bluuebunny/crossref_metadata_2025 huggingface dataset:
Microvascular abnormalities in sickle cell disease: a computer-assisted intravital microscopy study
Authors: Cheung Anthony T. W. , Chen Peter C. Y. , Larkin Edward C. , Duong Patricia L. , Ramanujam Sahana , Tablin Fern , Wun Ted
Published: October 2002

The conjunctival microcirculation of 18 homozygous sickle cell disease (SCD) patients during steady-state, painful crisis, and postcrisis conditions was recorded on high-resolution videotapes using intravital microscopy. Selected videotape sequences were subsequently coded, frame-captured, studied, and blindly analyzed using computer-assisted image analysis protocols. At steady-state (baseline), all SCD patients exhibited some of the following morphometric abnormalities: abnormal vessel diameter, comma signs, blood sludging, boxcar blood flow phenomenon, distended vessels, damaged vessels, hemosiderin deposits, vessel tortuosity, and microaneurysms. There was a decrease in vascularity (diminished presence of conjunctival vessels) in SCD patients compared with non-SCD controls, giving the bulbar conjunctiva a “blanched” avascular appearance in most but not all SCD patients during steady-state. Averaged steady-state red cell velocity in SCD patients was slower than in non-SCD controls. During painful crisis, a further decrease in vascularity (caused by flow stoppage in small vessels) and a 36.7% ± 5.2% decrease in large vessel (mostly venular) diameter resulted. In addition, the conjunctival red cell velocities either slowed significantly (6.6% ± 13.1%; P < .01) or were reduced to a trickle (unmeasurable) during crisis. The microvascular changes observed during crisis were transient and reverted to steady-state baseline after resolution of crisis. When combined, intravital microscopy and computer-assisted image analysis (computer-assisted intravital microscopy) represent the availability of a noninvasive tool to quantify microvascular abnormalities in vascular diseases, including sickle cell disease. The ability to identify and relocate the same conjunctival vessels for longitudinal studies uniquely underscores the applicability of this quantitative real-time technology.
Translocation of the tetraspanin CD63 in association with human eosinophil mediator release
Authors: Mahmudi-Azer Salahaddin , Downey Gregory P. , Moqbel Redwan
Published: October 2002

The tetraspanin CD63 (also known as LAMP-3) has been implicated in phagocytic and intracellular lysosome-phagosome fusion events. It is also present in eosinophils, with predominant expression on crystalloid granule membrane. However, its role in eosinophil function is obscure. We hypothesized that CD63 is associated with intracellular events involved in eosinophil activation and mediator release. We used a combination of confocal immunofluorescence microscopy, flow cytometry, and secretion assays, including β-hexosaminidase, eosinophil peroxidase, and RANTES, to examine CD63 expression, intracellular localization, and its association with cell activation and mediator release. In resting eosinophils, CD63 immunoreactivity was localized to plasma and crystalloid granule membranes. In interferon-γ (IFN-γ)– or C5a/CB–stimulated cells (10 minutes), intracellular CD63 appeared to shift to the cell periphery and plasma membrane, while stimulation with a cocktail of interleukin-3 (IL-3)/IL-5/granulocyte-macrophage colony-stimulating factor induced the appearance of discrete intracellular clusters of CD63 immunoreactivity. IFN-γ induced mobilization of CD63 to the cell periphery, which coincided with selective mobilization of RANTES prior to its release, implying CD63 association with piecemeal degranulation. Agonist-induced CD63 mobilization and cell surface up-regulation was associated with β-hexosaminidase, eosinophil peroxidase, and RANTES release. Dexamethasone as well as genistein (a broad-spectrum inhibitor of tyrosine kinases) inhibited agonist-induced intracellular mobilization of CD63 and RANTES together with cell surface up-regulation of CD63 and mediator release. This is the first report of an association between CD63 mobilization and agonist-induced selective mediator release, which may imply the involvement of CD63 in eosinophil activation and piecemeal degranulation.
Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34+ cells
Authors: Gur Hilit , Krauthgamer Rita , Berrebi Alain , Klein Tirza , Nagler Arnon , Tabilio Antonio , Martelli Massimo F. , Reisner Yair
Published: October 2002

Stem cell–dose escalation is one way to overcome immune rejection of incompatible stem cells. However, the number of hematopoietic precursors required for overcoming the immune barrier in recipients pretreated with sublethal regimens cannot be attained with the state-of-the-art technology for stem cell mobilization. This issue was addressed by the observation that cells within the human CD34+ population are endowed with veto activity. In the current study, we demonstrated that it is possible to harvest about 28- to 80-fold more veto cells on culturing of purified CD34+cells for 7 to 12 days with an early-acting cytokine mixture including Flt3-ligand, stem cell factor, and thrombopoietin. Analysis of the expanded cells with fluorescence-activated cell-sorter scanning revealed that the predominant phenotype of CD34+CD33− cells used at the initiation of the culture was replaced at the end of the culture by cells expressing early myeloid phenotypes such as CD34+CD33+ and CD34−CD33+. These maturation events were associated with a significant gain in veto activity as exemplified by the minimal ratio of veto to effector cells at which significant veto activity was detected. Thus, whereas purified unexpanded CD34+ cells exhibited veto activity at a veto-to-effector cell ratio of 0.5, the expanded cells attained an equivalent activity at a ratio of 0.125. The availability of novel sources of veto cells such as those in this study might contribute to the realization of immunologic tolerance in “minitransplants,” without any risk of graft-versus-host disease.
Janus kinase 3 inhibitor WHI-P131/JANEX-1 prevents graft-versus-host disease but spares the graft-versus-leukemia function of the bone marrow allografts in a murine bone marrow transplantation model
Authors: Uckun Fatih M. , Roers Bertram A. , Waurzyniak Barbara , Liu Xing-Ping , Cetkovic-Cvrlje Marina
Published: October 2002

