bluuebunny/crossref_metadata_2025 huggingface dataset:
Dysregulation of CD95/CD95 Ligand-Apoptotic Pathway in CD3+ Large Granular Lymphocyte Leukemia
Authors: Lamy Thierry , Liu Jin Hong , Landowski Terry H. , Dalton William S. , Loughran Thomas P.
Published: October 2019

CD95 (Fas)-induced apoptosis plays a critical role in the elimination of activated lymphocytes and induction of peripheral tolerance. Defects in CD95/CD95L (Fas-Ligand)-apoptotic pathway have been recognized in autoimmune lymphoproliferative diseases (ALPS) and lpr or gld mice and attributed to CD95 and CD95L gene mutations, respectively. Large granular lymphocyte (LGL) leukemia is a chronic disease characterized by a proliferation of antigen-activated cytotoxic T lymphocytes. Autoimmune features such as hypergammaglobulinemia, rheumatoid factor, and circulating immune complexes are common features in LGL leukemia and ALPS. Therefore, we hypothesize that expansion of leukemic LGL may be secondary to a defective CD95 apoptotic pathway. In this study, we investigated expression of CD95 and CD95L in 11 patients with CD3+ LGL leukemia and explored the apoptotic response to agonistic CD95 monoclonal antibody (MoAb). We found that leukemic LGL from each patient expressed constitutively high levels of CD95/CD95L, similar to those seen in normal activated T cells. However, cells from 9 of these 11 patients were totally resistant to anti-CD95–induced apoptosis. Similarly, cells were resistant to anti-CD3-MoAb–triggered cell death. Lack of anti-CD95–induced apoptosis was not due to mutations in the CD95 antigen. Leukemic LGL were not intrinsically resistant to CD95-dependent death, because LGL from all but 1 patient underwent apoptosis after phytohemagglutinin/interleukin-2 activation. The patient whose leukemic LGL were intrinsically resistant to CD95 had an aggressive form of LGL leukemia that was resistant to combination chemotherapy. These findings that leukemic LGL are resistant to CD95-dependent apoptosis despite expressing high levels of CD95 are similar to observations made in CD95L transgenic mice. These data suggest that LGL leukemia may be a useful model of dysregulated apoptosis causing human malignancy and autoimmune disease.
Inhibition of the Differentiation of Dendritic Cells From CD34+ Progenitors by Tumor Cells: Role of Interleukin-6 and Macrophage Colony-Stimulating Factor
Authors: Menetrier-Caux C. , Montmain G. , Dieu M.C. , Bain C. , Favrot M.C. , Caux C. , Blay J.Y.
Published: October 2019

The escape of malignant cells from the immune response against the tumor may result from a defective differentiation or function of professional antigen-presenting cells (APC), ie, dendritic cells (DC). To test this hypothesis, the effect of human renal cell carcinoma cell lines (RCC) on the development of DC from CD34+progenitors was investigated in vitro. RCC cell lines were found to release soluble factors that inhibit the differentiation of CD34+ cells into DC and trigger their commitment towards monocytic cells (CD14+CD64+CD1a−CD86−CD80−HLA-DRlow) with a potent phagocytic capacity but lacking APC function. RCC CM were found to act on the two distinct subpopulations emerging in the culture at day 6 ([CD14+CD1a−] and [CD14−CD1a+]) by inhibiting the differentiation into DC of [CD14+CD1a−] precursors and blocking the acquisition of APC function of the [CD14−CD1a+] derived DC. Interleukin-6 (IL-6) and macrophage colony-stimulating factor (M-CSF) were found to be responsible for this phenomenon: antibodies against IL-6 and M-CSF abrogated the inhibitory effects of RCC CM; and recombinant IL-6 and/or M-CSF inhibited the differentiation of DC similarly to RCC CM. The inhibition of DC differentiation by RCC CM was preceeded by an induction of M-CSF receptor (M-CSFR; CD115) and a loss of granulocyte-macrophage colony-stimulating factor receptor  (GM-CSFR; CD116) expression at the surface of CD34+cells, two phenomenon reversed by anti–IL-6/IL-6R and anti–M-CSF antibodies, respectively. Finally, a panel of tumor cell lines producing IL-6 and M-CSF induced similar effects. Taken together, the results suggest that the inhibition of DC development could represent a frequent mechanism by which tumor cells will escape immune recognition.
Somatic Fas Mutations in Non-Hodgkin’s Lymphoma: Association With Extranodal Disease and Autoimmunity
Authors: Grønbæk Kirsten , Straten Per thor , Ralfkiaer Elisabeth , Ahrenkiel Vibeke , Andersen Mette Klarskov , Hansen Niels Ebbe , Zeuthen Jesper , Hou-Jensen Klaus , Guldberg Per
Published: October 2019

