilon RI because their cross-linkage by dinitrophenyl bovine serum albumin after presensitization with IgE did not induce histamine production by hematopoietic progenitors. Among other aggregated isotypes tested, only IgG2a and, to a lesser extent, IgG1 had a consistent but lower effect, whereas IgM and IgA were completely inactive. The target cells of IL-3 and IgE in terms of histamine synthesis do not belong to mature bone marrow populations, especially mast cells. They copurify with hematopoietic progenitors in the low-density layers of a discontinuous Ficoll gradient where they represent around 5% of the cells, as determined by in situ hybridization. This percentage remained the same, regardless of whether the cells were stimulated by IgE or IL-3 alone or by a combination of both, suggesting a common responder cell. In accordance with this notion, histamine-producing cells could not be distinguished from each other on the basis of density, size and internal structure, or rhodamine (Rh) retention. Finally, the effect of IgE is not caused by the induction of IL-3 because anti-IL-3 antibodies did not abrogate the effect of IgE.
Variant Bernard-Soulier syndrome associated with a homozygous mutation in the leucine-rich domain of glycoprotein IX
Authors: Clemetson JM , Kyrle PA , Brenner B , Clemetson KJ
Published: October 2019

We describe a new variant of Bernard-Soulier syndrome. The patient (W.K.) showed the classic bleeding symptoms together with absence of platelet agglutination to restocetin plus von Willebrand factor, whereas aggregation to ADP, collagen, and arachidonic acid was normal. Platelets were markedly larger than normal and the patient had life- long thrombocytopenia. Surface-labeling of the platelets and two- dimensional gel electrophoresis showed reduced but detectable amounts of glycoprotein (GP) Ib-IX-V present;however, there was markedly less GPIX (2% +/- 1% of normal) than GPIb alpha, Ib beta, or V (7% +/- 2% of normal). This disproportion was confirmed by Western blotting. Sequence analysis was performed after polymerase chain reaction amplification of the coding region of the GPIX and GPI b alpha genes from the patient. A point mutation (A-->G) was found in GPIX converting 45Asn to Ser within the leucine-rich domain. No mutations were found in GPIb alpha. Both alleles of GPIX contained the same defect, which was confirmed by the appearance of a new cleavage site for the restriction enzyme Fnu4HI. This substitution did not affect glycosylation at the neighboring 44Asn as judged by the distribution on two-dimensional gels but did appear to change the conformation of the leucine-rich domain, thus reducing surface expression of the complex. The relationship between GPIb and GPV was not affected, indicating that GPIX does not regulate this. This homozygous mutation in GPIX indicates that, among other possible functions, the leucine-rich domains present on all components of GPIb- IX-V may play a role in the assembly and surface expression of the complex.
The structure of the urokinase-type plasminogen activator receptor gene
Authors: Casey JR , Petranka JG , Kottra J , Fleenor DE , Rosse WF
Published: October 2019

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that plays a central role in pericellular plasminogen activation. It contains 313 amino acid residues, including 28 cysteine residues in a pattern of three homologous repeats. The cysteine residue pattern suggests that uPAR belongs to a superfamily of proteins including CD59, murine Ly-6, and a variety of elapid snake venom toxins. A novel 1.7-kb uPAR cDNA was isolated that is missing exon 5 and that contains 380 bp not previously reported at the 5′ end. This cDNA was used to probe a human genomic library from which three clones were isolated and analyzed. The uPAR gene consists of 7 exons spread over 23 kb of genomic DNA. Exons 2, 4, and 6 code for homologous domains within the mature protein, as do exons 3, 5, and 7; CD59-like homologous pairs are encoded by exons 2–3, 4–5, and 6–7, respectively. The structure of the gene for uPAR further confirms the relationship of this molecule to the superfamily containing CD59, Ly-6, and the elapid snake venom toxins.
Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2
Authors: Barron-Casella EA , Kickler TS , Rogers OC , Casella JF
Published: October 2019