The purpose of the present study was to evaluate the effects of graft-versus-host disease (GVHD) prophylaxis with the Janus kinase 3 (JAK3) inhibitor WHI-P131/JANEX-1 on the graft-versus-leukemic (GVL) function of marrow allografts in mice undergoing bone marrow transplantation (BMT) after being challenged with an otherwise invariably fatal dose of BCL-1 leukemia cells. GVHD prophylaxis using WHI-P131 markedly improved the survival outcome after BMT. The probability of survival at 30 days after BMT was 11% ± 6% for vehicle-treated recipients (median survival time, 25 days) versus 63% ± 12% for recipients treated with WHI-P131 (median survival time, 36 days; P < .0001). Because WHI-P131 is devoid of antileukemic activity against BCL-1 leukemia cells, this marked improvement in survival outcome was due to reduced incidence of GVHD-associated fatalities combined with sustained GVL function of the allografts in the WHI-P131 group. Notably, adoptive transfer experiments demonstrated that the spleens of WHI-P131–treated allograft recipients contained less than 0.001% BCL-1 cells. Notably, GVHD prophylaxis with WHI-P131 plus methotrexate resulted in 100% survival of mice receiving allotransplants challenged with an otherwise invariably fatal dose of BCL-1 leukemia. Taken together, our results provide strong experimental evidence that GVHD prophylaxis using WHI-P131 does not impair the GVL function of the allografts and consequently contributes to an improved post-BMT survival outcome of the recipient mice.
Differential effects of 2C9*3 and 2C9*2 variants of cytochrome P-450 CYP2C9 on sensitivity to acenocoumarol
Authors: Hermida José , Zarza José , Alberca Ignacio , Montes Ramón , López Marı́a Luz , Molina Eva , Rocha Eduardo
Published: October 2002

The 2C9*3 and 2C9*2 polymorphisms of cytochrome P-450 CYP2C9 are associated with hypersensitivity to warfarin and bleeding. The effect of these polymorphisms on sensitivity to acenocoumarol is unknown. Three groups of patients, with low, medium, or high acenocoumarol-dose requirements, were studied. Age influenced the acenocoumarol sensitivity. Bearing the 2C9*3 allele was associated with the need for a lower acenocoumarol dose (odds ratio [OR], 6.02; 95% confidence interval [CI], 1.50-24.18); 80% of carriers of the 2C9*3 allele required a low dose. The 2C9*2 allele was associated with a lower acenocoumarol-dose requirement (OR, 2.70; 95% CI, 1.11-6.58) because of a reduced risk of the need for a high acenocoumarol dose (4.8% of the patients in the high-dose group carried the 2C9*2 allele versus 34.1% and 30.2%, respectively, in the medium-dose and low-dose groups). Therefore, carriers of 2C9*3 may need a low initial loading dose of acenocoumarol. Because acenocoumarol sensitivity with the 2C9*2 variant does not seem to be clinically relevant, the drug could be an alternative to warfarin in 2C9*2 carriers.
Induction of granulocytic differentiation by 2 pathways
Authors: Zhang Pu , Nelson Erik , Radomska Hanna S. , Iwasaki-Arai Junko , Akashi Koichi , Friedman Alan D. , Tenen Daniel G.
Published: October 2002

The CCAAT enhancer binding protein α (C/EBPα) transcription factor plays a critical role in granulocytopoiesis. Mice with a disruption of the C/EBPα gene demonstrate an early block in granulocytic differentiation, and disruption of C/EBPα function is a common theme in many types of human acute myelogenous leukemia, which is characterized by a block in myeloid development. To characterize further the nature of this block, we derived cell lines from the fetal liver of C/EBPα-deficient animals. These lines resembled morphologically the immature myeloid blasts observed in C/EBPα−/− fetal livers and did not express messenger RNA encoding early myeloid genes such as myeloperoxidase. Similarly, granulocytic markers such as Mac-1 and Gr-1 were not expressed; nor were erythroid and lymphoid surface antigens. Introduction of an inducible C/EBPα gene into the line revealed that conditional expression of C/EBPα induced the C/EBP family members C/EBPβ and C/EBPε and subsequent granulocyte differentiation. Similar results were obtained when C/EBPα−/− cells were stimulated with the cytokines interleukin-3 and granulocyte-macrophage colony-stimulating factor, but not with all-trans retinoic acid, supporting a model of at least 2 pathways leading to the differentiation of myeloid progenitors to granulocytes and implicating induction of other C/EBP family members in granulopoiesis.
c-kit associated with the transmembrane 4 superfamily proteins constitutes a functionally distinct subunit in human hematopoietic progenitors
Authors: Anzai Naoyuki , Lee Younghee , Youn Byung-S. , Fukuda Seiji , Kim Young-June , Mantel Charlie , Akashi Makoto , Broxmeyer Hal E.
Published: October 2002