Fas (APO-1/CD95) is a cell-surface receptor involved in cell death signaling. Germline mutations in the Fas gene have been associated with autoimmune lymphoproliferative syndrome, and somaticFas mutations have been found in multiple myeloma. We have examined the entire coding region and all splice sites of theFas gene in 150 cases of non-Hodgkin’s lymphoma. Overall, mutations were identified in 16 of the tumors (11%). Missense mutations within the death domain of the receptor were associated with retention of the wild-type allele, indicating a dominant-negative mechanism, whereas missense mutations outside the death domain were associated with allelic loss. Fas mutations were identified in 3 (60%) MALT-type lymphomas, 9 (21%) diffuse large B-cell lymphomas, 2 (6%) follicle center cell lymphomas, 1 (50%) anaplastic large cell lymphoma, and 1 unusual case of B-cell chronic lymphocytic leukemia with a marked tropism for skin. Among the 16 patients with somaticFas mutations, 15 showed extranodal disease at presentation, and 6 relapsed in extranodal areas. Ten of 13 evaluable patients showed features suggestive of autoreactive disease. Our data indicate that somatic disruption of Fas may play a role in the pathogenesis of some lymphomas, and suggest a link between Fas mutation, cancer and autoimmunity.

© 1998 by The American Society of Hematology.
High Efficiency Gene Transfer to Human Hematopoietic SCID-Repopulating Cells Under Serum-Free Conditions
Authors: Schilz Andrea J. , Brouns Gaby , Knöβ Heike , Ottmann Oliver G. , Hoelzer Dieter , Fauser Axel A. , Thrasher Adrian J. , Grez Manuel
Published: October 2019

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34+ cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45+ cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.

© 1998 by The American Society of Hematology.
Detection of a Complex That Associates With the Bβ Fibrinogen G−455-A Polymorphism
Authors: Brown Erika T. , Fuller Gerald M.
Published: October 2019

The promoter region of the Bβ fibrinogen gene containing the polymorphic site (G−455-A) shows an increase in fibrinogen levels for individuals containing an adenine rather than a guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were performed using specific DNA probes containing base sequences pertinent to the allelic site. Specific DNA binding proteins were detected and their binding characteristics were determined. Secondly, we placed DNA fragments containing different −455 nucleotide substitutions of the Bβ promoter upstream of a luciferase reporter gene and transfected them into HepG2 cells to determine their effect on transactivation. An adenine at position −455 resulted in greater luciferase activity than when a guanine was present. UV cross-linking bound protein to the DNA demonstrated a 47-kD protein binding preferentially to the site when a guanine rather than an adenine was present at −455. We hypothesize that a transactivation protein complex associates with the site, but its association is stronger when guanine is present, thereby slowing downstream Bβ gene transcription. These data provide the first molecular evidence that accounts for the increase in fibrinogen in individuals carrying this allele.

© 1998 by The American Society of Hematology.
Expansion of Philadelphia Chromosome–Negative CD3+CD56+ Cytotoxic Cells From Chronic Myeloid Leukemia Patients: In Vitro and In Vivo Efficacy in Severe Combined Immunodeficiency Disease Mice
Authors: Hoyle Christine , Bangs Charles D. , Chang Pearl , Kamel Onsi , Mehta Bela , Negrin Robert S.
Published: October 2019

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-γ (IFN-γ), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3+CD56+ cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3+ T cells (median 97%, range 90% to 99%). The CD3+CD56+subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-γ, and tumor necrosis factor- (TNF-), but not IL-4. Both the CD4+ and CD8+ subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 × 107autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus–associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = .03). This study shows that CIK cells, which are Ph chromosome–negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells.

© 1998 by The American Society of Hematology.
Cytokine-Specific Activation of Distinct Mitogen-Activated Protein Kinase Subtype Cascades in Human Neutrophils Stimulated by Granulocyte Colony-Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, and Tumor Necrosis Factor-
Authors: Suzuki Kenichi , Hino Masayuki , Hato Fumihiko , Tatsumi Noriyuki , Kitagawa Seiichi
Published: October 2019

To clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF– or TNF-induced superoxide (O2−) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF– or TNF-induced O2− release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.
Sequential Homoharringtonine and Interferon- in the Treatment of Early Chronic Phase Chronic Myelogenous Leukemia
Authors: O’Brien Susan , Kantarjian Hagop , Koller Charles , Feldman Eric , Beran Miloslav , Andreeff Michael , Giralt Sergio , Cheson Bruce , Keating Michael , Freireich Emil , Rios Mary Beth , Talpaz Moshe
Published: October 2019

