bluuebunny/crossref_metadata_2025 huggingface dataset:
Soluble thrombopoietin receptor (Mpl) and granulocyte colony- stimulating factor receptor directly stimulate proliferation of primitive hematopoietic progenitors of mice in synergy with steel factor or the ligand for Flt3/Flk2
Authors: Ku H , Hirayama F , Kato T , Miyazaki H , Aritomi M , Ota Y , D'Andrea AD , Lyman SD , Ogawa M
Published: October 2019

In an effort to establish the specificity of the thrombopoietin (TPO) effects on murine multipotential progenitors, we tested the effects of soluble TPO receptor (sTPOR; sMpl) on multilineage colony formation that was supported by a combination of TPO and steel factor (SF). Surprisingly, sTPOR did not suppress colony formation from primitive progenitors. This led to the discovery that sTPOR synergizes with SF or Flt3/Flk2 ligand (FL) to support the formation of various types of hematopoietic colonies including multilineage colonies. The colonies supported by the combination of sTPOR and SF were capable of expressing both myeloid and B-lymphoid potentials. Studies using micromanipulation and serum-free culture showed that the effects of sTPOR and SF on the primitive progenitors are direct, not mediated by contaminating stromal cells, and not dependent on factors present in the serum. TPOR belongs to the cytokine receptor group that includes granulocyte colony- stimulating factor receptor (G-CSFR) and erythropoietin receptor (EPOR). Therefore, we tested the effects of sG-CSFR and sEPOR on primitive progenitors. sG-CSFR, but not sEPOR, was able to synergize with SF or FL in supporting the proliferation of primitive progenitors. The direct effects of the soluble receptors appear to be mediated through interactions with their respective membrane-bound receptors expressed on the primitive hematopoietic progenitors.
Transplantation and gene transfer of the human glucocerebrosidase gene into immunoselected primate CD34+Thy-1+ cells
Authors: Donahue RE , Byrne ER , Thomas TE , Kirby MR , Agricola BA , Sellers SE , Gaudernack G , Karisson S , Lansdorp PM
Published: October 2019

In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.
Cloning and characterization of Fc alpha Rb, a novel Fc alpha receptor (CD89) isoform expressed in eosinophils and neutrophils
Authors: van Dijk TB , Bracke M , Caldenhoven E , Raaijmakers JA , Lammers JW , Koenderman L , de Groot RP
Published: October 2019

The Fc receptor for IgA (Fc alpha R, CD89) is a transmembrane glycoprotein found on monocytes, macrophages, neutrophils, and eosinophils. Here we describe the characterization of a novel isoform of the Fc alpha R cloned from a human eosinophil cDNA library. This clone, Fc alpha Rb, lacks the exon encoding the transmembrane/intracellular region of wild type Fc alpha R, which is replaced by 23 new amino acids. Expression of Fc alpha Rb mRNA could be detected in eosinophils and neutrophils. IIA1.6 murine pro-B cells transfected with Fc alpha Rb cDNA secrete high levels of the protein, but also a substantial amount of Fc alpha Rb is expressed at the cell membrane. Membrane-bound Fc alpha Rb binds IgA-coated beads equally well as wild type Fc alpha R. Surface expression is not affected by phosphatidyl inositol phospholipase C, indicating that glycosyl phosphatidyl inositol-linkage of Fc alpha Rb is not likely. In IIA1.6 cells expressing Fc alpha Rb and FcR gamma, which is necessary for signal transduction by wild type Fc alpha R, no tyrosine phosphorylation or Ca(2+)-mobilization could be observed after receptor cross-linking. These results indicate that Fc alpha Rb is likely to have a different function than wild-type Fc alpha R receptor.
Hepatosplenic T-cell lymphoma: a distinct clinicopathologic entity of cytotoxic gamma delta T-cell origin
Authors: Cooke CB , Krenacs L , Stetler-Stevenson M , Greiner TC , Raffeld M , Kingma DW , Abruzzo L , Frantz C , Kaviani M , Jaffe ES
Published: October 2019

We identified eight cases of T-cell lymphoma with evidence of a gamma delta phenotype over a 13-year period. Seven of these cases conformed to a distinct clinicopathologic entity of hepatosplenic gamma delta T- cell lymphoma. Nearly all of these patients were young adult males (five of seven), with a median age at presentation of 20 years. They presented with marked hepatosplenomegaly, without lymphadenopathy or significant peripheral blood lymphocytosis. Thrombocytopenia was seen in all patients, and five of seven were mildly anemic. The clinical course was aggressive, and despite multiagent chemotherapy, the median survival duration was less than 1 year. The morphologic findings were uniform; a monomorphic population of medium-sized lymphoid cells with moderately clumped chromatin and a rim of pale cytoplasm infiltrated the sinusoids of the spleen, liver, and bone marrow. The cells had a characteristic immunophenotype: CD2+, CD3+, CD4-, CD5-, CD7+, CD16+, CD57-, CD25-, T-cell receptor (TCR)delta +, beta F1-. CD8 was positive in four of seven cases tested, and CD56 was positive in five of six. All cases expressed the cytotoxic granule-associated protein, TIA1, but perforin was detected in only one case. All cases with assessable DNA had a TCR gamma gene rearrangement, and lacked Epstein-Barr virus sequences. Isochromosome 7q was identified in two cases with cytogenetic information. The one case of cutaneous gamma delta T-cell lymphoma differed in its clinical manifestations, histologic appearance, and immunophenotype. We conclude that hepatosplenic gamma delta T-cell lymphoma is a distinct clinicopathologic entity derived from cytotoxic gamma delta T cells, and should be distinguished from other lymphomas of T-cell and natural-killer cell (NK)-like T-cell derivation.
CD2 antigen expression on leukemic cells as a predictor of event-free survival after chemotherapy for T-lineage acute lymphoblastic leukemia: a Children's Cancer Group study
Authors: Uckun FM , Steinherz PG , Sather H , Trigg M , Arthur D , Tubergen D , Gaynon P , Reaman G
Published: October 2019