The transmembrane 4 superfamily (TM4SF) has come into prominence for its association with a wide range of cell surface molecules, especially integrins. We report that TM4SF molecules CD9, CD63, and CD81 are physically associated with c-kit receptor tyrosine kinase in the human factor–dependent myeloid cell line, MO7e. We characterized this complex using coimmunoprecipitation and colocalization methods. The c-kit coimmunoprecipitated with anti-TM4SF antibodies showed several distinct phenotypes compared to the total c-kit immunoprecipitated with anti–c-kit antibody. These included: (1) higher basal level of tyrosine phosphorylation without elevated kinase activity in the absence of Steel factor (SLF), (2) deficient enhancement of tyrosine phosphorylation and kinase activity in response to SLF, (3) elevated binding rate of SLF shown in chemical cross-linking studies, and (4) little internalization and degradation after SLF treatment. Cocapping studies in living cells showed that c-kit colocalized with TM4SF molecules after SLF stimulation, suggesting confirmation of the biochemical data obtained by the coimmunoprecipitation studies. Colocalization of c-kit with CD81 by SLF was also observed in cord blood CD34+ cells, suggesting the existence of functional units of c-kit in TM4SF complexes in primary hematopoietic cells. This suggests that some TM4SF members may negatively modulate function of c-kit receptor tyrosine kinase and thus regulate receptor sensitivity to SLF in hematopoietic progenitors.
Site-directed mutagenesis of platelet glycoprotein Ibα demonstrating residues involved in the sulfation of tyrosines 276, 278, and 279
Authors: Tait A. Sasha , Dong Jing-Fei , López José A. , Dawes Ian W. , Chong Beng H.
Published: October 2002

The interaction between platelet glycoprotein (GP) Ibα and von Willebrand factor (VWF) is essential for initiation of hemostasis. The sulfation of the 3 tyrosine residues 276, 278, and 279 in GPIbα is an important posttranslational modification that seems to promote the interaction with VWF. The environment where sulfation of tyrosines occurs has been proposed to contain highly acidic residues. This investigation has examined the highly acidic region from Asp249 to Asp287 in the mature GPIbα protein. Changes to most of the carboxylic acids in this region resulted in decreased reactivity to VWF. Only 3 mutants (Glu270Gln, Asp283Asn, Asp283Asn/Glu285Gln/Asp287Asn) resulted in the abolition of sulfation. Two novel mutations were also created. First, a deletion of the 7 amino acids from Tyr276 to Glu282 led to a loss of sulfation and totally abolished VWF binding in the presence of botrocetin. This confirms that it is these 3 tyrosines that undergo sulfation and that this region is crucial for botrocetin-mediated VWF binding. The second mutation involves changing the lysine residues at 253, 258, and 262 to alanine. This also led to distinct changes in VWF binding and abolition of sulfation.
Effect of DNA methylation and chromatin structure onITGAL expression
Authors: Lu Qianjin , Ray Donna , Gutsch David , Richardson Bruce
Published: October 2002

LFA-1 (CD11a/CD18, αLβ2) is an integrin expressed in a tissue-specific fashion and is important in inflammatory and immune responses. Promoter analysis has identified transcription factors that may be involved in CD11a expression, but the mechanisms contributing to its tissue-specific expression are incompletely characterized. In this report we have asked if DNA methylation and/or chromatin structure could contribute to tissue-specific CD11a expression. Bisulfite sequencing was used to compare methylation patterns in the promoter and 5′ flanking regions of the ITGAL gene, encoding CD11a, in normal human T cells, which express LFA-1, and fibroblasts, which do not. The region was found to be heavily methylated in fibroblasts but not T cells, and methylation correlated with an inactive chromatin configuration as analyzed by deoxyribonuclease 1 sensitivity. Patch methylation of the promoter region revealed that promoter activity was methylation-sensitive but that methylation of the 5′ flanking regions more than 500 base pairs 5′ to the transcription start site could also suppress promoter function. Treating fibroblasts with a DNA methylation inhibitor decreased ITGAL promoter methylation and increased CD11a messenger RNA. The results thus indicate that methylation and chromatin structure may contribute to the tissue-specific expression of CD11a.
The lymphoproliferative defect in CTLA-4–deficient mice is ameliorated by an inhibitory NK cell receptor
Authors: Chambers Cynthia A. , Kang Joonsoo , Wu Yongjian , Held Werner , Raulet David H. , Allison James P.
Published: October 2002

T-cell responses are regulated by activating and inhibiting signals. CD28 and its homologue, cytotoxic T-lymphocyte antigen 4 (CTLA-4), are the primary regulatory molecules that enhance or inhibit T-cell activation, respectively. Recently it has been shown that inhibitory natural killer (NK) cell receptors (NKRs) are expressed on subsets of T cells. It has been proposed that these receptors may also play an important role in regulating T-cell responses. However, the extent to which the NKRs modulate peripheral T-cell homeostasis and activation in vivo remains unclear. In this report we show that NK cell inhibitory receptor Ly49A engagement on T cells dramatically limits T-cell activation and the resultant lymphoproliferative disorder that occurs in CTLA-4–deficient mice. Prevention of activation and expansion of the potentially autoreactive CTLA-4−/− T cells by the Ly49A-mediated inhibitory signal demonstrates that NKR expression can play an important regulatory role in T-cell homeostasis in vivo. These results demonstrate the importance of inhibitory signals in T-cell homeostasis and suggest the common biochemical basis of inhibitory signaling pathways in T lymphocytes.
Evidence for distinct pathomechanisms in B-cell chronic lymphocytic leukemia and mantle cell lymphoma by quantitative expression analysis of cell cycle and apoptosis-associated genes
Authors: Korz Christian , Pscherer Armin , Benner Axel , Mertens Daniel , Schaffner Claudia , Leupolt Elke , Döhner Hartmut , Stilgenbauer Stephan , Lichter Peter
Published: October 2002

