bluuebunny/crossref_metadata_2025 huggingface dataset:
Leukemic burden in subpopulations of CD34+ cells isolated from the mobilized peripheral blood of alpha-interferon-resistant or -intolerant patients with chronic myeloid leukemia
Authors: Van den Berg D , Wessman M , Murray L , Tong J , Chen B , Chen S , Simonetti D , King J , Yamasaki G , DiGiusto R , Gearing D , Reading C
Published: October 2019

We attempted to determine the frequency of normal hematopoietic stem cells (HSC) and contaminating leukemic cells in mobilized peripheral blood (MPB) collected from chronic myeloid leukemia (CML) patients, intolerant of alpha-interferon or with interferon-resistant disease. A total of 14 MPB samples, six from patients in chronic phase (CP) and eight from patients in accelerated phase or blast crisis (AP/BC) were studied. Cytogenetic analysis of MPB collected from AP/BC patients showed that 100% of the cells were Ph+, whereas cells from four of five CP MPB were Ph-. By contrast, fluorescence in situ hybridization (FISH) analysis of CP MPB showed a mean frequency of 14.7% Ph+ cells, while AP/BC MPB contained 39.2% Ph+ cells. In an attempt to purify normal HSC, subpopulations of the MPB CD34+ cells were isolated based on expression of the Thy-1 antigen (CDw90). The mean Ph+ cell frequency as determined by FISH within the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- populations from CP patients was 19.2% and 33.9%, respectively. In the AP/BC patients, levels of residual leukemic cells were significantly greater with mean Ph+ cell frequencies of 59.2% and 72.7% for the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- fractions, respectively. The frequency of cobblestone area forming cells (CAFC) was used as a means of quantitating the numbers of functional HSC within these cell subpopulations. The mean CAFC frequency was 1 of 19 for the CD34+Thy- 1+Lin- cells as compared with 1 of 133 for the Thy-1-fraction indicating a higher frequency of primitive progenitor cells in the Thy- 1+ subpopulation. CD34+ cell subsets from two patients were also injected into SCID-hu bone assays to determine the in vivo behavior of these cell populations. After 8 weeks, multilineage donor engraftment was observed in these grafts. FISH analysis of the donor cells within the grafts showed that 55.3% and 60.0% of the cells were Ph+. We conclude that unfractionated MPB from this patient population is not leukemia-free and that the CD34+Thy-1+Lin- cell subpopulation, although predominantly enriched for normal HSC, still contains substantial numbers of residual leukemic cells.
High resolution HLA matching associated with decreased mortality after unrelated bone marrow transplantation
Authors: Speiser DE , Tiercy JM , Rufer N , Grundschober C , Gratwohl A , Chapuis B , Helg C , Loliger CC , Siren MK , Roosnek E , Jeannet M
Published: October 2019

As compared with related HLA-identical sibling donors, bone marrow transplantation (BMT) with phenotypically HLA ABDR-compatible unrelated donors is associated with increased mortality. This may be due to hidden HLA incompatibilities not detected by conventional typing. We have analyzed 44 unrelated patient-donor pairs who were matched for HLA- A, -B, and -DR by routine tissue typing. Our comprehensive HLA typing approach consisted of serology, cytotoxic T-cell precursor (CTLp) tests, T-cell cloning, oligotyping, and DNA sequencing. Using these techniques, we identified numerous HLA allele mismatches not detected by the previously applied routine typing. Twenty-four patient-donor pairs were highly matched and had a low CTLp frequency, whereas the remaining 20 pairs were allele-mismatched for HLA-A, -B, -C, -DR, -DQ antigens and/or had a positive result of the CTLp test. Patient and donor age, diagnosis, and treatment did not differ significantly between the matched and mismatched transplants. The probability for severe acute graft-versus-host disease grades III-IV was 21% in the matched and 47% in the mismatched patients (P = .0464). Transplant- related mortality was 21% and 57% (P = .0072) and actuarial patient survival rates at 3 years were 61% and 13% (P = .0005). We conclude that both HLA class I and class II allele mismatches between unrelated phenotypically ABDR-compatible patient-donor pairs are frequent and associated with increased incidence of posttransplant complications.
Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis
Authors: Tauchi T , Damen JE , Toyama K , Feng GS , Broxmeyer HE , Krystal G
Published: October 2019

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.
Maintenance of murine long-term repopulating stem cells in ex vivo culture is affected by modulation of transforming growth factor-beta but not macrophage inflammatory protein-1 alpha activities
Authors: Soma T , Yu JM , Dunbar CE
Published: October 2019

Transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-l alpha (MIP-1 alpha) are both well-described inhibitors of committed and multipotential hematopoietic progenitors. The effect of these cytokines; on true stem cell activity in ex vivo culture systems as assayed by murine long-term repopulating activity (LTRA) has not been examined. We studied the stem cell effects of the addition of these cytokines to ex vivo cultures containing interleukin-3 (IL-3), IL- 6, and stem cell factor (SCF), using the murine competitive repopulation assay. We also tested the impact of adding an anti-TGF- beta neutralizing antibody, to ask whether abrogation of autocrine/paracrine TGF-beta may protect or enhance the survival of LTRA during ex vivo culture. TGF-beta 1 had significant suppressive effects on both short- and long-term repopulating activities, and anti- TGF-beta antibody had enhancing effects compared with control cultures containing IL-3, IL-6, and SCF alone. MIP-1 alpha had no significant effects on either short- or long-term repopulating ability. These data suggest that abrogation of TGF-beta during suspension culture may allow enhanced survival or even expansion of primitive cells ex vivo, with implications for many applications, including gene therapy.
A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I
Authors: Niwa K , Takebe M , Sugo T , Kawata Y , Mimuro J , Asakura S , Sakata Y , Mizushima J , Maeda A , Endo H , Matsuda M
Published: October 2019

A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation.
Engagement of the adhesion receptor CD22 triggers a potent stimulatory signal for B cells and blocking CD22/CD22L interactions impairs T-cell proliferation
Authors: Tuscano J , Engel P , Tedder TF , Kehrl JH
Published: October 2019

The B-lymphocyte-restricted adhesion protein CD22 mediates sialic acid- dependent cell-cell interactions. Engagement of CD22 on B lymphocytes with a CD22 monoclonal antibody (MoAb) HB22.7 that blocks the binding of CD22 to its ligand(s) directly stimulated B-cell proliferation. In addition, the HB22.7 MoAb costimulated B-cell proliferation with either anti-IgM, interleukin-2 (IL-2), IL-4, or CD40 and triggered predominantly B-cell IgG secretion with IL-2. Even more striking levels of B-cell proliferation occurred with HB22.7 MoAb under culture conditions that enhanced B-B-cell interactions. In contrast, a nonblocking CD22 MoAb (CD22.5) poorly costimulated in similar experiments. The functional differences between the two antibodies likely result from differing abilities to trigger downstream signaling events as significant differences in CD22 tyrosine phosphorylation and the recruitment of the tyrosine kinase p53/56lyn and the tyrosine phosphatase SH-PTP1C were found. Besides their role in B-cell stimulation, CD22/CD22L interactions may also assist in regulating T- cell proliferation because inhibition of CD22-CD22L engagement with the HB22.7 MoAb impaired T-cell proliferation in a costimulatory assay. Thus, CD22/CD22L interactions result in stimulatory signals for both B and T lymphocytes.
A concurrent human herpesvirus-6 infection renders two human hematopoietic progenitor (TF-1 and KG-1) cell lines susceptible to human immunodeficiency virus type-1
Authors: Furlini G , Vignoli M , Ramazzotti E , Re MC , Visani G , La Placa
Published: October 2019

In human immunodeficiency virus type-1 (HIV-1) infected individuals, CD34+ hematopoietic stem/progenitor cells are profoundly impaired in their proliferation/differentiation capacities. The bulk of the available experimental evidence seems to indicate that hematopoietic progenitors are not susceptible to HIV-1 infection and their defects seem rather the consequence of direct or indirect negative influences of HIV-1-specific soluble proteins released by productively infected accessory cells. We have now shown that in the presence of a concurrent human herpesvirus-6 infection, two hematopoietic (TF-1 [erythromyeloid] and KG-1 [lymphomyeloid]) progenitor cell lines and human CD34+ hematopoietic progenitors isolated from the bone marrow of normal donors, became susceptible to HIV-1 infection and permissive to HIV-1 replication, although with a limited virus yield. These results suggest a further possible mechanism leading to hematopoietic derangement in HIV-1-infected subjects and may help to clarify the controversial issue of the susceptibility of human hematopoietic progenitors to HIV-1 infection.
Increased adhesion of erythrocytes to components of the extracellular matrix: isolation and characterization of a red blood cell lipid that binds thrombospondin and laminin
Authors: Hillery CA , Du MC , Montgomery RR , Scott JP
Published: October 2019

Red blood cell (RBC) adhesion to the vascular endothelium is increased in several pathologic conditions, including sickle cell disease and malaria. However, RBC interactions with components of the subendothelial matrix are not well-characterized. Under in vitro flow conditions of 1 dyne/cm2, washed RBCs bound to the purified adhesive molecules thrombospondin (TSP) and laminin. Sickle RBCs had the greatest adhesion of all tested RBCs. The adhesion of sickle RBCs to immobilized TSP was inhibited by the anionic polysaccharides high molecular weight (MW) dextran sulfate and chondroitin sulfate A, but not other anionic polysaccharides of similar structure and/or charge density. These data were consistent with the RBC adhesive molecule being a sulfated glycolipid. Therefore, TSP-binding lipids from normal and sickle RBCs were isolated and characterized. The TSP-binding lipid was purified by alkaline methanolysis, anion exchange chromatography and preparative thin layer chromatography (TLC). A homogeneous band on TLC was identified using a specific overlay TSP-binding assay. TSP binding to the purified lipid was stable to bass and neuraminidase treatment, labile to acid treatment, and was inhibited by high MW dextran sulfate, similar to that seen with intact RBCs binding to immobilized TSP under conditions of flow. In addition, soluble laminin bound to the purified RBC lipid. This acidic TSP- and laminin-binding lipid(s) isolated from both sickle and normal RBC membranes may contribute to erythrocyte interactions with the subendothelial matrix, hereby participating in the pathogenesis of vaso-occlusive diseases.
Fas-mediated apoptosis of CD4+ and CD8+ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists
Authors: Estaquier J , Tanaka M , Suda T , Nagata S , Golstein P , Ameisen JC
Published: October 2019