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.
Maturation of acute T-lymphoblastic leukemia cells after CD2 ligation and subsequent treatment with interleukin-2
Authors: Mentz F , Ouaaz F , Michel A , Blanc C , Herve P , Bismuth G , Debre P , Merle-Beral H , Mossalayi MD
Published: October 2019

In this study, we have investigated the ability of various cytokines to induce the maturation of acute lymphoblastic leukemia (T-ALL) cells with early T-cell phenotype. Leukemic blasts from 17 untreated T-ALL patients were assayed for their ability to acquire mature T-cell markers, CD3/T-cell receptor (TCR) in particular, after incubation with one or a combination of recombinant human interleukin-1 (IL-1), IL-2, IL-4, IL-7, and CD2-specific monoclonal antibody (MoAb). IL-7 or IL-2 induced the proliferation of some leukemic cells, whereas sequential cell treatment with CD2-MoAb and then IL-2 promoted CD3/TCR expression on nearly all CD2+ cells (15 of 16), except for 1 T-ALL that developed into CD3-CD16+CD56+ cells. Differentiation of T-ALL cells was also evidenced through the downregulation of CD34 precursor cell antigen, the generation of CD4+ and CD8+ cells from CD4+ CD8+ precursors, and the acquisition of mature T-cell functions. CD2 ligation induced a progressive increase of surface expression of IL-2 receptor alpha (IL- 2R alpha) and IL-2R beta and an accelerated in vitro death of leukemic cells. The ligation of IL-2R by IL-2 rescued T-ALL cells from death and promoted their progression toward more mature cells expressing extracellular CD3/TCR alpha beta complexes. Intracellular analysis indicates that TCR alpha transcription and membrane translocation of both TCR alpha and TCR beta were promoted in these conditions. Analysis of intracellular signals transduced during T-ALL differentiation indicated that CD2-ligation induced Ca2+ influx and that the ligation of CD2 and IL-2R induced distinct tyrosine phosphorylation patterns. The addition of inhibitors of tyrosine phosphorylation abolished T-ALL cell differentiation, which suggests the involvement of tyrosine kinases in this phenomenon. Together, we showed the constant maturation of leukemic early T cells after stimulation of surface CD2 and the high- affinity IL-2R.
High levels of the shed form of L-selectin are present in patients with acute leukemia and inhibit blast cell adhesion to activated endothelium
Authors: Spertini O , Callegari P , Cordey AS , Hauert J , Joggi J , von Fliedner V , Schapira M
Published: October 2019

L-selectin is expressed by most leukocytes and mediates the initial step of adhesion to vascular endothelium. A feature of this adhesion receptor is to be shed from the cell surface. We report here the presence of high levels of the shed form of L-selectin (sL-selectin) in plasma from patients with acute leukemia. We also show that sL-selectin purified from acute leukemia plasma exhibits functional activity. The mean (+/- 1 SD) plasma level of sL-selectin among 100 healthy individuals was 2.1 +/- 0.7 micrograms/mL. This value was increased (> 2 SD above the mean) in 63% of 58 patients with acute lymphoblastic leukemia (ALL) and 59% of 93 patients with acute myelogenous leukemia ([AML] P < .001). Repeated measurements in 24 patients showed normal- range levels in 16 of 16 patients in complete remission and high levels in eight of eight patients with therapy-resistant acute leukemia or leukemia relapse. Furthermore, elevated sL-selectin levels were detected in cerebrospinal fluid of three patients with ALL suffering from a relapse limited to the central nervous system. Epitope mapping with monoclonal antibodies demonstrated that L-selectin shedding from leukemic blasts was accompanied by conformational changes of its epidermal growth factor-like domain. A functional role for sL-selectin purified from leukemic plasma was supported by its ability to completely inhibit L-selectin-dependent adhesion of blast cells to tumor necrosis factor-alpha (TNF-alpha)-activated endothelium in vitro. These results suggest that sL-selectin may have an important role in the regulation of leukemic cell adhesion to endothelium. In addition, monitoring of the sL-selectin level may be useful for evaluating leukemia activity, in particular for the detection of leukemia relapse.
Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T-cell depletion after bone marrow transplantation?
Authors: Tiberghien P , Reynolds CW , Keller J , Spence S , Deschaseaux M , Certoux JM , Contassot E , Murphy WJ , Lyons R , Chiang Y
Published: October 2019