The B-cell lymphoproliferative malignancies B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) share characteristics, including overlapping chromosomal aberrations with deletions on chromosome bands 13q14, 11q23, 17p13, and 6q21 and gains on chromosome bands 3q26, 12q13, and 8q24. To elucidate the biochemical processes involved in the pathogenesis of B-CLL and MCL, we analyzed the expression level of a set of genes that play central roles in apoptotic or cell proliferation pathways and of candidate genes from frequently altered genomic regions, namely ATM, BAX, BCL2, CCND1, CCND3, CDK2,CDK4, CDKN1A, CDKN1B,E2F1, ETV5, MYC, RB1,SELL, TFDP2, TNFSF10, andTP53. Performing real-time quantitative reverse transcription polymerase chain reaction in a panel of patients with MCL and B-CLL and control samples, significant overexpression and underexpression was observed for most of these genes. Statistical analysis of the expression data revealed the combination of CCND1 and CDK4 as the best classifier concerning separation of both lymphoma types. Overexpression in these malignancies suggests ETV5 as a new candidate for a pathogenic factor in B-cell lymphomas. Characteristic deregulation of multiple genes analyzed in this study could be combined in a comprehensive picture of 2 distinctive pathomechanisms in B-CLL and MCL. In B-CLL, the expression parameters are in strong favor of protection of the malignant cells from apoptosis but did not provide evidence for promoting cell cycle. In contrast, in MCL the impairment of apoptosis induction seems to play a minor role, whereas most expression data indicate an enhancement of cell proliferation.
Randomized trial of different regimens of heparins and in vivo thrombin generation in acute deep vein thrombosis
Authors: Kakkar Vijay V. , Hoppenstead Debra A. , Fareed Jawed , Kadziola Zbigniew , Scully Mike , Nakov Roumen , Breddin Hans K.
Published: October 2002

Low-molecular-weight and unfractionated heparins are frequently used to treat venous thromboembolism, but it is not known whether they are equally effective in inhibiting in vivo generation of thrombin. In this multicenter trial, 1048 patients were randomized to intravenous unfractionated heparin (group A), twice daily low-molecular-weight heparin (reviparin) for 1 week (group B), or once daily reviparin for 4 weeks (group C). All patients received vitamin K antagonists. Blood samples withdrawn at the baseline and at weeks 1 and 3 were analyzed using markers of in vivo thrombin generation and other coagulation parameters. During the first 3 weeks symptomatic recurrent deep vein thrombosis–pulmonary embolism (DVT/PE) occurred in 17 (4.5%) of 375 patients in group A compared with 4 (1.0%) of 388 patients in group B, and 9 (2.4%) of 374 patients in group C. Forty percent of patients in group A, 53.4% in group B, and 53.5% in group C showed 30% or greater reduction in thrombus size assessed by venography. Patients in group B had significantly greater reduction in D-dimer, prothrombin fragments 1 and 2 (F1 + 2), endogenous thrombin potential (ETP), and thrombin-antithrombin (TAT) complexes compared to groups A and C. Greater release of tissue factor pathway inhibitor (TFPI) and reduction in levels of thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen were significantly more pronounced in group C patients. Reviparin administered twice daily plus vitamin K antagonist is more effective in inhibiting in vivo thrombin generation compared to intravenous unfractionated heparin plus vitamin K antagonist, and reviparin once daily produced significantly higher TFPI release and greater reduction in TAFI and fibrinogen levels.
A randomized comparison of nativeEscherichia coli asparaginase and polyethylene glycol conjugated asparaginase for treatment of children with newly diagnosed standard-risk acute lymphoblastic leukemia: a Children's Cancer Group study
Authors: Avramis Vassilios I. , Sencer Susan , Periclou Antonia P. , Sather Harland , Bostrom Bruce C. , Cohen Lewis J. , Ettinger Alice G. , Ettinger Lawrence J. , Franklin Janet , Gaynon Paul S. , Hilden Joanne M. , Lange Beverly , Majlessipour Fataneh , Mathew Pracad , Needle Michael , Neglia Joseph , Reaman Gregory , Holcenberg John S.
Published: October 2002

For this study, 118 children with standard-risk acute lymphoblastic leukemia (ALL) were given randomized assignments to receive native or pegylated Escherichia coli asparaginase as part of induction and 2 delayed intensification phases. Patients treated with pegaspargase had more rapid clearance of lymphoblasts from day 7 and day 14 bone marrow aspirates and more prolonged asparaginase activity than those treated with native asparaginase. In the first delayed intensification phase, 26% of native asparaginase patients had high-titer antibodies, whereas 2% of pegaspargase patients had those levels. High-titer antibodies were associated with low asparaginase activity in the native arm, but not in the pegaspargase arm. Adverse events, infections, and hospitalization were similar between arms. Event-free survival at 3 years was 82%. A population pharmacodynamic model using the nonlinear mixed effects model (NONMEM) program was developed that closely fit the measured enzyme activity and asparagine concentrations. Half-lives of asparaginase were 5.5 days and 26 hours for pegaspargase and native asparaginase, respectively. There was correlation between asparaginase enzymatic activity and depletion of asparagine or glutamine in serum. In cerebrospinal fluid asparagine, depletion was similar with both enzyme preparations. Intensive pegaspargase for newly diagnosed ALL should be tested further in a larger population.
Health-related quality of life of unrelated bone marrow donors in Japan
Authors: Nishimori Mina , Yamada Yoshitsugu , Hoshi Keiko , Akiyama Yuichi , Hoshi Yasutaka , Morishima Yasuo , Tsuchida Masahiro , Fukuhara Shunichi , Kodera Yoshihisa
Published: October 2002