We examined the prognostic impact of CD2 antigen expression for 651 patients with T-lineage acute lymphoblastic leukemia (ALL), who were enrolled in front-line Childrens Cancer Group treatment studies between 1983 and 1994. There was a statistically significant correlation between the CD2 antigen positive leukemic cell content of bone marrow and probability of remaining in bone marrow remission, as well as overall event-free survival (EFS) (P = .0003 and P = .002, log-rank tests for linear trend). When compared with patients with the highest CD2 expression level (> 75% positivity), the life table relative event rate (RER) was 1.22 for patients with intermediate range CD2 expression level (30% to 75% positivity) and 1.81 for “CD2-negative” patients (< 30% positivity). At 6 years postdiagnosis, the EFS estimates for the three CD2 expression groups (low positivity to high positivity) were 52.8%, 65.5%, and 71.9%, respectively. CD2 expression remained a significant predictor of EFS after adjustment for the effects of other covariates by multivariate regression, with a RER of 1.47 for CD2- negative patients (P = .04). Analysis of T-lineage ALL patients shows a significant separation in EFS after adjustment for the National Cancer Institute (NCI) age and white blood cell (WBC) criteria for standard and high-risk ALL (P = .002, RER = 1.67). The determination of CD2 expression on leukemic cells helped identify patients with the better and poorer prognoses in both of these risk group subsets. For standard risk T-lineage ALL, CD2-negative patients had a worse outcome (P = .0007, RER = 2.92) with an estimated 5-year EFS of 55.9% as compared with 78.3% for the CD2-positive patients. Thus, CD2 negativity in standard risk T-lineage ALL identified a group of patients who had a worse outcome than high-risk T-lineage ALL patients who were CD2 positive. The percentage of CD2 antigen positive leukemic cells from T- lineage ALL patients is a powerful predictor of EFS after chemotherapy. This prognostic relationship is the first instance in which a biological marker in T-lineage ALL has been unequivocally linked to treatment outcome.
Microsatellite instability and p53 mutations in therapy-related leukemia suggest mutator phenotype
Authors: Ben-Yehuda D , Krichevsky S , Caspi O , Rund D , Polliack A , Abeliovich D , Zelig O , Yahalom V , Paltiel O , Or R , Peretz T , Ben-Neriah S , Yehuda O , Rachmilewitz EA
Published: October 2019

During the last decade the frequency of therapy-related acute leukemia (t-leuk) and myelodysplastic syndrome (t-MDS) has been increasingly observed. Over the past 15 years, we treated 56 patients with t-leuk who had received prior chemotherapy (39%), radiotherapy (11%), or both (45%). The drugs received included alkylating agents and topoisomerase II inhibitors. The primary tumors included hematological malignancies (49%) and solid tumors such as breast or ovarian cancer. The median age at diagnosis of the primary tumor was relatively young (43 years +/- 18). Twelve patients had more than one primary tumor and 31 patients had a family history of malignancy. Karyotypic abnormalities were found in 91% of the patients. Prognosis was uniformly poor, with an overall median survival of 10 months. Twelve of the 18 patients examined (67%) had a multidrug resistance phenotype. P53 genes of the leukemic cells, as well as the original tumors, were analyzed in 21 patients using polymerase chain reaction (PCR) with single-stranded conformation polymorphism analysis followed by sequencing. P53 mutations were identified in 38% of these patients, a relatively high prevalence compared with other forms of MDS or de novo acute myeloid leukemia. Mutations were nongermline and restricted to the leukemic cells. We identified different p53 mutations in the various primary tumors of individual patients. The presence of a mutator phenotype was assessed by PCR analysis of microsatellites in eight loci (one trinucleotide repeat sequence, four dinucleotide, and three mononuclear repeat sequences). Microsatellite instability in two to seven loci were found in 15 of 16 (94%) of the patients. This instability is compatible with a mutator phenotype, which predisposes the patients to the development of malignancies including t-leuk.
Phorbol ester stimulation increases sickle erythrocyte adherence to endothelium: a novel pathway involving alpha 4 beta 1 integrin receptors on sickle reticulocytes and fibronectin
Authors: Kumar A , Eckmam JR , Swerlick RA , Wick TM
Published: October 2019