Homoharringtonine (HHT) is a novel plant alkaloid that produced a complete hematologic remission (CHR) in 72% of patients with late chronic phase chronic myelogenous leukemia (CML). Cytogenetic (CG) remissions were noted in 31%. In this study, six courses of HHT were administered to 90 patients with early chronic phase CML (< 1 year from diagnosis). Patients then received interferon- (IFN-) with a target dose of 5 MU/m2 daily. Results were compared with those in a prior group of patients treated with IFN-–based therapy between 1982 and 1990. Ninety-two percent of patients achieved CHR with HHT; CG responses were observed in 60% and were major in 27%. Both CHR and CG response rates were significantly higher than those seen in historical control patients after 6 months of IFN- therapy. After receiving HHT, patients required lower doses of IFN- to maintain a CHR. The median dose delivered was 2.4 MU/m2. This reduction in IFN- dose was associated with a lower incidence of myalgia and gastrointestinal (GI) disturbances than that seen in patients treated at the 5 MU/m2 dose. Overall, CG responses were seen in 66% of the patients who received HHT and IFN- compared with 61% of the historical control patients. HHT is a very effective treatment of early chronic phase CML, and ongoing trials are investigating the simultaneous administration of HHT and IFN-, as well as that of HHT and low-dose cytosine arabinoside in patients failing IFN- therapy.
The Impact of Obesity and Disease on Busulfan Oral Clearance in Adults
Authors: Gibbs John P. , Gooley Ted , Corneau Bruce , Murray Georgia , Stewart Patricia , Appelbaum Frederick R. , Slattery John T.
Published: October 2019

The apparent oral clearance (CL/F, mL/min) of busulfan was measured in 279 adolescent and adult patients. Significant (P< .05) determinants of CL/F by linear regression were: actual body weight (BW; r2 = 0.300), body surface area (BSA; r2 = 0.277), adjusted ideal body weight (AIBW; r2 = 0.265), and ideal body weight (IBW; r2= 0.173); whereas body mass index (BMI), height, age, gender, and disease were less important predictors. CL/F (mL/min) for normal weight patients (BMI, 18 to 27 kg/m2) was 16.2% lower (P< .001) than for obese patients (BMI, 27 to 35 kg/m2). Thus, expressing CL/F relative to BW did not eliminate statistically significant differences between normal and obese patients. However, busulfan CL/F expressed relative to BSA (110 ± 24 v 110 ± 24 mL/min/m2, P = 1.0) or AIBW (3.04 ± 0.65 v 3.19 ± 0.67 mL/min/kg, P = .597) were similar in normal and obese patients. Non-Hodgkin’s lymphoma patients (n = 10) had approximately 32% lower mean busulfan CL/F expressed relative to BW, BSA, or AIBW compared with patients with chronic myelogenous leukemia (n = 73). Routine dosing on the basis of BSA or AIBW in adults and adolescents does not require a specific accommodation for the obese. However, dosing based on BSA may be improved by considering CL/F differences in certain diseases. Adjusting dose for body size or disease does not diminish interpatient variability sufficiently to obviate plasma level monitoring in many indications.
Detailed Deletion Mapping of the Long Arm of Chromosome 6 in Adult T-Cell Leukemia
Authors: Hatta Yoshihiro , Yamada Yasuaki , Tomonaga Masao , Miyoshi Isao , Said Jonathan W. , Koeffler H. Phillip
Published: October 2019

Previously, we have found that the loss of heterozygosity (LOH) was frequently observed on chromosome 6q in acute/lymphoma-type adult T-cell leukemia (ATL), suggesting a putative tumor-suppressor gene for ATL may be present on chromosome 6q. To further define a region containing this gene, we performed fine-scale deletional mapping of chromosome 6q in 22 acute/lymphomatous ATL samples using 24 highly informative microsatellite markers. LOH was found in 9 samples (40.9%) at 1 or more of the loci examined. Of the 9 samples, 8 shared the same smallest commonly deleted region flanked by D6S1652 and D6S1644 (6q15-21). The genetic distance between these two loci is approximately 4 cM. These results suggest that a putative tumor-suppressor gene on chromosome 6q15-21 probably plays a very important role in the evolution of acute/lymphomatous ATL. Our map provides key information toward cloning the gene.
Reduced Expression of Adhesion Molecules and Cell Signaling Receptors by Chronic Lymphocytic Leukemia Cells With 11q Deletion
Authors: Sembries Sabine , Pahl Heike , Stilgenbauer Stephan , Döhner Hartmut , Schriever Folke
Published: October 2019

Deletions in chromosome bands 11q22-q23 were recently shown to be one of the most frequent chromosome aberrations in B-cell chronic lymphocytic leukemia (B-CLL). Patients suffering from B-CLL with 11q deletion are characterized by extensive lymphadenopathy, rapid disease progression, and short survival times. Phenotypic and functional characteristics of B-CLL cells with 11q deletion that may help to explain the pathophysiology of this entity are yet unknown. In the present study, B-CLL cells with (n = 19) and without (n = 19) 11q deletion were analyzed for their expression of functionally relevant cell surface molecules (n = 57). B-CLL cells with 11q deletion carried significantly lower levels of the adhesion molecules CD11a/CD18 (integrin L/β2), CD11c/CD18 (integrin X/β2), CD31 (PECAM-1), CD48, and CD58 (LFA-3). Furthermore, B-CLL cells with 11q deletion expressed less the cell signaling receptors CD45 (leukocyte common antigen [LCA]), CD6, CD35 (complement receptor 1), and CD39. Reduced CD45 levels and low-level expression of CD49d correlated with decreased overall survival. B-CLL cells with or without 11q deletion did not differ in their growth fractions, expression levels of transcription factor NF-κB, or their response to mitogenic stimuli. Decreased levels of functionally relevant adhesion molecules and of cell signaling receptors may contribute to the pathogenesis of the subgroup of B-CLL characterized by 11q22-q23 deletion.
Primary Plasma Cell Leukemia: Clinical, Immunophenotypic, DNA Ploidy, and Cytogenetic Characteristics
Authors: Garcı́a-Sanz R. , Orfão A. , González M. , Tabernero M.D. , Bladé J. , Moro M.J. , Fernández-Calvo J. , Sanz M.A. , Pérez-Simón J.A. , Rasillo A. , Miguel J.F. San
Published: October 2019