To promote bone marrow donation, both the safety and well-being of healthy unrelated volunteer donors must be protected. This prospective cohort study evaluated donors' health-related quality of life (HRQOL) and identified factors associated with it. Using the Medical Outcomes Study Short Form 36 Health Survey (SF-36) before bone marrow harvesting (BMH), and again 1 week and 3 months after the donors' discharge, we evaluated HRQOL of 565 donors (329 men, 236 women) registered with the Japan Marrow Donor Program (JMDP). We also examined the data routinely collected by the JMDP, such as BMH-related problems and other demographic and medical variables, to determine whether such data could be used to predict donors' HRQOL after discharge. Mean scores of all pre-BMH SF-36 subscales showed better functioning than the national norm. One week after discharge, mean scores on physical functioning (PF) and role-physical (RP) subscales, indicative of physical states, and bodily pain (BP) were approximately 1 SD lower than the national norm; however, mental health (MH) and general health perception (GH) remained above normal; the most frequent BMH-related problems were pain at the donation site and lower back pain, which were associated with lower PF, RP, and BP scores. Female gender and duration of procedure predicted lower PF, RP, and BP. Three months after discharge, mean scores of all SF-36 subscales had returned to baseline levels. These data show that the adverse effects of BMH on donors' HRQOL are transient and can be minimized by better management of pain.
Thrombocytopenia after second exposure to abciximab is caused by antibodies that recognize abciximab-coated platelets
Authors: Curtis Brian R. , Swyers Julia , Divgi Ajit , McFarland Janice G. , Aster Richard H.
Published: October 2002

Thrombocytopenia, often severe, occurs in 1% to 2% of patients given the fibrinogen receptor antagonist abciximab, a chimeric Fab fragment containing murine specificity-determining and human framework sequences. The cause of this complication has not yet been defined. Studies of 9 patients who developed profound thrombocytopenia (platelets <10 × 109/L [10 000/μL]) within a few hours of being given abciximab a second time showed that each had a strong immunoglobulin G (IgG) antibody that recognized platelets sensitized with abciximab. Five patients also had IgM antibodies. IgG antibodies reactive with abciximab-coated platelets were also found in 77 (74%) of 104 healthy subjects. However, the patient antibodies could be distinguished from “normal” ones in 2 ways: (1) only the patient antibodies reacted preferentially with platelets sensitized with the intact monoclonal antibody 7E3 from which the murine sequences in abciximab are derived; and (2) the “normal” antibodies could be inhibited by Fab fragments derived from normal human IgG, whereas the patient antibodies were relatively resistant to this treatment. The findings suggest that antibodies from the patients are specific for murine sequences in abciximab and are capable of causing life-threatening thrombocytopenia upon injection of this drug. The antibodies commonly found in healthy subjects are specific for the papain cleavage site of any Fab fragments and, although they react with abciximab-coated platelets, appear not to cause significant thrombocytopenia. It may be possible to identify patients at risk for developing thrombocytopenia if given abciximab by screening for antibodies that recognize 7E3-coated platelets.
The tumor vascular targeting agent combretastatin A–4-phosphate induces reorganization of the actin cytoskeleton and early membrane blebbing in human endothelial cells
Authors: Kanthou Chryso , Tozer Gillian M.
Published: October 2002

Combretastatin A–4-phosphate (CA-4-P) is a tubulin-binding compound currently in clinical trial as a tumor vascular-targeting agent. In endothelial cells, CA-4-P is known to cause microtubule depolymerization, but little is known about its subsequent effects on cell morphology and function. Here, we demonstrate that within minutes of endothelial cell exposure to CA-4-P, myosin light chain (MLC) was phosphorylated, leading to actinomyosin contractility, assembly of actin stress fibers, and formation of focal adhesions. These cytoskeletal alterations appeared to be a consequence of Rho activation, as they were abolished by either the Rho inhibitor C3 exoenzyme or Rho-kinase inhibitor Y-27632. In response to CA-4-P, some cells rapidly assumed a blebbing morphology in which F-actin accumulated around surface blebs, stress fibers misassembled into a spherical network surrounding the cytoplasm, and focal adhesions appeared malformed. Blebbing was associated with decreased cell viability and could be inhibited by Rho/Rho-kinase inhibitors or by blocking the CA-4-P–mediated activation of stress-activated protein kinase-2/p38. The extracellular-regulated kinases 1 and 2 (ERK-1/2) were shown to protect against blebbing since blebbing was attenuated on ERK-1/2 stimulation and was up-regulated by specific inhibition of ERK-1/2 activation. The use of MLC kinase (MLCK) and myosin adenosine triphosphatase inhibitors led us to propose a role for MLCK and myosin activity independent of MLC phosphorylation in regulating the blebbing process. CA-4-P–mediated contractility and blebbing were associated with a Rho-dependent increase in monolayer permeability to dextrans, suggesting that such functional changes may be important in the rapid response of the tumor endothelium to CA-4-P in vivo.
ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1–restricted CD8+ T-cell epitopes
Authors: Passoni Lorena , Scardino Antonio , Bertazzoli Carla , Gallo Barbara , Coluccia Addolorata M. L. , Lemonnier François A. , Kosmatopoulos Konstadinos , Gambacorti-Passerini Carlo
Published: October 2002

