bluuebunny/crossref_metadata_2025 huggingface dataset:
JunB gene expression is inactivated by methylation in chronic myeloid leukemia
Authors: Yang Ming-Yu , Liu Ta-Chih , Chang Jan-Gowth , Lin Pai-Mei , Lin Sheng-Fung
Published: April 2003

JunB is a component of the Jun family genes of the activating protein-1 transcription factors that are important in the control of cell growth and differentiation and neoplastic transformation. Recently, it was demonstrated that transgenic mice specifically lacking JunB expression in the myeloid lineage developed a myeloproliferative disease, eventually progressing to blast crisis that resembled human chronic myeloid leukemia (CML). To gain further insights into the role of JunB in human CML, we examined peripheral blood from 17 healthy individuals and CML patients (11 in blastic crisis and 21 in chronic phase) by real-time quantitative reverse transcription–polymerase chain reaction analysis for the expression of JunB. The results showed the expression levels of JunB were significantly impaired in CML cases (blastic crisis < chronic phase < normal). Mutational analysis of the whole gene and methylation analysis of cytosine-phosphate guanosine (CpG) sites at the promoter area were further performed to investigate the possible mechanisms. However, no mutation was found within the coding region or the 9 flanking evolutionarily conserved regions in all CML cases. Interestingly, in the promoter area of JunB gene, most of the CpG sites were methylated in CML cases; in contrast, none of these CpG sites were methylated in normal cases. Demethylation by treatment of hypermethylated K562 cells with 5′-aza-2′-deoxycytidine resulted in partial reactivation of JunB expression. Our results suggest that the down-regulated JunB expression in CML was due to the inactivation of JunB gene by methylation and the differential expression was correlated to the ratio of cells being methylated.
Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations
Authors: Mizuki Masao , Schwäble Joachim , Steur Claudia , Choudhary Chunaram , Agrawal Shuchi , Sargin Bülent , Steffen Björn , Matsumura Itaru , Kanakura Yuzuru , Böhmer Frank D. , Müller-Tidow Carsten , Berdel Wolfgang E. , Serve Hubert
Published: April 2003

The receptor tyrosine kinase Flt3 is expressed and functionally important in early myeloid progenitor cells and in the majority of acute myeloid leukemia (AML) blasts. Internal tandem duplications (ITDs) in the juxtamembrane domain of the receptor occur in 25% of AML cases. Previously, we have shown that these mutations activate the receptor and induce leukemic transformation. In this study, we performed genome-wide parallel expression analyses of 32Dcl3 cells stably transfected with either wild-type or 3 different ITD isoforms of Flt3. Comparison of microarray expression analyses revealed that 767 of 6586 genes differed in expression between FLT3-WT– and FLT3-ITD–expressing cell lines. The target genes of mutationally activated Flt3 resembled more closely those of the interleukin 3 (IL-3) receptor than those of ligand-activated Flt3. The serine-threonine kinase Pim-2 was up-regulated on the mRNA and the protein level in Flt3-ITD–expressing cells. Further experiments indicated that Pim-2 function was important for clonal growth of 32D cells. Several genes repressed by the mutations were found to be involved in myeloid gene regulation. Pu.1 and C/EBPα, both induced by ligand-activation of wild-type Flt3, were suppressed in their expression and function by the Flt3 mutations. In conclusion, internal tandem duplication mutations of Flt3 activate transcriptional programs that partially mimic IL-3 activity. Interestingly, other parts of the transcriptional program involve novel, IL-3–independent pathways that antagonize differentiation-inducing effects of wild-type Flt3. The identification of the transcriptional program induced by ITD mutations should ease the development of specific therapies.
Intravenous immunoglobulins induce the in vitro differentiation of human B lymphocytes and the secretion of IgG
Authors: de Grandmont Marie Joëlle , Racine Claudia , Roy Annie , Lemieux Réal , Néron Sonia
Published: April 2003