We report on a series of 26 patients diagnosed with primary (de novo) plasma cell (PC) leukemia (PCL) in whom we analyzed the clinicobiologic characteristics of the disease together with the immunophenotype, DNA cell content, proliferative index, and numeric chromosomal aberrations of the neoplastic PC, and compared them with 664 multiple myeloma (MM) patients at diagnosis. The median age, sex ratio, and bone lesion extension were similar, but PCL cases displayed a higher prevalence of clinical stage III, extramedullary involvement, and Bence Jones cases, with fewer IgA cases than for MM patients. In addition, according to several prognostic indicators (β2-microglobulin serum level, proportion of S-phase PCs, proteinuria, calcium serum level, lactate dehydrogenase [LDH] and renal function), the incidence of adverse prognostic factors was significantly higher in PCL versus MM. Immunophenotypic expression was similar for CD38, CD138, CD2, CD3, CD16, CD10, CD13, and CD15, but PCL differed from MM in the expression of CD56, CD9 HLA-DR, CD117, and CD20 antigens. Twenty-two PCL cases were diploid and one was hypodiploid, while most MM cases (57%) showed DNA hyperdiploidy. With the fluorescent in situ hydridization (FISH) technique, 12 of 13 PCL cases displayed the numeric aberrations, −13 (86%), ±1 (57%), +18 (43%), and −X in women (25%), but they lacked several numeric aberrations usually found in MM such as +3, +6, +9, +11, and +15. PCL cases had a lower overall response to therapy than MM cases (38% v 63%, P = .01332). Among PCL patients, a trend for a worse response was observed in cases treated with melphalan and prednisone (MP) versus polychemotherapy. Overall survival was significantly worse in PCL versus MM patients (8 v 36 months,P < .0001), but it was significantly better in PCL patients treated with polychemotherapy versus MP (18 v 3 months,P = .0137). By contrast, MM patients did not show significant differences in overall survival according to the treatment used, MP or polychemotherapy. Ten variables seemed to predict survival in PCL patients, but only the β2-microglobulin level and S-phase PCs retained an independent value in multivariate analysis. In summary, our study illustrates that PCs from PCL display singular phenotypic, DNA cell content, and cytogenetic characteristics that lead to a different disease evolution versus MM.
Treatment of Refractory and Relapsed Acute Myelogenous Leukemia With Combination Chemotherapy Plus the Multidrug Resistance Modulator PSC 833 (Valspodar)
Authors: Advani Ranjana , Saba Hussain I. , Tallman Martin S. , Rowe Jacob M. , Wiernik Peter H. , Ramek Joseph , Dugan Kathleen , Lum Bert , Villena Jenny , Davis Eric , Paietta Elisabeth , Litchman Manuel , Sikic Branimir I. , Greenberg Peter L.
Published: October 2019

A potential mechanism of chemotherapy resistance in acute myeloid leukemia (AML) is the multidrug resistance (MDR-1) gene product P-glycoprotein (P-gp), which is often overexpressed in myeloblasts from refractory or relapsed AML. In a multicenter phase II clinical trial, 37 patients with these poor risk forms of AML were treated with PSC 833 (Valspodar; Novartis Pharmaceutical Corporation, East Hanover, NJ), a potent inhibitor of the MDR-1 efflux pump, plus mitoxantrone, etoposide, and cytarabine (PSC-MEC). Pharmacokinetic (PK) interactions of etoposide and mitoxantrone with PSC were anticipated, measured in comparison with historical controls without PSC, and showed a 57% decrease in etoposide clearance (P = .001) and a 1.8-fold longer beta half-life for mitoxantrone in plasma (P < .05). The doses of mitoxantrone and etoposide were substantially reduced to compensate for these interactions and clinical toxicity and in Cohort II were well tolerated at dose levels of 4 mg/m2mitoxantrone, 40 mg/m2 etoposide, and 1 g/m2 C daily for 5 days. Overall, postchemotherapy marrow hypoplasia was achieved in 33 patients. Twelve patients (32%) achieved complete remission, four achieved partial remission, and 21 failed therapy. The PK observations correlated with enhanced toxicity. The probability of an infectious early death was 36% (4 of 11) in patients with high PK parameters for either drug versus 5% (1 of 20) in those with lower PK parameters (P = .04). P-gp function was assessed in 19 patients using rhodamine-123 efflux and its inhibition by PSC. The median percentage of blasts expressing P-gp was increased (49%) for leukemic cells with PSC-inhibitable rhodamine efflux compared with 17% in cases lacking PSC-inhibitable efflux (P = .004). PSC-MEC was relatively well tolerated in these patients with poor-risk AML, and had encouraging antileukemic effects. The Eastern Cooperative Oncology Group is currently testing this regimen versus standard MEC chemotherapy in a phase III trial, E2995, in a similar patient population.
Activation of Peripheral Blood T Cells by Interaction and Migration Through Endothelium: Role of Lymphocyte Function Antigen-1/Intercellular Adhesion Molecule-1 and Interleukin-15
Authors: Sancho David , Yáñez-Mó Marı́a , Tejedor Reyes , Sánchez-Madrid Francisco
Published: October 2019