Allogeneic bone marrow transplantation (BMT) is associated with a severe complication--graft-versus-host disease (GVHD). Although effectively preventing GVHD, ex vivo T-lymphocyte marrow depletion unfortunately increases graft rejection and reduces the graft-versus- leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir (GCV) if subsequent GVHD was to occur. Thus, one could preserve the beneficial effects of the T cells on engraftment and tumor control in patients not experiencing severe GVHD. To obtain T cells specifically depleted by GCV, we transduced primary T cells with a retroviral vector containing the HS-tk and neomycin resistance (NeoR) genes. Gene transfer was performed by coculturing PHA +/- CD3- or alloantigen-stimulated purified T cells on an irradiated retroviral vector producer cell line or by incubating the T cells in supernatant from the producer. Subsequent culture in G418 for 1 week allowed for the selection of transduced cells. GCV treatment of interleukin-2-responding transduced and selected cells resulted in greater than 80% growth inhibition, whereas GCV treatment of control cells had no effect. Similarly, the allogeneic reactivity of HS-tk-transduced cells was specifically inhibited by GCV. Combining transduced and nontransduced T cells did not show a bystander effect, thus implying that all of the cells inhibited by GCV were indeed transduced. Lastly, studies involving the transduction of the HUT-78 (T-lymphoma) cell line suggest that stable expression of HS-tk can be maintained over 3 months in vitro in the absence of G418. In summary, we have established the feasibility of generating HS-tk-transduced T cells for subsequent in vivo transfer with hematopoietic stem cells and, if GVHD occurs, specific in vivo GCV- induced T-cell depletion in allogeneic BMT recipients.
Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator
Authors: Li Q , Stamatoyannopoulos G
Published: October 2019

The human beta locus control region (LCR) consists of five DNAse I hypersensitive sites (HS), four of which are erythroid specific and one, the further upstream located 5′HS5, is constitutive. To characterize the function of 5′HS5 we analyzed globin gene expression of various constructs containing HS3 as an enhancer, HS5, and the beta gene as a reporter. Expression was analyzed in stably transfected MEL cells. We found that the enhancing effect of hypersensitive site 3 is blocked when the HS5 is interposed between HS3 and the beta globin gene. These data suggest that the human 5′HS5 has the properties of a chromatin insulator.
Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells [published erratum appears in Blood 1995 Feb 1;85(3):862]
Authors: Goodman S , Xiao X , Donahue RE , Moulton A , Miller J , Walsh C , Young NS , Samulski RJ , Nienhuis AW
Published: October 2019

Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.
Tumor necrosis factor alpha-induced endothelial tissue factor is located on the cell surface rather than in the subendothelial matrix
Authors: Mulder AB , Hegge-Paping KS , Magielse CP , Blom NR , Smit JW , van der Meer J , Hallie MR , Bom VJ
Published: October 2019

Because there is no consensus regarding the precise distribution of induced endothelial tissue factor (TF), we studied TF activity in and on tumor necrosis factor alpha-stimulated cultured human umbilical vein endothelial cells (ECs) and their underlying matrix. TF was mainly expressed on the cell surface. Only small traces were found on the apical surface suggesting that TF is predominantly located on the basolateral side of the cell membrane. The presence of TF on the cell surface was confirmed by flow cytometry. Subendothelial TF activity appeared to be dependent upon the procedure used to remove the stimulated EC monolayer. Whereas ammonium hydroxide or hypotonic lysis resulted in relatively high levels of matrix-associated TF, virtually no TF was found on the matrix after mild enzymatic detachment of stimulated ECs. Cell removal with EDTA resulted in intermediate levels of matrix-associated TF. Neither the enzymatic treatment nor EDTA degraded or removed this TF activity. Similar patterns were observed for matrix-associated TF antigen and EC surface markers. Electron microscopic analysis showed cell fragments on the matrix after monolayer lysis. The findings strongly suggest that induced endothelial TF associated with the subendothelial matrix actually represents TF on EC remnants.
In vivo neutrophil and lymphocyte function studies in a patient with leukocyte adhesion deficiency type II
Authors: Price TH , Ochs HD , Gershoni-Baruch R , Harlan JM , Etzioni A
Published: October 2019