Human immunodeficiency syndrome (HIV) infection leads to a progressive loss of T-cell-mediated immunity associated with T-cell apoptosis. We report here that CD4+ and CD8+ T cells from HIV-1-infected persons are sensitive to Fas (CD95/APO-1)-mediated death induced either by an agonistic anti-Fas antibody or by the physiologic soluble Fas ligand, although showing no sensitivity to tumor necrosis factor alpha-induced death. CD4+ and CD8+ T-cell apoptosis induced by Fas ligation was enhanced by inhibitors of protein synthesis and was prevented either by a soluble Fas receptor decoy or an antagonistic anti-Fas antibody. Fas- mediated apoptosis could also be prevented in a CD4+ or CD8+ T-cell- type manner (1) by several protease antagonists, suggesting the involvement of the interleukin-1beta (IL-1beta)-converting enzyme (ICE)- related cysteine protease in CD4+ T-cell death and of both a CPP32- related cysteine protease and a calpain protease in CD8+ T-cell death; and (2) by three cytokines, IL-2, IL-12, and IL-10, that exerted their effects through a mechanism that required de novo protein synthesis. Finally, T-cell receptor (TCR)-induced apoptosis of CD4+ T cells from HIV-infected persons involved a Fas-mediated death process, whereas TCR stimulation of CD8+ T cells led to a different Fas-independent death process. These findings suggest that Fas-mediated T-cell death is involved in acquired immunodeficiency syndrome (AIDS) pathogenesis and that modulation of Fas-mediated signaling may represent a target for new therapeutic strategies aimed at the prevention of CD4+ T-cell death in AIDS.
Continuous activation and deactivation of integrin CD11b/CD18 during de novo expression enables rolling neutrophils to immobilize on platelets
Authors: Sheikh S , Nash GB
Published: October 2019

In an in vitro flow model, unstimulated neutrophils rolled steadily over a surface coated with platelets, until superfusion of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) caused a dose-dependent (10(-11) to 10(-7) mol/L) transition from rolling to stationary attachment in seconds, followed more slowly by neutrophil shape change and spreading on the surface, However, at low concentrations of Ca2+ and Mg2+ (0.1 mmol/L and 0.05 mmol/L, respectively, rather than physiologic 1 mmol/L and 0.5 mmol/L), neutrophils first halted but then started to roll again and to detach from the surface over 5 to 10 minutes. At the low cation concentration, stopping was largely inhibited by antibodies to the neutrophil integrins CD18 or CD11b, but not CD11a. When neutrophils were pretreated with antibodies to CD11b or CD18 in 1 mmol/L Ca2+ 0.5 mmol/L Mg2+, stopping was not prevented but delayed. However, if antibodies were also included with the superfused fMLP, stopping was inhibited, and detachment followed. This indicates that CD11b/CD18 was newly expressed during shape change and mediated the second phase of neutrophil immobilization and spreading in a cation-dependent manner. Prestimulated neutrophils also bound to platelets and spread, but immobilization was blocked if they were perfused with antibody to CD18 or CD11b or with low Ca2+ and Mg2+. Examining the cation-dependence further, it was evident that the presence of Mg2+ was essential for integrin-mediated adhesion and that the Mg2+ concentration determined whether immobilization could be maintained or was transient. Continuous superfusion of fMLP was also essential for maintenance of stable adhesion and spreading. Thus, activation of constitutive CD11b/CD18 rapidly and reversibly converted rolling to stationary attachment, whereas maintenance of adhesion and neutrophil spreading required continual expression of additional CD11b/CD18 that was only functional at physiologic Mg2+. Continual activation and deactivation of CD11b/CD18 during de novo expression could mediate immobilization and onward migration of neutrophils in vivo, and activated platelets appear capable of supporting this process as well as endothelial cells.
Single human T cells stimulated in the absence of feeder cells transcribe interleukin-2 and undergo long-term clonal growth in response to defined monoclonal antibodies and cytokine stimulation
Authors: Sunder-Plassmann R , Breiteneder H , Zimmermann K , Strunk D , Majdic O , Knapp W , Holter W
Published: October 2019