Cell adhesion molecules have a key role in the migration of T cells to inflammatory foci. However, the effect of the endothelial-lymphocyte interaction on the activation of the latter cells remains unresolved. We have studied the effect of resting and stimulated endothelial cells (ECs) on the activation of peripheral blood T cells (PBTLs), as assessed by the expression of CD69 and CD25 activation antigens. The incubation of PBTLs with tumor necrosis factor-–activated EC monolayers, either alive or fixed, induced the expression of CD69 but not CD25, preferentially in the CD8+CD45RO+ cell subset. Furthermore, it induced the production of cytokines such as IFN-γ, but not that of interleukin-2 (IL-2) and IL-4. EC treated with other stimuli such as IL-1β, IFN-γ, or lipopolysaccharide also showed the same proactivatory effect on T cells. Lymphocyte activation was almost completely inhibited by blocking anti-CD18 and anti–intercellular adhesion molecule-1 (anti–ICAM-1) monoclonal antibodies (MoAbs), but only slightly affected by MoAbs against CD49d, vascular cell adhesion molecule-1, and anti–IL-15. In addition, the interaction of PBTL with immobilized ICAM-1 induced CD69 expression in the same memory T-cell subset. IL-15 induced T-cell activation with expression of CD69 and CD25, and production of IFN-γ, and its effect was additive with that triggered by cell adhesion to either EC or immobilized ICAM-1. The transmigration of PBTLs through either confluent EC monolayers or ICAM-1–coated membranes also induced efficiently the expression of CD69. When IL-15 was used as chemoattractant in these assays, a further enhancement in CD69 expression was observed in migrated cells. Together these results indicate that stimulated endothelium may have an important role in T-cell activation, through the lymphocyte function antigen-1/ICAM-1 pathway, and that IL-15 efficiently cooperates in this phenomenon. These observations could account for the abundance of CD69+ cells in the lymphocytic infiltrates of several chronic inflammatory diseases.
Fas-Independent and Nonapoptotic Cytotoxicity Mediated by a Human CD4+ T-Cell Clone Directed Against an Acute Myelogenous Leukemia–Associated DEK-CAN Fusion Peptide
Authors: Ohminami Hideki , Yasukawa Masaki , Kaneko Shin , Yakushijin Yoshihiro , Abe Yasuhito , Kasahara Yoshihito , Ishida Yasushi , Fujita Shigeru
Published: October 2019

The mechanism underlying the cytotoxicity mediated by a human CD4+ cytotoxic T-lymphocyte (CTL) clone directed against a peptide derived from the acute myelogenous leukemia-associated fusion protein, DEK-CAN, was investigated. A DEK-CAN fusion peptide-specific CD4+ Th0 CTL clone, designated HO-1, was established from the peripheral blood lymphocytes of a healthy individual. HO-1 exerted direct but not “innocent bystander” cytotoxicity within 2 hours. The cytotoxicity mediated by HO-1 was completely Ca2+-dependent. Because HO-1 lysed peptide-loaded Fas-deficient target cells derived from a patient with a homozygousFas gene mutation, its cytotoxicity appeared to be mediated by a Fas-independent pathway. In addition, its cytotoxicity was only partially inhibited by treatment with concanamycin A and strontium ions, which are inhibitors of the perforin-based cytotoxic pathway. Although membrane-bound type of tumor necrosis factor- (TNF-) was expressed on HO-1, an anti–TNF- antibody had no effect on HO-1–mediated cytotoxicity. HO-1 expressed mRNA for apoptosis-inducing mediators, including perforin, granzyme B, Fas ligand, TNF-, and lymphotoxin; however, no DNA fragmentation was detected in target cells incubated with HO-1 by 5-[125I]Iodo-2′-deoxyuridine release assay and agarose gel electrophoresis of DNA. Although it has been suggested that the Fas/Fas ligand system is the main pathway by which CD4+ CTL-mediated cytotoxicity is exerted in murine systems, HO-1 produced peptide-specific and HLA-restricted cytotoxicity via a Fas-independent and nonapoptotic pathway. The present study thus describes a novel mechanism of cytotoxicity mediated by CD4+ CTL.
A Reduced Sensitivity for Activated Protein C in the Absence of Factor V Leiden Increases the Risk of Venous Thrombosis
Authors: de Visser Marieke C.H. , Rosendaal Frits R. , Bertina Rogier M.
Published: October 2019