The therapeutic effects of intravenous immunoglobulins (IVIGs) in several autoimmune diseases are characterized by a decrease in pathologic autoantibody levels. Although little direct evidence has been reported in humans, the large repertoire of natural immunoglobulin G (IgG) antibodies in IVIGs is expected to be involved in the regulation of autoreactive B lymphocytes. In normal adult mice, IVIGs have been reported to modulate immature B cells as well as peripheral B lymphocytes through V-region connections. Studies with human serum also indicated that anti-idiotypic antibodies, present in IVIG preparations, could recognize both natural and pathologic autoantibodies. We have used an in vitro culture system to characterize the direct effect of IVIGs on human B lymphocytes. This in vitro culture system involves CD40 activation of B lymphocytes by its ligand CD154 in the presence of cytokines. In this system, addition of IVIGs decreased by 50% to 80% the expansion of B lymphocytes. This reduced expansion was due to a decrease in the proliferation rate. In addition, a portion of B lymphocytes was differentiated into IgG-secreting cells in the presence of IVIGs and the secreted IgGs were reactive with antigens such as nucleoprotamine, dsDNA, tetanus toxin, and human IgG F(ab′)2 fragments. These observations indicate that IVIGs can have direct effects on B lymphocytes and suggest that such IVIG regulation of B lymphocytes could be involved in the therapeutic effects of IVIGs in autoimmune diseases.
A model of APL with FLT3 mutation is responsive to retinoic acid and a receptor tyrosine kinase inhibitor, SU11657
Authors: Sohal Jastinder , Phan Vernon T. , Chan Philip V. , Davis Elizabeth M. , Patel Bhumi , Kelly Louise M. , Abrams Tinya J. , O'Farrell Anne Marie , Gilliland D. Gary , Le Beau Michelle M. , Kogan Scott C.
Published: April 2003

The PML-RARα fusion protein is central to the pathogenesis of acute promyelocytic leukemia (APL). Expression of this protein in transgenic mice initiates myeloid leukemias with features of human APL, but only after a long latency (8.5 months in MRP8 PML-RARAmice). Thus, additional changes contribute to leukemic transformation. Activating mutations of the FLT3 receptor tyrosine kinase are common in human acute myeloid leukemias and are frequent in human APL. To assess how activating mutations of FLT3 contribute to APL pathogenesis and impact therapy, we used retroviral transduction to introduce an activated allele of FLT3 into control and MRP8 PML-RARA transgenic bone marrow. Activated FLT3 cooperated with PML-RARα to induce leukemias in 62 to 299 days (median latency, 105 days). In contrast to the leukemias that arose spontaneously inMRP8 PML-RARA mice, the activated FLT3/PML-RARα leukemias were characterized by leukocytosis, similar to human APL with FLT3 mutations. Cytogenetic analysis revealed clonal karyotypic abnormalities, which may contribute to pathogenesis or progression. SU11657, a selective, oral, multitargeted tyrosine kinase inhibitor that targets FLT3, cooperated with all-trans retinoic acid to rapidly cause regression of leukemia. Our results suggest that the acquisition of FLT3 mutations by cells with a pre-existing t(15;17) is a frequent pathway to the development of APL. Our findings also indicate that APL patients with FLT3 mutations may benefit from combination therapy with all-trans retinoic acid plus an FLT3 inhibitor.
Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates
Authors: Devine Steven M. , Cobbs Carrington , Jennings Matt , Bartholomew Amelia , Hoffman Ron
Published: April 2003

Ex vivo–expanded mesenchymal stem cells (MSCs) were transduced with a green fluorescent protein (GFP) retroviral construct and subsequently infused into 3 adult baboons following lethal total body irradiation and hematopoietic support or without any prior conditioning. To study the long-term fate of these MSCs, necropsies were performed between 9 and 21 months following MSC infusion, and an average of 16 distinct tissues were recovered from each recipient and evaluated for the presence of the GFP transgene in purified genomic DNA by sensitive real-time polymerase chain reaction (PCR). Two baboons received autologous and one allogeneic GFP-transduced MSCs. Both allogeneic and autologous MSCs appeared to distribute in a similar manner. Gastrointestinal tissues harbored high concentrations of transgene per microgram of DNA. Additional tissues including kidney, lung, liver, thymus, and skin were also found to contain relatively high amounts of DNA equivalents. Estimated levels of engraftment in these tissues ranged from 0.1% to 2.7%. The nonconditioned recipient appeared to have less abundant engraftment. These data suggest that MSCs initially distribute broadly following systemic infusion and later may participate in ongoing cellular turnover and replacement in a wide variety of tissues.
A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow
Authors: Yahata Takashi , Ando Kiyoshi , Sato Tadayuki , Miyatake Hiroko , Nakamura Yoshihiko , Muguruma Yukari , Kato Shunichi , Hotta Tomomitsu
Published: April 2003