The two-signal model of T-cell activation postulates that T lymphocytes require at least two distinct signals for activation. This model has been established with bulk cultures of T cells in which T-cell-T-cell interaction can occur, possibly delivering further unrecognized costimulatory signals. The signal requirements of single T cells for the induction of clonal cell growth or the transcription of cytokines would best be studied in a cell cloning system in the absence of feeder cells; however, such an experimental system has not been reported so far. In this study, we report the long-term cloning of human resting peripheral blood CD4+CD45RO- T cells under feeder cell-free conditions in response to CD3 and CD28 stimulation in the presence of exogenous interleukin-2 (IL-2). Cloning efficiency ranged from 40% to 60% depending on the presence of additional cytokines IL-1 and IL-6. Single- call polymerase chain reaction showed that transcription of IL-2 occurred in cells stimulated through CD3 plus CD28 alone. T cells grown in response to CD3 plus CD28 plus IL-2 stimulation produced both IL-4 and interferon-gamma (IFN-gamma) on restimulation (Th0 cells) and could be functionally differentiated into Th1- or Th2-type cells by the addition of IFN-gamma or IL-4, respectively, during cell cloning. These data show on the single-cell level a two-signal model of T- cell activation for the transcription of IL-2. In addition, these experiments show that IFN-gamma and IL-4 exert their T-cell- differentiating effects directly on the T cell without any further need for antigen-presenting cells. Together, our experiments show the feasability of a defined long-term clonal cell culture system to study the growth and differentiation of human T lymphocytes.
True histiocytic lymphoma following therapy for lymphoblastic neoplasms
Authors: Soslow RA , Davis RE , Warnke RA , Cleary ML , Kamel OW
Published: October 2019

True histiocytic lymphomas (THLs) are rare tumors in which the malignant cells show morphologic and immunophenotypic evidence of histiocytic differentiation. We describe THLs that arose after therapy for one case of T-lineage lymphoblastic lymphoma (LyL) and two cases of acute lymphoblastic leukemia (ALL) (both CD10+, one pre-B phenotype). The lymphoblastic neoplasms were not unusual in any way, and responded well to standard therapy. The THLs arose 10 to 20 months after complete remission was achieved for the lymphoblastic neoplasms, at which time there was still no clinical or pathologic evidence of the lymphoblastic neoplasms. All three THLs exhibited clinical and morphologic features of malignancy. Neoplastic cells in the THLs had abundant eosinophilic vacuolated cytoplasm and pleomorphic nuclei, and expressed histiocytic antigens in the absence of lymphocyte-specific lineage markers. Because THLs are rare neoplasms, their occurrence after otherwise successful therapy for lymphoblastic neoplasms in these three cases may constitute a distinct clinicopathologic entity.
Selectins mediate eosinophil recruitment in vivo: a comparison with their role in neutrophil influx
Authors: Henriques GM , Miotla JM , Cordeiro SB , Wolitzky BA , Woolley ST , Hellewell PG
Published: October 2019

The role of selectins in mediating eosinophil recruitment in vivo was assessed in a model of lipopolysaccharide (LPS)-induced mouse pleurisy. LPS administration resulted in significant eosinophil influx at 24 hours, whereas neutrophil recruitment to the cavity peaked at 4 hours and persisted for 24 hours. The anti-L-selectin monoclonal antibody (MoAb) MEL-14 effectively inhibited (by 97%) eosinophil influx at 24 hours and also inhibited neutrophil recruitment at both times (75% to 95%). Eosinophil recruitment was partially reduced (54%) by the anti-P- selectin MoAb 5H1 but, in contrast, was unaffected by the anti-E- selectin MoAb 10E6. Neutrophil influx at 4 or 24 hours was not affected by the anti-P- or anti-E-selectin MoAbs. However, coadministration of anti-P-selectin and anti-E-selectin was very effective at inhibiting eosinophil influx at 24 hours (86%) and neutrophil influx at 4 (93%) and 24 hours (92%). These results show that all three selectins play a role in LPS-induced eosinophil and neutrophil recruitment in vivo, although P- and E-selectin show a degree of functional redundancy. The demonstration that P-selectin mediates eosinophil but not neutrophil influx suggests that suppressing the function of this adhesion molecule may be beneficial in blocking eosinophil accumulation in pleural inflammation.
Interferon-alpha and hydroxyurea in early chronic myeloid leukemia: a comparative analysis of the Italian and German chronic myeloid leukemia trials with interferon-alpha [letter]
Authors: Hasford J , Baccarani M , Hehlmann R , Anseri H , Tura S , Zuffa E
Published: October 2019

In 1994, the Italian and the German Chronic myeloid leukemia (CML) trials comparing interferon-alpha (IFN-alpha) with conventional chemotherapy were published. The survival advantage in favor of IFN- alpha compared with hydroxyurea (HU; 72 v 52 months) was significant in the Italian (P < .002), but not in the German trial (66 v 56 months, P < .44). We set up a collaborative study to identify the reasons for the different outcomes. There are major differences in the trial protocols concerning admission criteria, treatment strategy, and definitions. The German patients were older and more seriously sick. Fifty-two of the 327 patients in the German IFN and HU arms did not fulfil Italian admission criteria, and 41 of the 322 Italian patients did not fulfil German admission criteria. Using mutually uniform admission criteria, the median survival times of the IFN patients are 76 (Italian) and 72 (German) months (P = .56). The Italian group administered IFN combined with HU as needed, whereas the German group strictly used IFN as monotherapy with rerandomization to busulfan (BU) or HU after IFN resistance or intolerability. The differences seen between the Italian and the German trial results can be accounted for by objective differences in study design, especially the admission criteria, treatment strategy, and bias due to intention to treat analysis. The detailed analysis of the data suggests that the combination of IFN with HU as needed is more effective than either agent alone.
Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures
Authors: Taichman RS , Reilly MJ , Emerson SG
Published: October 2019