Activated protein C (APC) resistance caused by the factor V Leiden mutation is associated with an increased risk of venous thrombosis. We investigated whether a reduced response to APC, not due to the factor V point mutation, is also a risk factor for venous thrombosis. For this analysis, we used the Leiden Thrombophilia Study (LETS), a case-control study for venous thrombosis including 474 patients with a first deep-vein thrombosis and 474 age- and sex-matched controls. All carriers of the factor V Leiden mutation were excluded. A dose-response relationship was observed between the sensitivity for APC and the risk of thrombosis: the lower the normalized APC sensitivity ratio, the higher the associated risk. The risk for the lowest quartile of normalized APC-SR (<0.92), which included 16.5% of the healthy controls, compared with the highest quartile (normalized APC-SR > 1.05) was greater than fourfold increased (OR = 4.4; 95% confidence interval, 2.9 to 6.6). We adjusted for VIII:C levels, which appeared to affect our APC resistance test. The adjusted (age, sex, FVIII:C) odds ratio for the lowest quartile was 2.5 (95% confidence interval, 1.5 to 4.2). So, after adjustment for factor VIII levels, a reduced response to APC remained a risk factor. Our results show that a reduced sensitivity for APC, not caused by the factor V Leiden mutation, is a risk factor for venous thrombosis.
Detection of Kaposi’s Sarcoma Herpesvirus DNA Sequences in Multiple Myeloma Bone Marrow Stromal Cells
Authors: Chauhan Dharminder , Bharti Ajit , Raje Noopur , Gustafson Eric , Pinkus Geraldine S. , Pinkus Jack L. , Teoh Gerrard , Hideshima Teru , Treon Steve P. , Fingeroth Joyce D. , Anderson Kenneth C.
Published: October 2019

Whether Kaposi’s sarcoma herpesvirus (KSHV) is associated with multiple myeloma (MM) remains controversial. We assayed for KSHV DNA sequences in long-term bone marrow stromal cells (BMSCs) from 26 patients with MM and 4 normal donors. Polymerase chain reaction (PCR) using primers which amplify a KSHV gene sequence to yield a 233-bp fragment (KS330233 within open reading frame 26) was negative in all cases. Aliquots of these PCR products were used as templates in subsequent nested PCR, with primers that amplify a 186-bp product internal to KS330233. BMSCs from 24 of 26 (92%) patients with MM and 1 of 4 normal donors were KSHV PCR+. DNA sequence analyses showed interpatient specific mutations (2 to 3 bp). Both Southern blot and sequence analyses confirmed the specificity of PCR results. The presence of the KSHV gene sequences was further confirmed by amplifying T 1.1 (open reading frame [ORF] K7) and viral cyclin D (ORF 72), two other domains within the KSHV genome. Immunohistochemical studies of KSHV PCR+ MM BMSCs demonstrate expression of dendritic cell (DC) lineage markers (CD68, CD83, and fascin). Serological studies for the presence of KSHV lytic or latent antibodies were performed using sera from 53 MM patients, 12 normal donors, and 5 human immunodeficiency virus (HIV)/KSHV+ patients. No lytic or latent antibodies were present in sera from either MM patients or normal donors. Taken together, these findings show that KSHV DNA sequences are detectable in BMSCs from the majority of MM patients, but that serologic responses to KSHV are not present. Ongoing studies are defining whether the lack of antibody response is caused by the absence of ongoing infection, the presence of a novel viral strain associated with MM, or underlying immunodeficiency in these patients.
Rapid Death of Adoptively Transferred T Cells in Acquired Immunodeficiency Syndrome
Authors: Tan Rusung , Xu Xiaoning , Ogg Graham S. , Hansasuta Pokrath , Dong Tao , Rostron Tim , Luzzi Graz , Conlon Christopher P. , Screaton Gavin R. , McMichael Andrew J. , Rowland-Jones Sarah
Published: October 2019

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) probably play the major role in controlling HIV replication. However, the value of adoptive transfer of HIV-specific CTL expanded in vitro to HIV+ patients has been limited: this contrasts with the success of CTL therapy in treating or preventing Epstein-Barr virus and cytomegalovirus disease after bone marrow transplantation (BMT). We investigated the fate of expanded HIV-specific CTL clones in vivo following adoptive transfer to a patient with acquired immunodeficiency syndrome (AIDS). Two autologous CTL clones specific for HIV Gag and Pol were expanded to large numbers (>109) in vitro and infused into an HIV-infected patient whose viral load was rising despite antiretroviral therapy. The fate of one clone was monitored by staining peripheral blood mononuclear cells (PBMCs) with T-cell receptor–specific tetrameric major histocompatibility complex (MHC)-peptide complexes. Although the CTL transfer was well tolerated, there were no significant changes in CD4 and CD8 lymphocyte counts and virus load. By tracking an infused clone using soluble MHC-peptide complexes, we show that cells bearing the Gag-specific T-cell receptors were rapidly eliminated within hours of infusion through apoptosis. Thus, the failure of adoptively transferred HIV-specific CTL to reduce virus load in AIDS may be due to rapid apoptosis of the infused cells, triggered by a number of potential mechanisms. Further trials of adoptive transfer of CTL should take into account the susceptibility of infused cells to in vivo apoptosis.
Enhanced Myeloid Progenitor Cell Cycling and Apoptosis in Mice Lacking the Chemokine Receptor, CCR2
Authors: Reid Suzanna , Ritchie Alec , Boring Landin , Gosling Jennifa , Cooper Scott , Hangoc Giao , Charo Israel F. , Broxmeyer Hal E.
Published: October 2019