To measure the ability of human hematopoietic stem cells (HSCs), the SCID-repopulating cell (SRC) assay has been widely used. Conventionally, human HSCs are transplanted into a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse via a tail vein. However, those cells must go through various obstacles until they reach the mouse marrow environment, which could explain the generally low homing efficiency in this system. Thus, the capability of HSCs may not be studied accurately by this intravenous transplantation method. In our attempt to reveal actual SRC potential, ie, self-renewal and multilineage differentiation in recipient bone marrow, we introduced cells into mouse marrow directly (intrabone marrow [iBM]) to minimize the effect of factors that may interfere with the homing of HSCs and compared the results obtained by intravenous and iBM methods. When cord blood CD34+CD38− cells were transplanted in NOD/SCID mice by iBM, a 15-fold higher frequency of SRC, 1 in 44 CD34+CD38− cells, was achieved compared with 1 in 660 by the intravenous method. Furthermore, the iBM transplant showed high levels of engraftment in the secondary transplantation. Pretreatment of CD34+ cells with antibodies that block either very late antigen 4 (VLA-4) or VLA-5 reduced engraftment partially, whereas blockage of both molecules resulted in complete inhibition of engraftment, which suggests that VLA-4 and VLA-5 are involved in different processes in engraftment or have complementary roles. Our results indicate that the iBM injection strategy is a more sensitive and direct way to measure the capability of human SRCs and is useful to investigate the interaction of HSCs and marrow environment in vivo.
Hematopoietic capacity of connexin43 wild-type and knock-out fetal liver cells not different on wild-type stroma
Authors: Rosendaal Martin , Jopling Chris
Published: April 2003

In 1995 and 1997 we proposed that gap junctions between stromal and hematopoietic cells formed by connexin43 (Cx43) determine hematopoiesis. If this were the case, are the critical gap junctions in this regard those between hematopoietic and stromal cells, or those between stromal cells alone? To test the first possibility, we compared hematopoietic repopulating capacity between fetal liver hematopoietic cells expressing the different mouse Cx43 genotypes, wild type (WT), hemizygous, or knock-out (KO) on WT host mice stroma. We deleted host glucose phosphate isomerase 1a (Gpi-1a) stems and then raced identifiable Cx43 WT host fetal liver against congenic donor Cx43 WT, hemizygous, or KO cells in sets, comparing their capacity to form 5 end cells. Hematopoietic capacity did not differ between the Cx43 WT and KO genotypes. The role of Cx43 gap junctions in hematopoiesis remains uncertain.
Transformation of follicular lymphoma to diffuse large cell lymphoma is associated with a heterogeneous set of DNA copy number and gene expression alterations
Authors: Martinez-Climent Jose A. , Alizadeh Ash A. , Segraves Richard , Blesa David , Rubio-Moscardo Fanny , Albertson Donna G. , Garcia-Conde Javier , Dyer Martin J. S. , Levy Ronald , Pinkel Daniel , Lossos Izidore S.
Published: April 2003