Sickle-cell adherence to endothelium has been hypothesized to initiate or contribute to microvascular occlusion and pain episodes. Adherence involves plasma proteins, endothelial-cell adhesion molecules, and receptors on sickle erythrocytes. It has previously been reported that sickle reticulocytes express the alpha 4 beta 1 integrin receptor and bind to cytokine-activated endothelium via an alpha 4 beta 1/vascular-cell adhesion molecule-1 (VCAM-1) interaction. To elucidate other roles for alpha 4 beta 1 in sickle-cell adherence, the ability of activated alpha 4 beta 1 to promote adhesion to endothelium via a ligand different than VCAM-1 was explored. Adherence assays were performed under dynamic conditions at a shear stress of 1 dyne/cm2. Preincubation of sickle erythrocytes with phorbol 12,13-dibutyrate (PDBu) increased adherence of sickle cells eightfold as compared with untreated sickle cells. Normal erythrocytes, whether treated with PDBu or not, did not adhere to the endothelium. Activating anti-beta 1 antibodies 4B4 and 8A2 also increased the adhesion of sickle, but not normal, red blood cell (RBC) adhesion to endothelium. Anti-alpha 4 antibodies HP1/2 and HP2/1, inhibitory antibody 4B5, or an RGD peptide inhibited sickle-cell adherence induced by PDBu. Additional studies were undertaken to examine if fibronectin, a ligand for activated alpha 4 beta 1, was involved in PDBu-induced sickle erythrocyte adherence. Adherence of PDBu-treated sickle cells was completely inhibited by the CS-1 peptide of fibronectin. Fibronectin was detected on the surface of washed endothelium using an antifibronectin antibody in enzyme-linked immunosorbent assays. Antifibronectin antibody pretreatment of endothelial cells inhibited PDBu-induced adherence by 79% +/-17%. Incubation of sickle RBCs with exogenous fibronectin after PDBu treatment inhibited adherence 86% +/- 8%. Taken together, these data suggest that endothelial-bound fibronectin mediates adherence of PDBu- treated sickle cells. Interleukin-8 (IL-8), a chemokine released in response to bacterial infection, viral infection, or other injurious agents, and known to activate integrins, also increased adherence of sickle erythrocytes to endothelial cells via fibronectin. This novel adherence pathway involving sickle-cell alpha 4 beta 1 activated by PDBu or IL-8 may therefore be relevant in vivo at vascular sites that produce IL-8 or similar agonists in response to vascular injury or immune activation. These observations describe ways in which inflammation and immune responses cause vasoocclusive complications in sickle-cell disease.
Frequent clonal loss of heterozygosity but scarcity of microsatellite instability at chromosomal breakpoint cluster regions in adult leukemias
Authors: Pabst T , Schwaller J , Bellomo MJ , Oestreicher M , Muhlematter D , Tichelli A , Tobler A , Fey MF
Published: October 2019

Microsatellites are important highly polymorphic genetic markers dispersed in the human genome. Using a panel of 22 (CA)n repeat microsatellite markers mapped to recurrent breakpoint cluster regions specifically involved in leukemia, we investigated 114 adult leukemias (25 acute lymphocytic leukemia [ALL], 32 acute myeloid leukemia [AML], 36 chronic lymphocytic leukemia [CLL], and 21 chronic myeloid leukemia [CML] in chronic phase) for somatic mutations at these loci. In each patient, DNA from fresh leukemia samples was analyzed alongside normal constitutive DNA from buccal epithelium. We detected loss of heterozygosity (LOH) in 81 of 114 patients (ALL 16/25, AML 25/32, CLL 30/36, CML 10/21). Deletions were most often seen in ALL at 11q23 and 19p13; in AML at 8q22 and 11q23; in CLL at 13q14.3, 11q13, and 11q23; and in CML at 3q26. Only six deletions were reported in 74 karyotypes analyzed, whereas in these same cases, 91 LOH events were detected by microsatellites. Of 26 leukemias with a normal karyotype, 16 nevertheless showed at least one LOH by microsatellite analysis. Replication errors were found in 10 of 114 patients (8.8%). Thus, microsatellite instability is rare in leukemia in contrast to many solid tumors. Our findings suggest that in adult leukemia, LOH may be an important genetic event in addition to typical chromosomal translocations. LOH may point to the existence of tumor suppressor genes involved in leukemogenesis to a degree that has hitherto been underestimated.
Prognostic significance of BCL-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin's lymphoma: a British National Lymphoma Investigation Study
Authors: Hill ME , MacLennan KA , Cunningham DC , Vaughan Hudson B , Burke M , Clarke P , Di Stefano F , Anderson L , Vaughan Hudson G , Mason D , Selby P , Linch DC
Published: October 2019

The Bcl-2 protein is capable of preventing apoptosis, and in vitro evidence suggests a role in drug resistance. It is expressed and the gene is rearranged in a proportion of cases of large-cell non-Hodgkin's lymphoma (NHL), but the clinical significance of these findings is controversial. The purpose of this study was to determine the influence of both Bcl-2 expression and major breakpoint region (MBR) bcl-2 rearrangement in a large cohort of prospectively accrued patients with intermediate-grade B-cell NHL treated in a standardized manner. All patients with Working Formulation F, G, or H NHL treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy in British National Lymphoma investigation studies between July 1974 and April 1992 were considered for this study if the appropriate paraffin blocks were available. Paraffin sections from the diagnostic specimen were analyzed for evidence of MBR rearrangement using a polymerase chain reaction-based method, and for Bcl-2 expression using immunohistochemistry. Failure to achieve complete remission (CR), relapse, death from NHL, and deaths from all causes were used as end points to measure CR rate, actuarial relapse rate, actuarial survival from NHL, and actuarial overall survival. One hundred sixty-one suitable patients were identified and tested for the bcl-2 MBR translocation, with 27 (17%) found to be positive; 153 of these patients were tested with immunocytochemistry, and 84 (55%) showed evidence of Bcl-2 expression. For patients who achieved CR from the initial treatment, the relapse rate was significantly higher in those with Bcl-2 expression than in those without. In addition, multivariate analysis identified Bcl-2 expression as the only factor significantly related to relapse rate in the subjects measured. The cause-specific survival for NHL in the series as a whole was significantly lower in patients with Bcl-2 expression than in those without. MBR status had no significant influence on any of the outcome measures, but the number of MBR-positive patients was relatively small, and larger studies are required. In conclusion, in Working Formulation F, G, and H NHL of B-cell type, expression of Bcl-2 protein predicted independently for relapse.
Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery
Authors: Kook H , Goldman F , Padley D , Giller R , Rumelhart S , Holida M , Lee N , Peters C , Comito M , Huling D , Trigg M
Published: October 2019