Chemokines regulate hematopoiesis in part by influencing the proliferative status of myeloid progenitor cells (MPC). Human MCP-1/murine JE, a myelosuppressive chemokine, specifically binds C-C chemokine receptor 2 (CCR2). Transgenic mice containing a targeted disruption in CCR2 that prevents expression of CCR2 mRNA and protein and have MPC that are insensitive to inhibition by MCP-1 and JE in vitro were assessed for potential abnormalities in growth of bone marrow (BM) and spleen MPC. MPC in both unseparated and c-kit+lin− populations of BM from CCR2-deficient (−/−) mice were in a greatly increased proliferation state compared with CCR2 littermate control (+/+) mice, an effect not apparent with progenitors from spleens of CCR2 (−/−) mice. Increased cycling status of CCR2 (−/−) BM MPC did not result in increased numbers of nucleated cells or MPC in BM or spleens of CCR2 (−/−) mice. Possible reasons for this apparent discrepancy were highlighted by flow cytometric analysis of c-kit+lin− BM cells and colony formation by MPC subjected to delayed addition of growth factors. The c-kit+lin− population of BM cells from CCR2 (−/−) mice had a significantly higher percentage of apoptotic cells than those from CCR2 (+/+) BM. However, elevated apoptosis was not associated with decreased numbers of c-kit+lin− cells. The increased percentage of apoptotic c-kit+lin− cells was due to elevated apoptosis within the c-kitdimlin−, but not the c-kitbrightlin−, subpopulations of cells. Consistent with enhanced apoptosis of phenotypically defined cells, MPC from CCR2 (−/−) BM and purified c-kit+lin− cells demonstrated decreased cell survival in vitro upon delayed addition of growth factors. The data suggest that signals received by CCR2 limit proliferation of progenitor cells in the BM, but also enhance survival of these cells.
H-Ras Is Involved in the Inside-out Signaling Pathway of Interleukin-3–Induced Integrin Activation
Authors: Shibayama Hirohiko , Anzai Naoyuki , Braun Stephen E. , Fukuda Seiji , Mantel Charlie , Broxmeyer Hal E.
Published: October 2019

The proto-oncogene product, p21ras, has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)–induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; 4β1 integrins) and VLA-5 (5β1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras–transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras–transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras–transfected Baf3 cells. Anti-β1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras–transfected Baf3 cells as much as the other types of H-Ras–transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti–phospho-MAPK antibody, but not adhesion of any type of H-Ras–transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3–induced VLA-4 and VLA-5 activation in Baf3 cells.
In Vitro Hematopoietic and Endothelial Cell Development From Cells Expressing TEK Receptor in Murine Aorta-Gonad-Mesonephros Region
Authors: Hamaguchi Isao , Huang Xu-Ling , Takakura Nobuyuki , Tada Jun-ichi , Yamaguchi Yuji , Kodama Hiroaki , Suda Toshio
Published: October 2019

Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34+ and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1+ endothelial cells on the OP9 stromal layer, while TEK− cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.
Reduced Folate Carrier Expression in Acute Lymphoblastic Leukemia: A Mechanism for Ploidy but not Lineage Differences in Methotrexate Accumulation
Authors: Belkov Vladimir M. , Krynetski Eugene Y. , Schuetz John D. , Yanishevski Yuri , Masson Eric , Mathew Susan , Raimondi Susana , Pui Ching-Hon , Relling Mary V. , Evans William E.
Published: October 2019