Genomic aberrations in a series of paired biopsy samples from patients who presented initially with follicle center lymphoma (FCL) and subsequently transformed to diffuse large B-cell lymphoma (DLBCL) were measured by array comparative genomic hybridization (CGH). The consequences of these aberrations on gene expression were determined by comparison with expression analysis on these specimens using cDNA microarrays. A heterogeneous pattern of acquired genomic abnormalities was observed upon transformation, some of which were recurrent in small subsets of patients. Some of the genomic aberration acquired upon transformation, such as gain/amplification of 1q21-q24, 2p16 (REL/BCL11A gene loci), 3q27-q29 (including theBCL6 locus), 7q11.2-q22.1, 12pter-q12, 18q21 (including theBCL2 locus) and Xq, and deletion of 6q22-q24, 13q14-q21 and 17p13 (P53 locus) have been previously implicated in the FCL/DLBCL pathogenesis. In addition, novel genomic imbalances not previously reported in association with FCL transformation, such as overrepresentation of 4p12-pter, 5p12-p15, 6p12.3-p21, 9p23, 9q13-q31, 16q, 17q21, and loss of 1p36.3, 4q21-q23, 5q21-q23, 9q31-qter, 11q24-q25, and 15q23, were identified. We observed a differential expression profile of many genes within regions of gain and deletion upon transformation, including novel target genes associated with FCL transformation. However, other genes did not show deregulated expression despite their location within these areas. In summary, the combination of array CGH and expression analysis provides a more comprehensive picture of the transformation of FCL to DLBCL. This process is associated with the acquisition of a variable spectrum of genomic imbalances affecting recurrent chromosomal areas that harbor overexpressed or underexpressed genes targeted upon transformation.
Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis
Authors: Jones Dylan T. , Ganeshaguru Kanagasabai , Anderson Robert J. , Jackson Trevor R. , Bruckdorfer K. Richard , Low Sylvia Y. , Palmqvist Lars , Prentice H. Grant , Hoffbrand A. Victor , Mehta Atul B. , Wickremasinghe R. Gitendra
Published: April 2003

Activation of the phosphatidylinositol 3- kinase/AKT pathway antagonizes apoptosis in diverse cellular systems. We previously showed that human plasma activated AKT and potently blocked the ability of chlorambucil or gamma radiation to induce apoptosis of B-chronic lymphocytic leukemia (CLL) cells. Here we report experiments that identify albumin as the major component of plasma that blocks CLL cell killing by chlorambucil or radiation. Intact plasma depleted of albumin by chromatography on Cibacron blue–Sepharose or plasma from a subject with analbuminemia failed either to activate AKT or to protect CLL cells from chlorambucil-induced apoptosis. Both functions were restored by re-addition of albumin. The protective action of albumin as well as AKT activation was compromised by the binding of lipids. Fluorescence-activated cell sorter (FACScan) analysis demonstrated the uptake of fluoresceinated albumin by CLL cells. Accumulation of albumin in intracellular vesicles was also shown by confocal microscopy. Indirect inhibition of AKT activation by the phosphatidylinositol 3-kinase inhibitor LY294002 reversed the blockade of chlorambucil-induced killing by plasma albumin. The data suggest that activation of AKT consequent to binding of albumin by CLL cells blocks chlorambucil- and radiation-induced apoptosis. Strategies designed to block albumin-induced antiapoptotic signaling may, therefore, be of value in enhancing cytotoxic drug action on CLL cells.
The incidence of heparin-induced thrombocytopenia in hospitalized medical patients treated with subcutaneous unfractionated heparin: a prospective cohort study
Authors: Girolami Bruno , Prandoni Paolo , Stefani Piero M. , Tanduo Cinzia , Sabbion Paola , Eichler Petra , Ramon Roberto , Baggio Giovannella , Fabris Fabrizio , Girolami Antonio
Published: April 2003