We investigated in vivo neutrophil and lymphocyte function in a patient who lacks Sialyl-Lewis-X, a ligand for the selectin family of leukocyte adhesion molecules (leukocyte adhesion deficiency II, LAD II). As assessed by skin chamber and skin window techniques, in vivo chemotaxis of neutrophils was markedly impaired (less than 6% of normal values). A marginal pool was present as determined by an increase in circulating neutrophils after epinephrine injection and calculated recovery of infused radiolabeled autologous neutrophils. Kinetic studies showed a reduced half-life of 3.2 hours (normal 7 hours) and markedly increased turnover rate (cells/kg/d) of approximately eight times the normal value. A normal antibody response to the T-cell-dependent antigen bacteriophage phi X174 showed that T/B-cell interaction is not affected in LAD II. These findings provide direct evidence that the selectin family and its ligands play an important role in neutrophil function.
Developmental regulation of human gamma- and beta-globin genes in the absence of the locus control region
Authors: Starck J , Sarkar R , Romana M , Bhargava A , Scarpa AL , Tanaka M , Chamberlain JW , Weissman SM , Forget BG
Published: October 2019

Two lines of transgenic mice carrying a normal 40-kb Kpn I beta-globin cluster transgene lacking the locus control region (LCR) were analyzed for the expression of human gamma- and beta-globin genes during mouse development. After RNase protection assays, the ratios of human G gamma- , A gamma-, or beta-mRNAs relative to endogenous mouse zeta + alpha mRNAs were obtained for each stage of development. The two gamma transgenes were expressed in day-11.5 blood (embryonic stage) and day- 13.5 blood (early fetal stage), but their expression was markedly decreased by day 16.5 of fetal life. Expression of the beta transgene was essentially absent at day 13.5, appeared at a low level by day 16.5, and was maximal by day 18.5, reaching a level similar to that observed in adult mice. Therefore, developmentally regulated expression of the human gamma- and beta-globin transgenes was obtained in the absence of the LCR. The relative expression of human gamma- and beta- globin genes was also examined in mice carrying 40-kb Kpn I beta- cluster transgenes with two different base substitutions associated with nondeletion forms of hereditary persistence of fetal hemoglobin (HPFH), -202 C-->G G gamma HPFH and -117 G-->A A gamma HPFH. The ratio of G gamma- to beta-globin transcripts was markedly increased in red blood cells of adult mice from three different lines carrying the transgene with the -202 G gamma HPFH mutation. This result confirms our previous preliminary results (Tanaka et al: Ann NY Acad Sci, 612:167, 1990) indicating that the -202 G gamma HPFH phenotype was reproduced in transgenic mice. The relatively low levels of G gamma-mRNA expression in adult mice carrying the non-HPFH transgene excludes a major influence of the 3′ beta-globin enhancer, present upstream of the G gamma gene because of the tandem repeat insertion, as a factor in the persistent G gamma gene expression observed in blood of adult mice carrying the -202 G gamma HPFH transgene. This conclusion is also supported by the fact that, in mice carrying the -117 A gamma HPFH transgene, G gamma-globin mRNA was detected in blood of adult animals only at low levels similar to that observed in the non-HPFH lines. However, the A gamma-HPFH phenotype was not reproduced in the transgenic lines carrying the -117A gamma HPFH mice.
Effect of low- and intermediate-purity clotting factor therapy on progression of human immunodeficiency virus infection in congenital clotting disorders. Transfusion Safety Study Group
Authors: Gjerset GF , Pike MC , Mosley JW , Hassett J , Fletcher MA , Donegan E , Parker JW , Counts RB , Zhou Y , Kasper CK
Published: October 2019