We prospectively studied immune reconstitution in 102 children who underwent T-lymphocyte depleted bone marrow transplants using either closely matched unrelated donors or partially matched familial donors by assaying total lymphocyte counts (TLC), T-cell subsets, B cells, and natural killer cells. TLC, CD3+, and CD4+ T-cell counts remained depressed until 2 to 3 years posttransplant, whereas CD8+ T-cell counts normalized by 18 months, resulting in an inverted CD4:CD8 ratio until 12 months posttransplant. Although the percentage of NK cells was elevated early posttransplant, their absolute numbers remained normal. CD20+ B cells were depressed until 12 to 18 months posttransplant. Factors affecting immunophenotypic recovery were analyzed by nonparametric statistics. Younger patients tended to have higher TLC posttransplant. Higher marrow cell doses were not associated with hastened immunophenotypic recovery. Graft-versus-host disease (GVHD) and/or its treatment significantly delayed the immune reconstitution of CD3+, CD4+, and CD20+ cells. The presence of cytomegalovirus was associated with increased CD8+ counts and a decrease in the percentages of CD4+ and CD20+ cells.
Granulocyte colony-stimulating factor recruitment of CD34+ progenitors to peripheral blood: impaired mobilization in chronic granulomatous disease and adenosine deaminase--deficient severe combined immunodeficiency disease patients
Authors: Sekhsaria S , Fleisher TA , Vowells S , Brown M , Miller J , Gordon I , Blaese RM , Dunbar CE , Leitman S , Malech HL
Published: October 2019

Peripheral blood (PB) CD34+ cells mobilized by granulocyte colony- stimulating factor (G-CSF) administration are potentially useful for transplantation and as a target of gene transfer for therapy of hematopoietic disorders. Efficient harvest and planning for clinical use of PB CD34+ cells ideally requires foreknowledge of the expected mobilization kinetics and yield. We developed a sensitive flow cytometric assay for accurately enumerating CD34+ cells throughout the range seen at baseline to peak mobilization. We used this assay to assess the kinetics of G-CSF-mediated mobilization of CD34+ cells to PB in normal volunteers and in patients with chronic granulomatous disease (CGD) or adenosine deaminase (ADA)-deficient severe combined immunodeficiency disease (SCID). Two dose levels of G-CSF were examined (5 and 10 micrograms/kg/d for 7 days). Both doses were well tolerated. For normal subjects and patients an increase in PB CD34+ cells was first detected only preceding the third dose of G-CSF (day 3), peaked transiently on day 5 or 6, and then decreased thereafter despite additional doses of G-CSF. With 32 normal volunteers mean peak CD34+ cell counts were 57 and 76 cells/mm2 of blood (5 and 10 micrograms doses, respectively), whereas for 18 CGD patients the mean peaks were 31 and 40 cells/mm2 of blood. For 2 ADA-deficient SCID patients studied at a G-CSF dose of 5 micrograms/kg/d, the average peak was 16 cells/mm2 of blood. For both of these patient groups mobilization of CD34+ cells to PB was impaired compared with similarly treated normal subjects (P < .05). By contrast to the kinetics of the CD34+ cell mobilization, the absolute neutrophil count (ANC) increased markedly by 6 hours after the first dose of G-CSF and then increased steadily through day 8. At days 5 and 6 (peak mobilization of CD34+ cells) the mean ANC of CGD and ADA patients was only slightly lower ( < or = 15%) than that seen with normal subjects, whereas the difference in CD34+ cell mobilization was > 48%. Thus, ANC is not a reliable surrogate to predict peak PB CD34+ cell counts and direct enumeration of PB CD34+ counts should be undertaken in decisions regarding timing and duration of apheresis to harvest a specific number of these cells. Finally, unexpected, but significant differences in the PB CD34+ cell mobilization between normal subjects and patients with inherited disorders can occur and underscores the importance of establishing the expected mobilization of PB CD34+ cells in the planning of treatment approaches using these cells.
Interferon-gamma suppresses T-cell proliferation to mitogen via the nitric oxide pathway during experimental acute graft-versus-host disease
Authors: Krenger W , Falzarano G , Delmonte J Jr , Snyder KM , Byon JC , Ferrara JL
Published: October 2019