Hematopoietic stem cell differentiation occurs in direct proximity to osteoblasts within the bone marrow cavity. Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently, we showed that human osteoblasts produce a variety of cytokine mRNAs including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6. We examined here the ability of osteoblasts to support the development of hematopoietic colonies from progenitors as well the ability to maintain long-term culture-initiating cells (LTC-IC) in vitro. Examination of the hematopoietic cells recovered after 2 weeks of culture showed that osteoblasts support the maintenance of immature hematopoietic phenotypes. In methylcellulose assays, osteoblasts stimulate the development of hematopoietic colonies to a level at least 10-fold over controls from progenitor cells. Using limiting dilutional bone marrow cultures, we observed an activity produced by osteoblasts resulting in an threefold to fourfold expansion of human LTC-IC and progenitor cells in vitro. Thus, the presence of hematopoietic stem cells in close proximity to endosteal surfaces in vivo may be due in part to a requirement for osteoblast-derived products.
The promoter and 5' flanking sequences controlling human B29 gene expression
Authors: Thompson AA , Wood WJ Jr , Gilly MJ , Damore MA , Omori SA , Wall R
Published: October 2019

The product of the B-cell-specific B29 gene (B29, Ig beta, CD79b) is essential for Ig-mediated B-cell activation via the B-cell antigen receptor complex (BCR) on human and murine B lymphocytes. To better understand the regulation of this pivotal gene, we have analyzed the human genomic DNA sequence upstream of the B29 ATG start codon for transcriptional control activity. The human B29 gene lacks either a TATA or a CAAT box and transcription is initiated at multiple sites. The minimal promoter of the human B29 gene is contained within a 193-bp region 5′ of these multiple start sites. This minimal promoter exhibits B-cell-specific activity and contains SP1, ETS, OCT, and IKAROS/LYF-1 transcription factor motifs. All these motifs are strikingly conserved in sequence and placement relative to the previously characterized murine B29 promoter. Additional upstream gene segments dramatically affected B29 minimal promoter activity. A newly identified motif called the B29 conserved sequence (BCS), found upstream of both human and murine B29 promoters, appears to stimulate B29 transcription through a novel mechanism. A single BCS had little effect either on the minimal B29 promoter or on a heterologous promoter. Instead, the BCS stimulated transcription by counteracting 5′ negative regulatory DNA sequences that block the activity of the B29 minimal promoter in its absence. These findings indicate that B29 gene expression is controlled by the complex interplay of positive and negative regulatory elements.
BCL-2 but not its Epstein-Barr virus-encoded homologue, BHRF1, is commonly expressed in posttransplantation lymphoproliferative disorders [see comments]
Authors: Murray PG , Swinnen LJ , Constandinou CM , Pyle JM , Carr TJ , Hardwick JM , Ambinder RF
Published: October 2019

Posttransplantation lymphoproliferative disease (PTLD) is virtually always associated with Epstein-Barr virus (EBV) infection. BCL-2 and other proteins that confer resistance to apoptosis have been implicated in the pathogenesis of a variety of malignancies including lymphomas. One EBV protein, BHRF1, is a homologue of BCL-2, whereas another, the latency membrane protein 1 (LMP-1), upregulates BCL-2 expression in vitro. In the present study, we used immunohistochemistry to study the expression of these viral and cellular proteins as well as a variety of other EBV-encoded proteins in PTLD. BHRF1 was not detected in any PTLD specimen, whereas BCL-2 was shown in 12 of 17 lesions examined. With one exception, all LMP1-positive cases also expressed BCL-2 and the absence of LMP1 was always associated with a lack of BCL-2 expression. The results do not support a role for the EBV homologue of BCL-2 in PTLD, but they do support a role for viral induction of BCL-2 expression.
Hypoxic induction of gene expression in chronic granulomatous disease- derived B-cell lines: oxygen sensing is independent of the cytochrome b558-containing nicotinamide adenine dinucleotide phosphate oxidase
Authors: Wenger RH , Marti HH , Schuerer-Maly CC , Kvietikova I , Bauer C , Gassmann M , Maly FE
Published: October 2019

Reduced oxygenation of a variety of cells results in transcriptional upregulation of several genes, including the hematopoietic hormone erythropoietin, the angiogenic vascular endothelial growth factor (VEGF), and glycolytic enzymes such as aldolase. Recently, the heme protein cytochrome b558 of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has been proposed as a key component of the oxygen-sensing mechanism. Cytochrome b558 consists of the p22phox and gp91phox subunits and is essential for superoxide generation in phagocytes and B lymphocytes. Mutations in these subunits result in cytochrome b558-negative chronic granulomatous disease (cytb- CGD), an inherited disorder in humans characterized by reduced microbicidal activity due to deficient superoxide generation. To test whether NADPH oxidase is involved in oxygen sensing, we exposed wild- type B-cell lines as well as cytb- CGD-derived B cell lines, deficient in either p22phox or gp91phox, to hypoxia (1% oxygen) or CoCl2 (100 mumol/L) and compared the mRNA levels of VEGF and aldolase with the untreated controls. Northern blot analysis revealed unimpaired basal and inducible expression of VEGF and aldolase mRNA in all four cytb- CGD-derived B-cell lines compared with wild-type cells. Furthermore, reconstitution of cytochrome b558 expression in cytb- CGD-derived B cells by transfection with p22phox or gp91phox expression vectors did not modify VEGF and aldolase mRNA expression. Thus, cytochrome b558 of the NADPH oxidase complex appears not to be essential for hypoxia- activated gene expression and can be excluded as a candidate for the putative universal oxygen sensor.
Rapid mobilization of hematopoietic progenitor cells in rhesus monkeys by a single intravenous injection of interleukin-8
Authors: Laterveer L , Lindley IJ , Heemskerk DP , Camps JA , Pauwels EK , Willemze R , Fibbe WE
Published: October 2019