Low- and intermediate-purity clotting-factor therapies are believed to accelerate human immunodeficiency virus (HIV) progression in hemophiliacs through adverse immune effects of the other plasma proteins in the preparations. To investigate this postulate, we evaluated data from six clinical centers that observed persons with congenital factor deficiencies at 6-month intervals. The present analysis is based on HIV-infected subjects who received intermediate purity factor VIII or factor IX concentrates, or cryoprecipitate. For long-term outcome, we classified 374 subjects by the type and amount of treatment during our first year of observation, and determined the subsequent rate of progression to a CD4 count less than 200 cells/microL. A second analysis of this group used a repeated-measures, random-effect model that allowed for individual differences in CD4 decline. Finally, we compared short-term rates of change in CD4 count in each treatment interval of 525 subjects with the type and amount of factor therapy received in the same interval. There was no overall or dose-related deleterious effect of any form of treatment on CD4 trend. The CD4 decrease was less when cryoprecipitate was administered alone or combined with concentrate, but not significantly so. Our results counter the assertion that low- and intermediate-purity products accelerate the rate of CD4 decrease in HIV-1-infected hemophiliacs.
Fractionated total-body irradiation and high-dose etoposide as a preparatory regimen for bone marrow transplantation for 94 patients with chronic myelogenous leukemia in chronic phase
Authors: Snyder DS , Negrin RS , O'Donnell MR , Chao NJ , Amylon MD , Long GD , Nademanee AP , Stein AS , Parker PM , Smith EP
Published: October 2019

Ninety-four consecutive patients with chronic myelogenous leukemia in first clinical chronic phase, median age of 34.0 years (range, 6.8 to 52.4 years), with a histocompatible sibling donor, were treated with fractionated total body irradiation (1,320 cGy) and high-dose etoposide (60 mg/kg) followed by allogeneic bone marrow transplantation (BMT). The median time from diagnosis to BMT was 7.0 months (range, 2.3 to 72.0 months). Sixty patients were treated before BMT with hydroxyurea alone, four patients with busulfan alone, one patient with interferon alone, and the other 29 patients were treated with various combinations of these drugs. Cumulative probabilities of overall survival, event- free survival, and relapse at 5 years were 73%, 64%, and 14%, respectively. The median follow-up time for surviving patients was 38 months, ranging from 12 to 88 months. By stepwise Cox regression analysis, significant prognostic variables were age at transplant, acute graft-versus-host disease > or = grade II, cytomegalovirus- associated interstitial pneumonitis, and years from diagnosis to BMT.
Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor
Authors: Tian SS , Lamb P , Seidel HM , Stein RB , Rosen J
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that stimulates proliferation and differentiation of progenitor cells of neutrophils by signaling through its receptor (G-CSFR). Although the G- CSFR belongs to the cytokine receptor superfamily, which lacks an intracellular kinase domain, G-CSF-induced tyrosine phosphorylation of cellular proteins is critical for its biologic activities. We report here that JAK1 and JAK2 tyrosine kinases are tyrosine phosphorylated in response to G-CSF induction. We also demonstrate that the DNA-binding protein STAT3 (also called the acute-phase response factor [APRF], activated by interleukin-6) is an early target of G-CSF-induced tyrosine phosphorylation. G-CSF induces two DNA-binding complexes; the major complex contains tyrosine phosphorylated STAT3 protein and the minor complex appears to be a heterodimer of the STAT1 (previously p91, a component of DNA-binding complexes activated by interferons) and STAT3 proteins. Antiphosphotyrosine antibody interferes with the DNA binding activity of activated STAT3, indicating that tyrosine phosphorylation of STAT3 is important for the DNA binding activity. These results identify a signal transduction pathway activated in response to G-CSF and provide a mechanism for the rapid modulation of gene expression by G-CSF.
Retroviral vector design for long-term expression in murine hematopoietic cells in vivo
Authors: Correll PH , Colilla S , Karlsson S
Published: October 2019