Oncogenic anaplastic lymphoma kinase (ALK) fusion proteins (NPM/ALK and associated variants) are expressed in about 60% of anaplastic large cell lymphomas (ALCLs) but are absent in normal tissues. In this study, we investigated whether ALK, which is expressed at high levels in lymphoma cells, could be a target for antigen-specific cell-mediated immunotherapy. A panel of ALK-derived peptides was tested for their binding affinity to HLA-A*0201 molecules. Binding peptides were assessed for their capacity to elicit a specific immune response mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A*0201 transgenic mice, and in vitro in the peripheral blood lymphocytes (PBLs) from healthy donors. Two HLA-A*0201–restricted CTL epitopes, p280-89 (SLAMLDLLHV) and p375-86 (GVLLWEIFSL), both located in the ALK kinase domain were identified. The p280-89– and p375-86–induced peptide-specific CTL lines were able to specifically release interferon-γ (IFN-γ) on stimulation with ALK peptide-pulsed autologous Epstein-Barr virus–transformed B cells (LCLs) or T2 cells. Anti-ALK CTLs lysed HLA-matched ALCL and neuroblastoma cell lines endogenously expressing ALK proteins. CTL activity was inhibited by anti-HLA-A2 monoclonal antibody CR11.351, consistent with a class I–restricted mechanism of cytotoxicity. These results show the existence of functional anti-ALK CTL precursors within the peripheral T-cell repertoire of healthy donors, clearly indicating ALK as a tumor antigen and ALK-derived peptides, p280-89 and p375-86, as suitable epitopes for the development of vaccination strategies.
Effects of administration of progenipoietin 1, Flt-3 ligand, granulocyte colony-stimulating factor, and pegylated granulocyte-macrophage colony-stimulating factor on dendritic cell subsets in mice
Authors: O'Keeffe Meredith , Hochrein Hubertus , Vremec David , Pooley Joanne , Evans Robert , Woulfe Susan , Shortman Ken
Published: October 2002

We studied the effects of administration of several cytokines, including progenipoietin-1 (ProGP-1), Flt-3 ligand (FL), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor in a pegylated form (pGM-CSF), on dendritic cell (DC) populations in mouse spleen. ProGP-1 produced the most striking increase in overall DC numbers, apparently more than its constituent FL and G-CSF components. However, the expansion in DC numbers was strongly subpopulation selective, with ProGP-1 and FL producing selective expansion of CD8+ DCs, whereas pGM-CSF produced selective expansion of CD8− DCs. Surprising differences were observed between the effects of murine and human recombinant FL preparations on murine DCs. Many of the biologic functions of the DC subpopulations expanded by cytokines remained intact, including the capacity of the ProGP-1– and FL-expanded CD8+ DCs to produce the T-helper-1–biasing cytokine interleukin 12 (IL-12). However, the expanded DCs from all but G-CSF–treated mice were deficient in the ability to make interferon γ, and the CD8+ DCs produced with pGM-CSF treatment had an abrogated capacity to form bioactive IL-12. Such selective expansion of DC populations and alterations in their cytokine-secretion capacity have implications for clinical use of the studied cytokines in immune modulation.
Negative effect of CTLA-4 on induction of T-cell immunity in vivo to B7-1+, but not B7-2+, murine myelogenous leukemia
Authors: LaBelle James L. , Hanke Carrie A. , Blazar Bruce R. , Truitt Robert L.
Published: October 2002

B7 molecules provide important costimulatory signals to T cells, and B7 genes have been introduced into B7-negative tumor cells to enhance their immunogenicity. However, the role of B7 molecules in inducing tumor immunity is controversial because of conflicting results and reports of differential signaling through the B7 molecules and their ligands CD28 and CTLA-4. In this study, we compared the effect of B7-1 (CD80) and B7-2 (CD86) on the induction of T-cell immunity to C1498, a murine myelogenous leukemia. When cultured with exogenous cytokines in vitro, C1498/B7-1 and C1498/B7-2 induced syngeneic CD8+ T cells to kill parental C1498. In vivo, C1498/B7-1 grew progressively after subcutaneous injection, whereas C1498/B7-2 completely regressed after transient growth in naive mice. Spontaneous rejection of C1498/B7-2 resulted in immunity to challenge doses of C1498 and C1498/B7-1. Antibody-depletion studies in vivo showed that CD8+ T cells rejected C1498/B7-2, whereas only natural killer cells affected the growth of C1498/B7-1. Two approaches were used to determine whether preferential interaction of B7-1 with CTLA-4 contributed to the failure of C1498/B7-1 to activate CD8+ T cells in vivo. First, CTLA-4 specific monoclonal antibody was used to block B7-1–CTLA-4 interaction. Second, CTLA-4 deletional mutant (−/−) bone marrow chimeras were used as tumor hosts. In both systems, there was a significant increase in the rate of rejection of C1498/B7-1 tumors. Resistance to C1498/B7-1 in CTLA-4−/− hosts was mediated by CD8+ T cells. Blocking or deletion of CTLA-4 did not affect the growth of parental C1498, indicating that B7-1 was important for the induction of CD8+ T-cell immunity in the absence of CTLA-4.
Oncogenesis of multiple myeloma: 14q32 and 13q chromosomal abnormalities are not randomly distributed, but correlate with natural history, immunological features, and clinical presentation
Authors: Avet-Loiseau Hervé , Facon Thierry , Grosbois Bernard , Magrangeas Florence , Rapp Marie-José , Harousseau Jean-Luc , Minvielle Stéphane , Bataille Régis
Published: October 2002