Interleukin-8 (IL-8) is a chemoattractant cytokine involved in chemotaxis and activation of neutrophils. Because in vivo administration of IL-8 induces mobilization of hematopoietic stem cells in mice, we assessed the mobilizing properties of IL-8 in rhesus monkeys. Recombinant human IL-8 was administered as a single intravenous injection at doses of 10, 30, and 100 micrograms/kg to rhesus monkeys (age, 2 to 3 years; weight, 2.5 to 4.5 kg). Venous blood samples were obtained at time intervals ranging from 1 to 480 minutes after IL-8 administration. Cell counts, colony-forming unit-Mix assays, and fluorescence-activated cell sorter analysis were performed. Plasma was harvested to assess IL-8 levels. A time-controlled bolus intravenous injection of 100 micrograms IL-8 per kilogram of body weight resulted in peak IL-8 plasma levels up to 5 micrograms/mL. The calculated half-time life of free IL-8 was 9.9 +/- 2.2 minutes. IL-8 injection resulted in instant neutropenia that was due to pulmonary sequestration, as shown using 99mTc-labeled leukocytes. Within 30 minutes after IL-8 injection, neutrophilia developed with counts up to 10-fold greater than baseline levels. The numbers of hematopoietic progenitor cells (HPCs) increased from 45 +/- 48/mL to 1,382 +/- 599/mL of blood at 30 minutes after injection of 100 micrograms IL-8 per kilogram of bodyweight (mean +/- SD, n = 8). Individual animals showed 10- to 100-fold increase in numbers of circulating HPCs that returned to almost pretreatment values (92 +/- 52 CFU/mL) at 240 minutes after the injection of IL-8. Immunophenotyping showed no significant changes in lymphocyte (sub)populations. A second bolus injection of IL-8 with an interval of 72 hours resulted in similar numbers of mobilized stem cells as observed after the first injection, showing that no tachyphylaxis had occurred. We conclude that IL-8 induces mobilization of HPCs from the bone marrow of rhesus monkeys in a rapid and reproducible fashion. Therefore, IL-8 may be a potentially useful cytokine in the setting of blood stem cell transplantation.
Stable clonal expansion of T cells induced by bone marrow transplantation
Authors: Masuko K , Kato S , Hagihara M , Tsuchida F , Takemoto Y , Izawa K , Kato T , Yamamori S , Mizushima Y , Nishioka K , Tsuji K , Yamamoto K
Published: October 2019

The immune mechanisms of T cells regeneration after bone marrow transplantation (BMT) and the factors maintaining allogeneic marrow graft in the host are still unknown. To pursue this issue, we analyzed T-cell clonality of peripheral blood lymphocytes (PBLs) in BMT recipients, using reverse transcription polymerase chain reaction with T-cell receptor (TCR) V beta gene segment-specific primers and single- strand conformation polymorphism. PBLs from patients and donors showed a heterogeneous T-cell population with oligoclonal accumulations of CD8+ T cells. When PBLs were cultured in HLA-matched mixed lymphocytes reaction in vitro, no distinct clonal expansion was observed. However, after BMT, oligoclonal expansions were induced in the recipients in vivo, without a restriction of TCR V beta gene usage. Although part of the expansion was transient, the majority was repeatedly detected even several months later. Our results suggested that certain in vivo mechanisms maintain a stable clonal expansion of distinct T cells in marrow recipients. We also found in a single patient with graft-versus- host disease a replacement of expanded clones by other clones during follow-up. Diminishing numbers of accumulation clones were found in long-term marrow recipients, indicating a general tendency for clonal expansion to subside progressively. Considered together, our data suggest the involvement of clonally expanded T cells in lymphoid regeneration and in acute and chronic immune responses after BMT.
Expression of the p53 tumor suppressor gene induces differentiation and promotes induction of differentiation by 1,25-dihydroxycholecalciferol in leukemic U-937 cells
Authors: Ehinger M , Bergh G , Olofsson T , Baldetorp B , Olsson I , Gullberg U
Published: October 2019