A series of retroviral vectors containing the human glucocerebrosidase (GC) cDNA driven by various promoters have been constructed in an attempt to discover which vector design can most efficiently transduce murine hematopoietic stem cells (HSCs) and drive expression of the transferred gene in hematopoietic cells of mice reconstituted with the transduced stem cells. The simplest vector, LG, in which the GC gene is driven by the viral LTR, was the most efficient vector at infecting HSCs, with an average viral copy number in hematopoietic tissues of 3 copies/cell in recipient mice. In general, the viral vectors that contained any additional promoters or enhancers to drive expression of either the GC gene or a selectable marker gene (Neo) had lower titers and/or transduced HSCs at a lower efficiency. This was seen most markedly when the human phosphoglycerate (PGK) promoter was used to drive the human GC cDNA. Despite repeated attempts to obtain a high titer producer clone, this virus consistently produced low titers and subsequently resulted in the lowest proviral copy numbers in long-term reconstituted mice. Only the viral LTR and PGK promoter were capable of driving significant levels of human GC RNA in hematopoietic cells of long-term reconstituted mice, with a much lower level of RNA generated by an internal herpes TK or SV40 immediate early promoter. Insertion of the internal transcription unit in the opposite orientation relative to the viral LTRs had a detrimental effect on gene expression. The levels of RNA generated by a hybrid LTR containing the myeloproliferative sarcoma virus enhancer were higher in bone marrow-derived macrophages than in nonadherent cells of the bone marrow when compared with the LG vector. The presence of an internal promoter to drive expression of the human GC cDNA did not seem to have a detrimental effect on expression levels from the viral LTR. In fact, in the presence of an internal TK or PGK promoter expression from the LTR was increased despite the presence of lower proviral copy numbers. Insertion of a second gene (Neo) into the vector had a negative impact on long-term expression in hematopoietic cells in vivo; however, this seems to be due solely to the lower transduction efficiency of this vector. Overall, the highest levels of GC activity in macrophages of long-term reconstituted mice were generated by the LG vector; however, these levels were variable.(ABSTRACT TRUNCATED AT 400 WORDS)
The role of amino-terminal residues of the heavy chain of factor IXa in the binding of its cofactor, factor VIIIa
Authors: Hamaguchi N , Bajaj SP , Smith KJ , Stafford DW
Published: October 2019

The purpose of this study is to determine which residues of the factor IXa heavy chain are important for interaction with the cofactor of factor IXa, factor VIIIa. Because the monoclonal antibody (MoAb) FXC008 inhibits interaction between factors IXa and VIIIa, and because it also reacts with residues 181–310 of the factor IXa heavy chain, we used the computer-modelled structure of the factor IXa heavy chain to select charged surface residues likely to interact with FXC008 and/or factor VIIIa. We made mutations in the region of residues 181–310 of the heavy chain of factor IX, and replaced these amino acids individually with those located at the same position in factor X. The mutated factor IX retained complete clotting activity and thus interacted normally with factor VIIIa. Five mutant proteins (factor IXK214F, factor IXK228R, factor IXE240Q, factor IXK247V, and factor IXN260K) reacted with heavy chain-specific MoAbs FXC008 and A-5. Neither factor IXD276K nor factor IXR248H bound to FXC008. Factor IXR252V had reduced affinity to FXC008. Our results suggest the following: (1) factor IXa residues 214, 228, 240, 247, 248, 252, 260, and 276 are not involved in specific interaction with factor VIIIa; and (2) the FXC008 and factor VIIIa binding sites may not share critical residues.
Fetal thrombocytopenia: a retrospective survey of 5,194 fetal blood samplings
Authors: Hohlfeld P , Forestier F , Kaplan C , Tissot JD , Daffos F
Published: October 2019