The development of graft-versus-host disease (GVHD) is associated with long-lasting and profound deficits in immune function that lead to increased morbidity and mortality after bone marrow transplantation (BMT). We investigated a mechanism of T-cell immunodeficiency in response to mitogen or alloantigen in an experimental model of acute GVHD by analyzing the roles of two immunosuppressive moieties: interferon gamma (IFN-gamma) and nitric oxide (NO). Splenocytes from mice with GVHD did not proliferate either to the T-cell mitogen, concanavalin A (Con A), or to host alloantigens, but only mitogen- activated cultures produced increased levels of NO. The abrogation of NO synthesis with LG-mono-methyl-arginine (NMMA) restored mitogen- induced proliferation but not the response to host antigens. The mechanism of impared proliferation to mitogen was dependent on IFN- gamma because blockade of this cytokine in culture inhibited NO production and restored proliferation to Con A to levels similar to those in transplanted control mice without GVHD. NMMA did not substantially reduce IFN-gamma levels, demonstrating that NO acted distally to IFN-gamma in the pathway of immunosuppression in response to mitogen. Furthermore, the prevention of IFN-gamma production in vivo after allogeneic BMT, by transplantation of polarized type 2 donor T cells (secreting interleukin-4 but not IFN-gamma), also prevented NO production and restored splenocyte responses to mitogen. Our data demonstrate the existence of NO-dependent and NO-independent pathways involved in suppression of T-cell proliferation during acute GVHD. Excess NO synthesis appears to be one mechanism by which IFN-gamma induces immunodeficiency after allogeneic BMT.
TEL-AML1 translocations with TEL and CDKN2 inactivation in acute lymphoblastic leukemia cell lines
Authors: Kim DH , Moldwin RL , Vignon C , Bohlander SK , Suto Y , Giordano L , Gupta R , Fears S , Nucifora G , Rowley JD
Published: October 2019

The t(12;21) (p 13; q22) results in the fusion of the TEL gene located on chromosome 12 with the AML1 gene located on the derivative chromosome 21. Because this translocation is difficult to detect using standard cytogenetic techniques, 27 previously karyotyped B-lineage acute lymphoblastic leukemia (ALL) cell lines were evaluated for the presence of the TEL-AML1 fusion using the reverse transcriptase- polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and cDNA sequencing. Six cell lines expressed the TEL-AML1 chimeric transcript by RT-PCR and the t(12;21) was confirmed by FISH analysis with probes for TEL, AML1, and chromosome 12. While only one of the 6 cell lines with the t(12;21) lost the der(12)t(12;21)-encoded AML1-TEL fusion transcript, 4 cell lines lacked expression of the nontranslocated allele of TEL and 5 cell lines lacked expression of CDKN2. Moreover, in 2 patients (1 with the TEL-AML1 transcript and 1 without), TEL expression was lost with disease progression; le, TEL was expressed in the initial cell lines (established at diagnosis or first relapse) whereas TEL was not expressed in the cell lines established from these patients in late-stage disease. These data show the coexistence of multiple genetic defects in childhood B-lineage ALL Cell lines with t(12;21) will facilitate the study of TEL-AML1 and AML1-TEL fusion proteins as well as TEL and CDKN2 gene inactivation in leukemia transformation and progression.
Mechanism of transcriptional activation of the immediate early gene Egr- 1 in response to PIXY321
Authors: Mignacca RC , Lee HJ , Kwon EM , Sakamoto KM
Published: October 2019

Studies with the granulocyte-macrophage colony-stimulating factor (GM- CSF)/interleukin-3 (IL-3) fusion protein, PIXY321, demonstrated enhanced biological activity of this molecule in comparison with GM-CSF or IL-3 alone or in combination. Experiments were performed to study the mechanisms resulting in PIXY321-induced egr-1 expression in human myeloid leukemic cells (TF-1). Transfections of egr-1 promoter constructs revealed that PIXY321 stimulation resulted in fourfold induction of the -116 and -600 nucleotide (nt) constructs. We transfected a -116 nt construct containing a deletion of the cyclic AMP response element (CRE) or mutation in the serum response element (SRE) and demonstrated that both the SRE and CRE are necessary for maximal induction. However, PIXY321 stimulation resulted in 2.5-fold induction of a SRE-CRE-containing construct (P < .05), suggesting that the SRE and CRE are sufficient for PIXY321 responsiveness. Electrophoretic mobility shift assays (EMSA) revealed that the CRE binding protein (CREB) was phosphorylated on serine 133 in PIXY321-stimulated but not - unstimulated extracts from cells cultured in GM-CSF. By Western analysis and EMSA, CREB was constitutively phosphorylated in TF-1 cells grown on PIXY321 before growth factor and serum starvation. However, in TF-1 cells grown on GM-CSF before starvation, CREB phosphorylation was observed 10 minutes after PIXY321 stimulation. Further-more, ENSAs with PIXY321-stimulated and -unstimulated extracts demonstrated the presence of specific proteins that recognize the SRE. Our data demonstrate that transcriptional regulation of egr-1 by PIXY321 is mediated by the CRE and SRE.
Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6
Authors: Nurden P , Humbert M , Piotrowicz RS , Bihour C , Poujol C , Nurden AT , Kunicki TJ
Published: October 2019

The sequence beta (3)203–228 is involved, in a yet undetermined manner, in alpha IIb beta 3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for beta (3)211–221, binds to alpha IIb beta 3 on adenosine diphosphate (ADP)-activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding- site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of alpha IIb beta 3. Its binding to thrombin- stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing with P-selectin and suggesting the presence of alpha IIb beta 3-ligand complexes in the alpha-granule membrane. There was little binding of AP6 to surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to alpha IIb beta 3 within the platelet. Thus, our results provide evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand in nonactivated platelets; (2) thrombin- induced activation of alpha IIb beta 3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound alpha IIb beta 3 to the surface.
Decreased interleukin-12 (IL-12) from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon- gamma, natural killer, and lymphokine-activated killer activity by IL- 12 in cord blood mononuclear cells
Authors: Lee SM , Suen Y , Chang L , Bruner V , Qian J , Indes J , Knoppel E , van de Ven C , Cairo MS
Published: October 2019