Multiple myeloma (MM) is a plasma-cell malignancy characterized by marked epidemiological, biological, and clinical heterogeneity. The goal of this study was to find a genetic basis for this heterogeneity. Using fluorescence in situ hybridization, we analyzed a prospective cohort of 901 patients with various plasma-cell disorders—monoclonal gammopathies of undetermined significance, smoldering MM, MM, and primary plasma-cell leukemia—for genetic abnormalities involving the 13q14 and 14q32 chromosomal regions; the patients were consecutively enrolled in the Intergroupe Francophone du Myélome clinical trials, We performed statistical analyses comparing these chromosomal abnormalities in terms of immunological (ie, immunoglobulin types and light-chain subtypes) and clinical status and, to some exent, prognostic features. It was found that 14q32 translocations and del(13) are the most frequent chromosomal abnormalities, observed in 75% and 45% of the patients, respectively, and are not randomly distributed, but interconnected. Second, correlations between them allowed us to define 4 major genetic categories of patients: (1) patients lacking any 14q32 abnormality (25%) and generally also lacking del(13); (2) patients presenting either t(4;14) or t(14;16), almost always associated with a del(13) (15% of patients); (3) patients with other 14q32 abnormalities and presenting del(13) (25%); and (4) patients with other 14q32 abnormalities but not presenting del(13) (35%). Third, we show that this genetic stratification is highly correlated with immunological status and clinical presentation and with some major prognostic factors. For the first time, this study gives genetic support to the heterogeneity observed in patients with MM and demontrates that the 14q32 and 13q chromosomal abnormalities are not randomly distributed. The strong correlations we found might be the basis for a novel genetic classification of MM, as has been previously demonstrated for leukemias and lymphomas. Furthermore, our study supports different models for MM oncogenesis.
Retinoic acid–induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27Kip1
Authors: Dimberg Anna , Bahram Fuad , Karlberg Inger , Larsson Lars-Gunnar , Nilsson Kenneth , Öberg Fredrik
Published: October 2002

All-trans retinoic acid (ATRA) is a potential therapeutic agent for the treatment of hematopoietic malignancies, because of its function as an inducer of terminal differentiation of leukemic blasts. Although the efficacy of ATRA as an anticancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), the molecular mechanisms of ATRA-induced cell cycle arrest of myeloid cells have not been fully investigated. In this study, we show that the onset of ATRA-induced G0/G1 arrest of human monoblastic U-937 cells is linked to a sharp down-regulation of c-Myc and cyclin E levels and an increase in p21WAF1/CIP1 expression. This is followed by an increase in p27Kip1 protein expression due to enhanced protein stability. The importance of an early decrease in Myc expression for these events was demonstrated by the failure of a U-937 subline with constitutive exogenous expression of v-Myc to cell cycle arrest and regulate cyclin E and p27Kip1 in response to ATRA. Preceding the initiation of G1 arrest, a transient rise in retinoblastoma protein (pRb), p107, and cyclin A levels was detected. Later, a rapid fall in the levels of cyclins A and B and a coordinate dephosphorylation of pRb at Ser780, Ser795, and Ser807/811 coincided with the accumulation of cells in G1. These results thus identify a decrease in c-Myc and cyclin E levels and a posttranscriptional up-regulation of p27Kip1 as important early changes, and position them in the complex chain of events regulating ATRA-induced cell cycle arrest of myeloid cells.
Human eosinophils produce neurotrophins and secrete nerve growth factor on immunologic stimuli
Authors: Kobayashi Hitoshi , Gleich Gerald J. , Butterfield Joseph H. , Kita Hirohito
Published: October 2002

Neurotrophins, such as nerve growth factor (NGF) and neurotrophin-3 (NT-3), are essential for development, function, and survival of peripheral sympathetic and sensory neurons. Most eosinophilic leukocytes in the human body are localized in mucosal tissues; however, the roles of eosinophils in human diseases are not fully understood. We found that human eosinophils constitutively express messenger RNA for NGF and NT-3, synthesize and store these proteins intracellularly, and continuously replenish them. Incubation of eosinophils with a transcription inhibitor, actinomycin D, for 8 hours completely depletes intracellular NGF and NT-3. New synthesis of NGF is enhanced by Fc-receptor–mediated stimuli, such as immunoglobulin (Ig)A and IgG immune complexes; in contrast, production of NT-3 is not affected by these stimuli. Notably, supernatants of eosinophils stimulated with IgA immune complex and interleukin 5 promote neurite extension of the PC-12 pheochromocytoma cell line; this effect is abolished by pretreatment of the supernatants with anti-NGF–neutralizing antibody. By enzyme-linked immunosorbent assay, substantial amounts of NGF protein are also detected in the supernatants of stimulated eosinophils. Furthermore, in patients with seasonal allergic rhinitis, the concentrations of NGF in nasal secretions correlate with the magnitudes of eosinophilic inflammation in the airway, suggesting a potential clinical implication of eosinophil NGF. Our observations propose a new pathologic mechanism by which eosinophils may contribute to enhanced neurologic responses in patients with allergic diseases and other eosinophilic disorders. Alternatively, eosinophils may play important roles in maintenance and restoration of homeostatic functions of mucosal tissues through the pleitropic activities of NGF.
Campath-1H and fludarabine in combination are highly active in refractory chronic lymphocytic leukemia
Authors: Kennedy Ben , Rawstron Andy , Carter Chris , Ryan Mary , Speed Kevin , Lucas Guy , Hillmen Peter
Published: October 2002