Fetal platelet counts were retrospectively studied in a series of 5,194 consecutive fetal blood samplings (FBS). The mean value was 245 +/- 65 x 10(9)/L, without significant variation between 17 and 41 weeks' gestation. After exclusion of false thrombocytopenia due to contamination with amniotic fluid, 247 fetuses had platelet counts less than 150 x 10(9)/L. In 70 cases, thrombocytopenia was due to congenital infectious diseases (toxoplasmosis, rubella, and cytomegalovirus). It was related to immune causes in 45 cases: anti-HPA-1a (n = 23), anti- HPA-5b (n = 2) or possible anti-HLA (n = 2) alloimmunizations, and immune thrombocytopenic purpura (n = 18). Chromosomal abnormality was the etiology in 43 cases (trisomy 13, 18, and 21, Turner's syndrome, triploidy), and other disorders (multiple birth defects, intrauterine growth retardation, rhesus disease, and gestational thrombocytopenia) in 62 cases. No specific cause for the low platelet count could be established in 27 fetuses (range, 115 to 149 x 10(9)/L). Severe thrombocytopenia (< or = 50 x 10(9)/L) occurred mainly in immune cases (16%), congenital infectious diseases (7%), and chromosomal abnormalities (1%). Diagnosis, prognosis, and management of fetal thrombocytopenia are presented in the different clinical situations. In this series, FBS was never associated with serious bleeding, and no fetal exsanguination was observed.
A novel membrane antigen selectively expressed on terminally differentiated human B cells
Authors: Goto T , Kennel SJ , Abe M , Takishita M , Kosaka M , Solomon A , Saito S
Published: October 2019

A monoclonal antibody (MoAb) that defines a novel terminal B-cell- restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenstrom's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig- secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non- plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.
Transforming growth factor-beta relieves stem cell factor-induced proliferation of myelogenous leukemia cells through inhibition of binding of the transcription factor NF-jun
Authors: Sott C , Dorner B , Karawajew L , Herrmann F , Brach MA
Published: October 2019

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth factor-stimulated hematopoiesis in normal and leukemic conditions. Using the factor-dependent myelogenous leukemia cell lines GF-D8 and Mo7, we show that TGF-beta interferes with stem cell factor (SCF)-induced proliferation by downmodulating c-jun gene expression. The ability of SCF to induce accumulation of c-jun transcripts was abolished when TGF-beta was present in culture. Transcriptional nuclear run-on assays indicated that TGF-beta relieved the capacity of SCF to enhance the transcriptional rate of the c-jun gene. Deletion analysis of the c-jun promoter furthermore showed that SCF was activating the c- jun promoter via the NF-jun transcription factor. Gel mobility shift assays showed that SCF increased the binding activity of NF-jun to its recognition site within 5 to 15 minutes. Binding activity peaked at 1 hour after exposure to SCF and declined to starting levels within 4 hours. The ability of SCF to enhance NF-jun binding activity was also dose-dependent in the range of 5 to 100 ng/mL. Exposure of GF-D8 and Mo7 cells to TGF-beta before the addition of SCF antagonized SCF- induced NF-jun binding. Moreover, whereas SCF was capable of functionally activating a heterologous promoter containing the NF-jun binding site, pretreatment of GF-D8 cells with TGF-beta abolished transcriptional activation of this heterologous promoter. These findings indicate that SCF-mediated activation of c-jun via NF-jun is crucial for the SCF-inducible proliferative response and is inhibited by TGF-beta. In additional experiments, the antisense technique was used. Treatment of GF-D8 and Mo7 cells with an antisense oligodeoxyribonucleotide directed against the translation initiation site of c-jun abolished the capacity of SCF to induce a proliferative response, whereas sense and nonsense oligomers had no effect. Taken together, our data indicate that the counteracting modulation of the binding activity of NF-jun by SCF and TGF-beta regulates the expression of the c-jun gene and thereby the proliferative state of the GF-D8 and Mo7 target.