Interleukin-12 (IL-12) is a critical cytokine regulating natural killer (NK) and T-cell function. We hypothesized that the impaired ability of cord blood (CB) to produce normal adult levels of IL-12 in response to stimulation may contribute to the immaturity of CB immunity. Furthermore, exogenous IL-12 may compensate for the immaturity in CB cellular immunity and have the potential for immunotherapy post cord blood transplantation. We compared the expression and production of IL- 12 from activated cord versus adult mononuclear cells (MNC), regulatory mechanisms associated with IL-12 expression in CB MNC, and the effects of IL-12 on induction of CB interferon (IFN)-gamma production, NK, and lymphokine-activated killer (LAK) cytotoxicity. Northern analysis and enzyme-linked immunosorbent assay were performed in lipopolysaccharide (LPS)-stimulated CB and adult peripheral blood (APB) MNC. IL-12 mRNA expression was induced within 6 hours with LPS (10 micrograms/ml) and reached peak levels at 12 hours in both CB and APB MNC. However, IL-12 mRNA expression and protein accumulation in CB MNC were 35.8% +/- 4.84% (12 hours, n = 11, P < .05), and 17.6% +/- 1.7% (24, 72, 96 hours, n = 9, P < .05) respectively, when compared with APB MNC. Nuclear run-on assays showed no differences between CB and APB MNC in both the basal levels of transcription and the degree of transcriptional activation. However, the half-life of IL-12 p40 mRNA was approximately threefold lower in activated CB MNC than in activated APB MNC (CB: 114 +/- 3.0 minutes v APB: 353 +/- 7.8 minutes, n = 3, P < .05). Exogenous IL-12 (10 U/mL) induced a significant increase of IFN-gamma from both CB and APB MNC (24 hours, 72 hours, P < .05, n = 3). The stimulated CB IFN- gamma level reached comparable levels produced by unstimulated APB. IL- 12 treatment also significantly enhanced CB NK cytotoxicity against K562 and NB-100 cell lines to the comparable levels of APB (P < .05, n = 4). CB MNC was more responsive to IL-12 stimulation with respect to IFN-gamma production, NK, and LAK cytotoxicity when compared with APB. The present study suggests that IL-12 mRNA and protein expression is decreased in activated CB. This discrepancy in IL-12 production is secondary, at least in part, to the altered posttranscriptional regulation. The impaired, ability of CB MNC to produce IL-12 in response to stimulation may contribute to the decrease in IFN-gamma production and NK cytotoxicity. However, IL-12 enhanced IFN-gamma and NK activity in CB MNC up to the comparable levels of APB MNC. These findings suggest that reduced expression and production of IL-12 from activated CB may contribute to the immaturity in CB cellular immunity and contribute, in part, to decreased graft-versus-host disease following CB stem cell transplantation.
Prevention of allogeneic marrow graft rejection by donor T cells that do not recognize recipient alloantigens: potential role of a veto mechanism
Authors: Martin PJ
Published: October 2019

Clinical trials and experimental studies have demonstrated that donor T cells can play a critical role in preventing allogeneic marrow graft rejection. Results of a previous study showed that donor T cells were most effective for preventing rejection when they recognize an alloantigen expressed by recipient T cells and can cause graft-versus- host disease (GVHD). The present study examined models where marrow graft rejection can be prevented by donor T cells that do not recognize host alloantigens and cannot cause GVHD. Donor T cells prevented rejection of major histocompatibility complex (MHC) class I and II- disparate F1 marrow in parental recipients prepared with > or = 800 cGy total body irradiation (TBI) but not in those prepared with < or = 750 cGy TBI. In recipients prepared with high TBI exposures, rejection was mediated entirely by host CD8 cells. With lower TBI exposures, rejection was mediated by host CD4 cells and CD8 cells. These observations suggested the hypothesis that donor T cells prevent rejection mediated by host effectors that recognize donor MHC class I alloantigens but do not prevent rejection mediated by host effectors that recognize donor class II alloantigens. Consistent with this hypothesis, further experiments showed that F1 donor T cells can prevent rejection of MHC class I-disparate marrow in irradiated parental recipients but have no detectable effect on rejection of MHC class II-disparate marrow. We propose that the expression of MHC class I molecules on donor T cells makes it possible for these cells to inactivate the host response against donor class I alloantigens through a veto mechanism, whereas the absence of MHC class II molecules on murine T cells explains why these cells cannot inactivate the host response against donor class II alloantigens. Finally, donor CD4 cells and CD8 cells were equivalently effective for preventing rejection of F1 marrow in parental recipients, suggesting that veto activity is not restricted solely to the CD8 subset of murine T cells. A veto mechanism could enable donor T cells to prevent allogeneic marrow graft rejection without causing GVHD.
PI 3-kinase activation in BCR/abl-transformed hematopoietic cells does not require interaction of p85 SH2 domains with p210 BCR/abl
Authors: Jain SK , Susa M , Keeler ML , Carlesso N , Druker B , Varticovski L
Published: October 2019