Methotrexate (MTX) is one of the most active and widely used agents for the treatment of acute lymphoblastic leukemia (ALL). To elucidate the mechanism for higher accumulation of MTX polyglutamates (MTX-PG) in hyperdiploid ALL and lower accumulation in T-lineage ALL, expression of the reduced folate carrier (RFC) was assessed by reverse transcription-polymerase chain reaction in ALL blasts isolated from newly diagnosed patients. RFC expression exhibited a 60-fold range among 29 children, with significantly higher expression in hyperdiploid B-lineage ALL (median, 11.3) compared with nonhyperdiploid ALL (median, 2.1; P < .0006), but no significant difference between nonhyperdiploid B-lineage and T-lineage ALL. Furthermore, mRNA levels of RFC (mapped by FISH to chromosome 21) were significantly related to chromosome 21 copy number (P = .0013), with the highest expression in hyperdiploid ALL blasts with 4 copies of chromosome 21. To assess the functional significance of gene copy number, MTX-PG accumulation was compared in ALL blasts isolated from 121 patients treated with either low-dose MTX (LDMTX; n = 60) or high-dose MTX (HDMTX; n = 61). After LDMTX, MTX-PG accumulation was highest in hyperdiploid B-lineage ALL with 4 copies of chromosome 21 (P = .011), but MTX-PG accumulation was not significantly related to chromosome 21 copy number after HDMTX (P = .24). These data show higher RFC expression as a mechanism for greater MTX accumulation in hyperdiploid B-lineage ALL and indicate that lineage differences in MTX-PG accumulation are not due to lower RFC expression in T-lineage ALL.
Expression and Function of Leptin Receptor Isoforms in Myeloid Leukemia and Myelodysplastic Syndromes: Proliferative and Anti-Apoptotic Activities
Authors: Konopleva Marina , Mikhail Adel , Estrov Zeev , Zhao Shourong , Harris David , Sanchez-Williams Gisela , Kornblau Steven M. , Dong Joan , Kliche Kay-Oliver , Jiang Shuwei , Snodgrass H. Ralph , Estey Elihu H. , Andreeff Michael
Published: October 2019

The receptor for the gene product of the obesity gene, leptin, was recently reported to be expressed on murine and human hematopoietic progenitor cells. Therefore, we studied the expression of the leptin receptor, OB-R, in normal myeloid precursors, human leukemia cell lines, and primary leukemic cells using reverse-transcriptase polymerase chain reaction. In normal hematopoiesis, OB-R was expressed in CD34+ cells. Normal promyelocytes (CD34−33+ and CD34−13+) expressed only very low levels of the short, presumably nonsignaling isoform. Both the long and short isoforms of OB-R were expressed in 10 of 22 samples from patients with newly diagnosed primary or secondary acute myeloid leukemia (AML), with a higher incidence of the long isoform in primary AML (87.6% v28.6%; P = .01). The incidence of OB-R expression was higher in recurrent than in newly diagnosed AML (P < .001), and samples from four patients with refractory AML showed strong expression of both isoforms. Both OB-R isoforms were also expressed in newly diagnosed and recurrent acute promyelocytic leukemia cells but were essentially absent in samples of chronic or acute lymphocytic leukemia. In vitro growth of myeloid leukemic cell lines and of blasts from 14 primary AMLs demonstrated that recombinant human leptin alone induced low level proliferation, significantly (P < .05) increased proliferation induced by recombinant human granulocyte colony-stimulating factor, interleukin 3, and stem cell factor in a subset of AML and increased colony formation (P < .005). Also, leptin reduced apoptosis induced by cytokine withdrawal in MO7E and TF-1 cells. Serum leptin levels correlated only with body mass index (P < .001) and gender (P = .03). Results confirm the reported expression of leptin receptor in normal CD34+ cells and demonstrate the frequent expression of leptin receptors in AML blasts. While normal promyelocytes lack receptor expression, leukemic promyelocytes express both isoforms. We also demonstrate proliferative effects of leptin alone and in combination with other physiologic cytokines, and anti-apoptotic properties of leptin. These findings could have implications for the pathophysiology of AML.
A Randomized, Double-Blind, Placebo-Controlled Study With Pegylated Recombinant Human Megakaryocyte Growth and Development Factor (PEG-rHuMGDF) as an Adjunct to Chemotherapy for Adults With De Novo Acute Myeloid Leukemia
Authors: Archimbaud Eric , Ottmann Oliver G. , Yin John A. Liu , Lechner Klaus , Dombret Herve , Sanz Miguel A. , Heil Gerhard , Fenaux Pierre , Brugger Wolfram , Barge Alan , O’Brien-Ewen Caroline , Matcham James , Hoelzer Dieter
Published: October 2019

To determine the safety, biologic, and clinical benefits of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF; Amgen, Thousand Oaks, CA) after myelosuppressive chemotherapy in acute myeloid leukemia (AML), 108 adult patients with de novo AML were randomized to receive either PEG-rHuMGDF (2.5 μg/kg/d or 5 μg/kg/d) for up to 21 doses (group A), a single dose of 2.5 μg/kg PEG-rHuMGDF, 7 daily doses of 2.5 μg/kg PEG-rHuMGDF (group B), or placebo. The greatest biologic activity was seen in group A with a median peak platelet count of 1,084 × 109/L, occurring at a median 9 days after the last dose of study drug, compared with 517 × 109/L and 390 × 109/L in group B and placebo group, respectively. Thrombocytosis (platelets >1,000 × 109/L) was seen at rates of 52%, 8%, and 9% in groups A, B, and placebo, respectively, but were not associated with any adverse event. There was no effect on median time to transfusion independent platelet recovery (≥20 × 109/L). The median time to neutrophil recovery (≥500/μL) and red blood cell transfusion requirements were similar in all groups, and there was no apparent stimulation of leukemia. PEG-rHuMGDF was biologically active and well tolerated. Further investigation of dose and scheduling is required, specifically earlier dosing before and during chemotherapy.