Although heparin-induced thrombocytopenia (HIT) is a known complication of intravenous unfractionated heparin (UFH), its incidence in medical patients treated with subcutaneous UFH is less well defined. To determine the incidence of HIT in this category of patients, the platelet count was performed at baseline and then every 3 ± 1 days in 598 consecutive patients admitted to 2 medical wards and treated with subcutaneous UFH for prophylactic (n = 360) or therapeutic (n = 238) indications. The diagnosis of HIT was accepted in the case of a platelet drop of 50% or more and either the demonstration of heparin-dependent antibodies or (when this search could not be performed) the combination of the following features: (1) the absence of any other obvious clinical explanation for thrombocytopenia, (2) the occurrence of thrombocytopenia at least 5 days after heparin start, and (3) either the normalization of the platelet count within 10 days after heparin discontinuation or the earlier patient's death due to an unexpected thromboembolic complication. HIT developed in 5 patients (0.8%; 95% CI, 0.1%-1.6%); all of them belonged to the subgroup of patients who received heparin for prophylactic indications. The prevalence of thromboembolic complications in patients with HIT (60%) was remarkably higher than that observed in the remaining 593 patients (3.5%), leading to an odds ratio of 40.8 (95% CI, 5.2-162.8). Although the frequency of HIT in hospitalized medical patients treated with subcutaneous heparin is lower than that observed in other clinical settings, this complication is associated with a similarly high rate of thromboembolic events.
Discrimination of polycythemias and thrombocytoses by novel, simple, accurate clonality assays and comparison with PRV-1 expression and BFU-E response to erythropoietin
Authors: Liu Enli , Jelinek Jaroslav , Pastore Yves D. , Guan Yongli , Prchal Jaroslav F. , Prchal Josef T.
Published: April 2003

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD, IDS, and MPP1 genes, which together were informative in about 65% of female subjects. To increase our ability to detect clonality, we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these, all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis, whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly, interferon α therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET, and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus, these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels.
The tetrapeptide AcSDKP, an inhibitor of primitive hematopoietic cell proliferation, induces angiogenesis in vitro and in vivo
Authors: Liu Jian-Miao , Lawrence Françoise , Kovacevic Milica , Bignon Jérôme , Papadimitriou Evangelia , Lallemand Jean-Yves , Katsoris Panagiotis , Potier Pierre , Fromes Yves , Wdzieczak-Bakala Joanna
Published: April 2003

The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), purified from bone marrow and constitutively synthesized in vivo, belongs to the family of negative regulators of hematopoiesis. It protects the stem cell compartment from the toxicity of anticancer drugs and irradiation and consequently contributes to a reduction in marrow failure. This current work provides experimental evidence for another novel biologic function of AcSDKP. We report that AcSDKP is a mediator of angiogenesis, as measured by its ability to modulate endothelial cell function in vitro and angiogenesis in vivo. AcSDKP at nanomolar concentrations stimulates in vitro endothelial cell migration and differentiation into capillary-like structures on Matrigel as well as enhances the secretion of an active form of matrix metalloproteinase-1 (MMP-1). In vivo, AcSDKP promotes a significant angiogenic response in the chicken embryo chorioallantoic membrane (CAM) and in the abdominal muscle of the rat. Moreover, it induces the formation of blood vessels in Matrigel plugs implanted subcutaneously in the rat. This is the first report demonstrating the ability of AcSDKP to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo.
Recombinant P-selectin glycoprotein ligand–1 directly inhibits leukocyte rolling by all 3 selectins in vivo: complete inhibition of rolling is not required for anti-inflammatory effect
Authors: Hicks Anne E. R. , Nolan Sarah L. , Ridger Victoria C. , Hellewell Paul G. , Norman Keith E.
Published: April 2003

Selectin-dependent leukocyte rolling is one of the earliest steps of an acute inflammatory response and, as such, contributes to many inflammatory diseases. Although inhibiting leukocyte rolling with selectin antagonists is a strategy that promises far-reaching clinical benefit, the perceived value of this strategy has been limited by studies using inactive, weak, or poorly characterized antagonists. Recombinant P-selectin glycoprotein ligand–1–immunoglobulin (rPSGL-Ig) is a recombinant form of the best-characterized selectin ligand (PSGL-1) fused to IgG, and is one of the best prospects in the search for effective selectin antagonists. We have used intravital microscopy to investigate the ability of rPSGL-Ig to influence leukocyte rolling in living blood vessels and find that it can reduce rolling dependent on each of the selectins in vivo. Interestingly, doses of rPSGL-Ig required to reverse pre-existing leukocyte rolling are 30-fold higher than those required to limit inflammation, suggesting additional properties of this molecule. In support of this, we find that rPSGL-Ig can bind the murine chemokine KC and inhibit neutrophil migration toward this chemoattractant in vitro.
Antithrombin reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation through promotion of prostacyclin production
Authors: Mizutani Akio , Okajima Kenji , Uchiba Mitsuhiro , Isobe Hirotaka , Harada Naoaki , Mizutani Sachiko , Noguchi Takayuki
Published: April 2003