BCR/abl is a chimeric oncogene implicated in the pathogenesis of human chronic myelogenous leukemia. Expression of the BCR/abl gene induces hematologic malignancies in transgenic mice and transformation of interleukin-3-dependent hematopoietic cells. The mechanism of BCR/abl- mediated transformation of hematopoietic cells is poorly understood and involves activation of at least two signaling pathways, p21ras and PI 3- kinase. Here we report that PI 3,4-P2 and PI 3,4,5-P3, the enzymatic products of PI 3-kinase, accumulate in metabolically labeled transformed hematopoietic cells, in contrast to our previous report on the lack of accumulation of PI 3-kinase products in nontransformed NIH 3T3 fibroblasts that express p210 BCR/abl. Transformed cells also have increased PI 3-kinase activity in total cell extracts and membrane fractions. Activation of PI 3-kinase occurs by occupancy of SH2 domains of PI 3-kinase regulatory subunit, p85, by phosphorylated YXXM motifs. Therefore, we investigated whether BCR/abl binds to p85 and whether this binding is mediated by interaction of p85 SH2 domains with YXXM motif of BCR/abl. Association of p210 BCR/abl with p85 in immune complexes and with p85 SH2 domains was evident in hematopoietic cells that express the wt p210 BCR/abl. However, the binding of BCR/abl to p85 SH2 domains was abolished in cells expressing mutant, temperature- sensitive (ts) p210 BCR/abl in which the tyrosine in the YXXM motif of p210 BCR/abl was replaced by histidine. Despite lack of direct interaction with p85 SH2 domains, expression of ts p210 BCR/abl resulted in rapid, time-dependent activation of total and membrane- associated PI 3-kinase and increased PI 3-kinase activity in anti-P-tyr and anti-abl immunoprecipitates. These data suggest that BCR/abl- induced activation of PI 3-kinase in hematopoietic cells does not require binding of p85 SH2 domains to BCR/abl gene product and involves interaction with other tyrosine phosphorylated intermediate proteins.
Targeted disruption of the murine tissue factor gene results in embryonic lethality
Authors: Toomey JR , Kratzer KE , Lasky NM , Stanton JJ , Broze GJ Jr
Published: October 2019

Tissue factor (TF) is an integral membrane glycoprotein that is believed to be the physiologic initiator of the blood coagulation cascade. Disruption of the mouse tissue factor gene leads to embryonic lethality between days E9.5-E11.5 of gestation. On E9.5, TF(-/-) embryos appear indistinguishable from their TF(+/+) and TF(+/-) littermates. By E10.5, TF(-/-) embryos are severely growth retarded, appear nearly bloodless, and are in most cases dead. Initial observations suggest that TF(-/-) embryos are dying of circulatory failure. Approximately 15% of the TF(-/-) embryos survive beyond E10.5, but none complete gestation. Heterozygotes appear normal and free of bleeding complications.
Nerve growth factor is involved in the supportive effect by bone marrow- -derived stromal cells of the factor-dependent human cell line UT-7 [published erratum appears in Blood 1996 Oct 1;88(7):2818]
Authors: Auffray I , Chevalier S , Froger J , Izac B , Vainchenker W , Gascan H , Coulombel L
Published: October 2019

We previously demonstrated that murine MS-5 and SI/SI4 cell lines induce the proliferation of human factor-dependent UT-7 cells in the absence of normally required human cytokines and also stimulate the differentiation of CD34+/CD38-LTC-ICs. We report in this study that the effect of MS-5 cells on UT-7 cells can be completely explained by the synergistic action of nerve growth factor (NGF) and stem cell factor (SCF) produced by these murine stromal cells. Purified murine NGF was able to support short-term clone formation and long-term growth of UT-7 cells in suspension cultures as efficiently as rhu-granulocyte- macrophage colony-stimulating factor. NGF action was mediated through the TrkA receptor, in which messenger RNA (mRNA) was easily detected in UT-7 cells by Northern blot. MS-5 cells strongly expressed NGF mRNA in Northern blot, and direct implication of MS-5-derived NGF in the induction of UT-7 cells proliferation was demonstrated in inhibition assays with an anti-NGF monoclonal antibody (MoAb) that neutralized by 84% +/- 4.1% (n = 5) UT-7 clone formation. However, NGF did not act alone, and several arguments demonstrated the synergistic action of MS- 5-derived SCF: (1) an anti-c-kit partially inhibited UT-7 cells clone formation in coculture assays, (2) SCF and NGF synergized in an H3-TdR incorporation assay, and (3) the stimulatory effect of 10x-concentrated MS-5 supernatant was completely inhibited by an anti-c-kit but not by an anti-NGF, and levels of soluble NGF (1.2 ng/mL) detected by enzyme- linked immunosorbent assay in 10x supernatant of MS-5 cells cultures were below the biologically active concentrations. In contrast, although MS-5 cells also promoted the differentiation of very primitive CD34+/CD38- human stem cells both in colony assays and long-term cultures, we could not incriminate MS-5-derived NGF in the observed effect: an anti-NGF MoAb did not inhibit the synergistic effect of MS-5 cells in colony assays or long-term cultures nor did soluble muNGF duplicate MS-5 effect and survival of CD34+/CD38- clonogenic progenitor cells promoted by MS-5 was unaffected by an anti-NGF and was not induced by soluble NGF alone or combined with SCF. In contrast, NGF in synergy with SCF supported the short-term maintenance of high numbers of CD34+/CD38+ mature erythroid progenitors probably through an indirect mechanism implying macrophages. These results suggest that NGF, in which the primary target cells are outside the hematopoietic system, is present in the marrow environment and might act at some steps of hematopoietic stem cell development. These results also underline that the response of cell lines and normal stem cells to stromal cells is mediated by different pathways.
A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa (beta 3) in a Chinese patient with Glanzmann's thrombasthenia
Authors: Grimaldi CM , Chen F , Scudder LE , Coller BS , French DL
Published: October 2019