Leukemic U-937 cells, which lack normal p53, were stably transfected with a temperature-sensitive mutant of p53 to investigate the consequences for growth and differentiation. On induction of wild-type p53 activity at the permissive temperature, some of these cells underwent maturation as judged by the capacity for oxidative burst and the appearance of monocyte related cell surface molecules. Moreover, wild-type p53-expressing cells were more sensitive than p53-negative control cells to induction of differentiation by 1,25- dihydroxycholecalciferol; a twofold to fourfold increase of the fraction of cells showing signs of terminal maturation was observed when wild-type p53-expressing cells were incubated with 1,25- dihydroxycholecalciferol at concentrations that only slightly affected control cells. Whereas wild-type p53 activity per se induced maturation of certain cells, other underwent cell death judging from the reduced capability to exclude trypan blue and the appearance of fragmented DNA in flow cytometric analysis. The p53-induced cell death could be inhibited by incubation with 1,25-dihydroxy-cholecalciferol, but not all-trans retinoic acid. Thus, 1,25-dihydroxycholecalciferol, seemed to increase the survival of wild-type p53-expressing cells and to cooperate with wild-type p53 to induce differentiation. The data imply that p53-mediated maturation in U-937 cells depends on optimal regulation of signals for differentiation, survival and proliferation, and suggest a role for p53 in the differentiation induction of leukemic cells.
Consistent amounts of acute leukemia-associated P190BCR/ABL transcripts are expressed by chronic myelogenous leukemia patients at diagnosis
Authors: Saglio G , Pane F , Gottardi E , Frigeri F , Buonaiuto MR , Guerrasio A , de Micheli D , Parziale A , Fornaci MN , Martinelli G , Salvatore F
Published: October 2019

In chronic myelogenous leukemia (CML), the Philadelphia (Ph) chromosome translocation results in the formation of BCR/ABL genes, normally transcribed in two types of hybrid transcripts with a b2a2 or b3a2 BCR/ABL junction, which give origin to 210-kD fusion proteins (P210). A third type of BCR/ABL (with e1a2 type of junction) has been identified in approximately 50% of the Ph-positive acute lymphoblastic leukemia (Ph+ALL) cases and results in the production of a BCR/ABL protein of 190 kD (P190). The presence of this transcript has been associated almost exclusively with the presence of an acute leukemia phenotype. By contrast, here we describe that in addition to transcripts with the b2a2 and b3a2 types of junction corresponding to the P210 proteins, virtually all CMLs at diagnosis bear also BCR/ABL transcripts showing the e1a2 type of junction, which correspond to the acute leukemia- associated P190 protein. With a quantitative polymerase chain reaction assay we found that the amount of the e1a2 mRNA present in CMLs in chronic phase, although in absolute amount much lower than that present in Ph+ ALLs, represents in most cases approximately 20% to 30% of the total BCR/ABL transcripts. Moreover, using a novel and very sensitive Western blot technique, we detected relevant amounts of P190 protein in addition to P210 from peripheral cells of two of the patients.
Induction of cyclophosphamide-resistance by aldehyde-dehydrogenase gene transfer
Authors: Magni M , Shammah S , Schiro R , Mellado W , Dalla-Favera R , Gianni AM
Published: October 2019

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cell lines in vitro, we tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. We have cloned a full- length human ALDH1 cDNA and used retroviral vectors to transduce it into human (U937) and murine (L1210) hematopoietic cell lines that were then tested for resistance to maphosphamide, an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced L1210 and U937 cells (50% inhibition concentration [IC50], approximately 13 mumol/L). The resistant phenotype was specifically caused by ALDH1 overexpression as shown by its reversion by disulfiram, a specific ALDH1 inhibitor. ALDH1 transduction into peripheral blood human hematopoietic progenitor cells also led to significant increases (4- to 10-fold; IC50, approximately 3 to 4 mumol/L) in cyclophosphamide resistance in an in vitro colony-forming assay. These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.
Phase II trial of 2-chlorodeoxyadenosine for the treatment of cutaneous T-cell lymphoma [see comments]
Authors: Kuzel TM , Hurria A , Samuelson E , Tallman MS , Roenigk HH Jr , Rademaker AW , Rosen ST
Published: October 2019

We investigated the efficacy of 2-chlorodeoxyadenosine (2-CdA) therapy in patients with mycosis fungoides (MF) and the Sezary syndrome (SS). Between February 1991 and November 1993, 21 patients with relapsed or refractory MF/SS were treated with 2-CdA. 2-CdA was administered by continuous intravenous infusion at a dose of 0.1 mg/kg/d for 7 days initially (13 patients), but was subsequently reduced to 5 days (nine patients) due to hematologic toxicity. All patients had failed to respond to at least one prior treatment for MF/SS (median number of total prior therapies, five; median number of systemic prior therapies, three) and had an Eastern Cooperative Oncology Group performance status of two or better. Cycles were administered at 28-day intervals. Assessable patients received at least 5 days of 2-CdA. Fourteen patients received more than one cycle of 2-CdA. An overall response rate of 28% was achieved. Three patients (14%) had a complete response with a median duration of 4.5 months (range, 2.5 to 16). Three (14%) had a partial response with a median duration of 2 months (range, 2 to 4). Fifteen patients (72%) had no response. The most significant toxicities encountered were bone marrow suppression (62% of patients) and infectious complications (62% of patients). Thirty-eight percent of patients experienced no toxicity from 2-CdA. 2-CdA has activity as a single agent in patients with previously treated relapsed MF/SS. Studies in less heavily pretreated individuals with 2-CdA alone or in combination will be undertaken.
Differential effects of nitric oxide on erythroid and myeloid colony growth from CD34+ human bone marrow cells
Authors: Shami PJ , Weinberg JB
Published: October 2019