Antithrombin (AT) supplementation in patients with severe sepsis has been shown to improve organ failures in which activated leukocytes are critically involved. However, the precise mechanism(s) for the therapeutic effects of AT is not well understood. We examined in rats whether AT reduces ischemia/reperfusion (I/R)–induced renal injury by inhibiting leukocyte activation. AT markedly reduced the I/R-induced renal dysfunction and histologic changes, whereas neither dansyl glutamylglycylarginyl chloromethyl ketone–treated factor Xa (DEGR-F.Xa), a selective inhibitor of thrombin generation, nor Trp49-modified AT, which lacks affinity for heparin, had any effect. Renal tissue levels of 6-keto-PGF1α, a stable metabolite of prostacyclin (PGI2), increased after renal I/R. AT enhanced the I/R-induced increases in renal tissue levels of 6-keto-PGF1α, whereas neither DEGR-F.Xa nor Trp49-modified AT had any effect. AT significantly inhibited I/R-induced decrease in renal tissue blood flow and the increase in the vascular permeability. Ischemia/reperfusion-induced increases in renal tissue levels of tumor necrosis factor-α, cytokine-induced neutrophil chemoattractant, and myeloperoxidase were significantly inhibited in animals given AT. Pretreatment of animals with indomethacin reversed the effects induced by AT. Iloprost, an analog of PGI2, produced effects similar to those induced by AT. These observations strongly suggest that AT reduces the I/R-induced renal injury by inhibiting leukocyte activation. The therapeutic effects of AT might be mainly mediated by PGI2released from endothelial cells through interaction of AT with cell surface glycosaminoglycans.
Monoclonal T-cell expansions in asymptomatic individuals and in patients with large granular leukemia consist of cytotoxic effector T cells expressing the activating CD94:NKG2C/E and NKD2D killer cell receptors
Authors: Bigouret Valérie , Hoffmann Till , Arlettaz Lionel , Villard Jean , Colonna Marco , Ticheli André , Gratwohl Alois , Samii Kaveh , Chapuis Bernard , Rufer Nathalie , Roosnek Eddy
Published: April 2003

We have analyzed the phenotype, cytokine profile, and mitotic history (telomere length) of monoclonal T-cell expansions in 5 CD3+ T-cell large granular lymphocyte (TLGL) leukemia patients by fluorescence activated cell sorting (FACS) and single-cell polymerase chain reaction (PCR). We confirm that the common phenotype of TLGL leukemia is CD3+CD8+CD45RA+CD27−CD94+(CD57+). Interestingly, the C-type lectin-like type killer cell receptor CD94 was invariably associated with the activating form of its signal-transducing molecule NKG2. Furthermore, when judged by criteria such as interferon gamma (IFN-γ)/tumor necrosis factor (TNF) production, expression of granzyme, FasL, and NKG2D, the TLGL cells had all the features of a cytotoxic effector T cell. Telomere shortening in TLGL cells was in the normal range for CD8+ T cells, indicating that they had not divided significantly more than chronically stimulated CD8+ T cells in healthy individuals. In 25 of 27 controls, cells with a TLGL phenotype occurred at low (1%-3%) frequencies. However, in the other 2 individuals (ages 28-36 years), large stable (> 3 years) monoclonal expansions of CD3+CD8+CD45RA+CD27−CD57+CD94+ NKG2C+ were found which rendered these controls phenotypically indistinguishable from TLGL leukemia patients. We believe that the TLGL clonopathy, rather than being of a neoplastic nature, is more likely an extreme manifestation of the large and stable clonal size characteristic of CD8+ effector cells. Such a TLGL clone consisting of cells without any particular pathologic trait might exist in a considerable number of individuals. Clinical symptoms may occur in individuals in whom the TLGL clone encounters antigen and is triggered to produce large amounts of effector molecules that dysregulate the immune system, which could manifest itself as autoimmunity or as a FasL-mediated neutropenia.
Features of the overexpressed V1-69 genes in the unmutated subset of chronic lymphocytic leukemia are distinct from those in the healthy elderly repertoire
Authors: Potter Kathleen N. , Orchard Jenny , Critchley Eustacia , Mockridge C. Ian , Jose Annette , Stevenson Freda K.
Published: April 2003