A 20-year-old woman from a consanguineous family in the Hunan Province of the People's Republic of China was diagnosed as having Glanzmann's thrombasthenia based on (1) nearly a lifelong history of epistaxis, gum bleeding, petechiae, and purpura; (2) severe menorrhagia resulting in anemia and need for whole-blood transfusion; (3) normal coagulation assays; (4) prolonged bleeding time; (5) absent clot retraction; (6) decreased glass bead retention; (7) absent platelet aggregation in response to adenine diphosphate, epinephrine, and collagen; and (8) normal initial slope of platelet aggregation in response to ristocetin, but with a diminished maximal extent. The patient's platelets had a decreased level of platelet fibrinogen, but the deficiency was not as severe as in other Glanzmann's thrombasthenia patients. As judged by monoclonal antibody binding studies, surface glycoprotein (GP) IIb/IIIa (alpha IIb beta 3) expression was less than 15% of normal and alpha v beta 3 vitronectin receptor expression was 15% to 19% of normal, suggesting that the defect was in GPIIIa (beta 3). Immunoblotting of platelet lysates demonstrated decreased levels of GPIIb (approximately 30% to 35% of normal) and GPIIIa (approximately 10% of normal), and the GPIIb had undergone normal maturational processing into GPIIb heavy and light chains. Sequence analysis of the patient's GPIIIa RNA identified a G to A mutation at nucleotide 1219, predicting a Cys to Tyr substitution at residue 374. The patient's parents, who are first cousins, are asymptomatic and have only minor reductions in platelet aggregation. Direct sequencing of polymerase chain reaction-amplified cDNA and GPIIIa exon VIII indicated that the patient is homozygous and her parents are heterozygous for the mutation. Transient transfection studies in Chinese hamster ovary cells indicated that the mutation results in an 85% to 90% reduction in GPIIb/IIIa surface expression, but these cells retain the ability to mediate adhesion to immobilized fibrinogen. The relative preservation of platelet fibrinogen despite the very low level of platelet surface GPIIb/IIIa expression in this patient raises some interesting questions regarding the mechanism of fibrinogen uptake and the pathophysiology of Glanzmann's thrombasthenia.
Molecular basis of protein S deficiency in three families also showing independent inheritance of factor V leiden
Authors: Beauchamp NJ , Daly ME , Cooper PC , Makris M , Preston FE , Peake IR
Published: October 2019

The molecular basis of type I or III Protein S deficiency has been investigated in three kindred also showing independent inheritance of factor V (FV) Leiden. A T to C transition in codon 570 (Met-->Thr) was identified in the propositi and shown to segregate with protein S deficiency in all but one of the affected members of two kindred. This individual was heterozygous for a second transition (C to T) causing substitution of serine 624 by leucine. A second member of the same family, with markedly reduced free protein S levels when compared with affected relatives, was heterozygous for both mutations. Haplotype analysis of individuals with the mutated ATG570ACG allele in the two kindred suggested they may have been related by a common ancestor. A G to A transition resulting in substitution of cysteine 145 by tyrosine was detected in the third kindred. All mutations are believed to interfere with protein S binding to C4b-binding protein resulting in reduced free protein S levels. Of the five individuals studied who had experienced thrombotic events, three had combined protein S deficiency and FV Leiden reemphasising the importance of FV Leiden as an additional risk factor for thrombosis in protein S deficiency.
The CD43 130-kD peripheral T-cell activation antigen is downregulated in thymic positive selection
Authors: Ellies LG , Tao W , Fellinger W , Teh HS , Ziltener HJ
Published: October 2019

Specific glycoforms of CD43, the major O-glycosylated cell-surface protein on T lymphocytes, can affect cell adhesion according to the types of carbohydrate side chains carried. In the peripheral immune system, CD43 130 kD, which carries core 2 O-glycan structures on its surface, is an activation antigen expressed on both CD4 and CD8 single- positive (SP) T cells. We have previously shown that the 115-kD resting and 130-kD activation glycoforms of murine CD43 are differentially regulated on peripheral SP T cells. In this study, we used transgenic mice expressing T-cell receptors (TCRs) specific for antigens presented by class I and class II major histocompatibility complex (MHC) molecules to determine whether CD43 glycoforms are involved in thymocyte differentiation. Positive selection in these mice results in an increase in the production of CD8 and CD4 SP T cells, respectively, which express the transgenic TCR. Positive selection is also accompanied by the upregulation of TCR, CD69, and CD5. Using these markers to define stages of thymocyte maturation, we found that CD43 130 kD was downregulated in the positive selection of CD4 CD8 double- positive thymocytes expressing a class I but not class II MHC- restricted TCR. These data suggest that core 2 glycosyltransferase (C2GnT) modulated expression of CD43 glycoforms may be involved in thymic selection events.