Nitric oxide (NO) is a reactive molecule with numerous physiologic and pathophysiologic roles affecting the nervous, cardiovascular, and immune systems. In previous work, we have demonstrated that NO inhibits the growth and induces the monocytic differentiation of cells of the HL- 60 cell line. We have also demonstrated that NO inhibits the growth of acute nonlymphocytic leukemia cells freshly isolated from untreated patients and increases monocytic differentiation antigens in some. In the present work, we studied the effect of NO on the growth and differentiation of normal human bone marrow cells in vitro. Mononuclear cells isolated from human bone marrow were cultured in semisolid media and treated with the NO-donating agents sodium nitroprusside (SNP) or S- nitroso-acetyl penicillamine (SNAP) (0.25 to 1 mmol/L). Both agents decreased colony-forming unit-erythroid (CFU-E) and colony-forming unit- granulocyte macrophage (CFU-GM) formation by 34% to 100%. When CD34+ cells were examined, we noted that these cells responded to SNP and SNAP differently than did the mononuclear cells. At a concentration range of 0.25 to 1 mmol/L, SNP inhibited the growth of CFU-E by 30% to 75%. However, at the same concentration range, SNP increased the number of CFU-GM by up to 94%. At concentrations of 0.25 to 1 mmol/L, SNAP inhibited the growth of CFU-E by 33% to 100%. At a concentration of 0.25 mmol/L, SNAP did not affect CFU-GM. At higher concentrations, SNAP inhibited the growth of CFU-GM. Although SNP increased intracellular levels of cGMP in bone marrow cells, increasing cGMP in cells by addition of 8-Br-cGMP (a membrane permeable cGMP analogue) did not reproduce the observed NO effects on bone marrow colonies. These results demonstrate that NO can influence the growth and differentiation of normal human bone marrow cells. NO (generated in the bone marrow microenvironment) may play an important role modulating the growth and differentiation of bone marrow cells in vivo.
A1, a Bcl-2 family member, prolongs cell survival and permits myeloid differentiation
Authors: Lin EY , Orlofsky A , Wang HG , Reed JC , Prystowsky MB
Published: October 2019

A1, a bcl-2 family member, has been identified as a hematopoietic- specific, early inducible gene. In this study it is shown that stable transfection of A1 into an interleukin-3 (IL-3)-dependent myeloid precursor cell line, 32D c13, leads to a retardation of IL-3 withdrawal- induced cell death similar to that observed with transfection of bcl-2. However, unlike bcl-2. A1 expression permits the accumulation of differentiated myeloid cells both before and after IL-3 withdrawal. Total cell accumulation, on the other hand, is considerably greater after IL-3 deprivation in the bcl-2 transfectant than in A1-expressing cells. Cells cotransfected with the two genes behave similarly to cells singly transfected with bcl-2, except that viability following IL-3 withdrawal is somewhat further enhanced. These results suggest that these two proteins have distinct roles that may be related to the divergent regulation of their expression during myeloid differentiation.
The eosinophil-specific cell surface antigen, EOS47, is a chicken homologue of the oncofetal antigen melanotransferrin
Authors: McNagny KM , Rossi F , Smith G , Graf T
Published: October 2019

The EOS47 antigen is a 100-kD cell surface glycoprotein selectively expressed by avian retrovirus-transformed eosinophils and their precursors. We have purified the EOS47 protein to homogeneity and used peptide sequence information to clone EOS47-encoding cDNAs. The open reading frames from these cDNAs predict a 738 amino acid protein with homology to human melanotransferrin, a membrane-found, transferrin-like protein that is expressed at high levels by a subset of melanomas, tumor cell lines, fetal intestine, and liver, but not by most normal adult tissues. The predicted protein sequence of EOS47 displays a 61% sequence identity with melanotransferrin and conservation of all 28 cysteine residues, indicating a similar tertiary structure. The finding that EOS47 lacks several of the iron-coordinating amino acids present in all transferrins suggests that it may be impaired in its ability to bind iron. In nonhematopoietic tissues, EOS47 is expressed at high levels by epithelial brush borders of small intestine and kidney and at lower levels by cells lining the sinusoids of the liver. Within hematopoietic tissues, EOS47 is restricted to a subpopulation of cells (1% to 5%) in bone marrow and early spleen and fluorescence-activated cell sorting of EOS47+ cells leads to a dramatic ( > 30-fold) enrichment of peroxidase+ eosinophils. In contrast, peripheral blood eosinophils are EOS47-, suggesting that the antigen is expressed by newly formed eosinophils and that expression ceases shortly before these cells emigrate from the bone marrow into the peripheral blood. Our results show that melanotransferrin is a stage-specific marker of eosinophils and should be useful for their isolation and further characterization.