Campath-1H (alemtuzumab) is the most effective monoclonal antibody in single-agent use in B-cell chronic lymphocytic leukemia (CLL) with reported response rates of 33% to 70%. Combination therapy is now the conventional treatment for most hematologic malignancies. Monoclonal antibody treatments may sensitize tumor cells to subsequent chemotherapy. We report the combination of Campath-1H with fludarabine in patients with CLL refractory to each agent used singly. Six patients who had received a median of 8 courses of fludarabine (range, 4-10 courses) and 16 weeks of Campath-1H (range, 8-32 weeks) were treated. Five patients responded, including one who had a complete response by National Cancer Institute criteria. The responses observed were better in each patient than responses after each agent used singly. Complete morphologic bone marrow responses were seen in 3 patients, including eradication of disease measured by sensitive flow cytometry in 2. Campath-1H combined with fludarabine is a highly promising novel therapy for refractory CLL.
Etanercept, a soluble tumor necrosis factor receptor, palliates constitutional symptoms in patients with myelofibrosis with myeloid metaplasia: results of a pilot study
Authors: Steensma David P. , Mesa Ruben A. , Li Chin-Yang , Gray Leigh , Tefferi Ayalew
Published: October 2002

Patients with myelofibrosis with myeloid metaplasia (MMM) often experience debilitating constitutional symptoms such as drenching night sweats, profound fatigue, unexplained fevers, and unintentional weight loss. Tumor necrosis factor (TNF) contributes to organ fibrosis and hypercatabolic symptoms in a variety of disease states. We conducted an open-label pilot study of etanercept, a soluble TNF receptor, administered at a dose of 25 mg subcutaneously twice weekly for up to 24 weeks in 22 patients with MMM. Of 20 evaluable patients, 12 (60%) experienced an improvement in constitutional symptoms, and 4 (20%) had an objective response (improvement in peripheral cytopenias or spleen size). The degree of marrow fibrosis was unchanged, and only minor changes in overall marrow cellularity were observed. Toxicity was mild, with injection site reactions (20%) and minor infections (10%) as the most common side effects. One patient developed reversible pancytopenia. Etanercept may be useful for palliation of constitutional symptoms in MMM.
Allogeneic blood cell transplantation following reduced-intensity conditioning is effective therapy for older patients with myelofibrosis with myeloid metaplasia
Authors: Devine Steven M. , Hoffman Ron , Verma Amit , Shah Rajul , Bradlow Basil A. , Stock Wendy , Maynard Vera , Jessop Ellen , Peace David , Huml Marianne , Thomason Deana , Chen Yi-Hsiang , van Besien Koen
Published: October 2002

Standard myeloablative conditioning prior to allogeneic hematopoietic stem cell (HSC) transplantation has been associated with significant toxicity in patients older than 45 years of age with myelofibrosis with myeloid metaplasia (MMM). We sought to evaluate the efficacy of a reduced-intensity conditioning regimen for allogeneic HSC transplantation in this setting. A regimen consisting of fludarabine (30 mg/m2 intravenously daily for 5 days) and melphalan (70 mg/m2 intravenously daily for 2 days) followed by transplantation of filgrastim-mobilized peripheral blood cells from HLA-identical siblings was administered to 4 older patients (median age, 56 years; range, 48-58 years) with advanced MMM. All patients achieved prompt neutrophil and platelet engraftment and have experienced a significant regression of splenomegaly and bone marrow fibrosis. All now have normal bone marrow cellularity. With a median follow-up of 13 months (range, 11-19 months), all 4 patients are alive with stable full-donor hematopoietic chimerism. These results support the feasibility and effectiveness of reduced-intensity conditioning prior to allogeneic HSC transplantation for older patients with advanced MMM.
Somatically mutated Ig VH3-21 genes characterize a new subset of chronic lymphocytic leukemia
Authors: Tobin Gerard , Thunberg Ulf , Johnson Anna , Thörn Ingrid , Söderberg Ola , Hultdin Magnus , Botling Johan , Enblad Gunilla , Sällström Jan , Sundström Christer , Roos Göran , Rosenquist Richard
Published: October 2002

Recent studies on the immunoglobulin variable heavy chain (IgVH) genes have revealed that B-cell chronic lymphocytic leukemia (B-CLL) consists of at least 2 clinical entities with either somatically mutated or unmutated VH genes. We have analyzed the VH gene mutation status and VH gene usage in 119 B-CLL cases and correlated them to overall survival. A novel finding was the preferential use of the VH3-21 gene in mutated cases, whereas biased VH1-69 gene usage was found in unmutated cases as previously reported. Interestingly, the subset of mutated cases using the VH3-21 gene displayed distinctive genotypic/phenotypic characteristics with shorter average length of the complementarity determining region 3 and clonal expression of λ light chains. In addition, this mutated subset showed significantly shorter survival than other mutated cases and a similar clinical course to unmutated cases. We therefore suggest that B-CLL cases with mutated VH3-21 genes may constitute an additional entity of B-CLL.
Missense mutations of the WASP gene cause intermittent X-linked thrombocytopenia
Authors: Notarangelo Lucia D. , Mazza Cinzia , Giliani Silvia , D'Aria Chiara , Gandellini Francesca , Ravelli Chiara , Locatelli Maria Grazia , Nelson David L. , Ochs Hans D. , Notarangelo Luigi D.
Published: October 2002

Mutations of the WASP gene have been previously shown to be responsible for classical Wiskott-Aldrich syndrome, isolated X-linked thrombocytopenia, and severe, congenital X-linked neutropenia. We report herewith 2 families in which affected males had a history of intermittent thrombocytopenia with consistently reduced platelet volume, in the absence of other major clinical features, and carried missense mutations of the WASP gene that allowed substantial protein expression. This observation broadens the spectrum of clinical phenotypes associated with WASP gene defects, and it indicates the need for molecular analysis in males with reduced platelet volume, regardless of the platelet number.