Chronic lymphocytic leukemia (CLL) comprises 2 subsets, distinguished by expression of unmutated or mutated VHgenes, with the former having a worse prognosis. Biased usage of the V1-69 gene is found in unmutated cases and is combined with selected D gene segments and JH6. It is controversial whether this is a CLL-associated feature or mirrors the normal B-cell pattern. Since CLL is a disease of the elderly, where changes in the B-cell repertoire may occur, we have analyzedV1-69 usage in the elderly (older than 75 years) population. Using monoclonal antibody (MoAb) G6, specific for 51p1-related V1-69 alleles, we found no increased expression with age. In 51p1-encoded immunoglobulin M (IgM), complementarity-determining region 3 (CDR3) length and frequency of D and JH genes were similar to those in the healthy young and distinct from those in CLL. These findings support the concept that CLL arises from B cells driven by antigen/superantigen and is not a stochastic event in the elderly B-cell population.
Cell death induced by granzyme C
Authors: Johnson Hillary , Scorrano Luca , Korsmeyer Stanley J. , Ley Timothy J.
Published: April 2003

Although the functions of granzymes A and B have been defined, the functions of the other highly expressed granzymes (Gzms) of murine cytotoxic lymphocytes (C, D, and F) have not yet been evaluated. In this report, we describe the ability of murine GzmC (which is most closely related to human granzyme H) to cause cell death. The induction of death requires its protease activity and is characterized by the rapid externalization of phosphatidylserine, nuclear condensation and collapse, and single-stranded DNA nicking. The kinetics of these events are similar to those caused by granzyme B, and its potency (defined on a molar basis) is also equivalent. The induction of death did not involve the activation of caspases, the cleavage of BID, or the activation of the CAD nuclease. However, granzyme C did cause rapid mitochondrial swelling and depolarization in intact cells or in isolated mitochondria, and this mitochondrial damage was not prevented by cyclosporin A pretreatment. These results suggest that granzyme C rapidly induces target cell death by attacking nuclear and mitochondrial targets and that these targets are distinct from those used by granzyme B to cause classical apoptosis.
Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk
Authors: Wolberg Alisa S. , Monroe Dougald M. , Roberts Harold R. , Hoffman Maureane
Published: April 2003

Individuals with elevated prothrombin levels are at increased risk of venous thrombosis. To understand the mechanism behind this observation, we studied the effect of prothrombin concentration on thrombin generation and fibrin clot structure. The pattern of thrombin generation was directly related to the prothrombin level at all concentrations tested. From 0% to 300% of normal plasma levels of prothrombin, increasing the prothrombin concentration increased the initial rate, peak, and total amount of thrombin generated. Importantly, fibrin clot structure was also affected by the prothrombin concentration. Fibrin clots made from prothrombin concentrations less than 10% of plasma levels were weak and poorly formed. Fibrin clots made at 10% to 100% of plasma levels of prothrombin had similar fiber structures (mass-to-length ratio; μ). However, the fiber mass-to-length ratio decreased with increasing prothrombin levels more than 100% of plasma levels, in a dose-dependent manner. These results suggest that increased levels of prothrombin alter thrombin generation and clot structure. Specifically, elevated prothrombin levels produce clots with reduced fibrin mass-to-length ratios compared with normal clots. We hypothesize that this alteration in fibrin clot structure is an important determinant of the risk of thrombosis.
Distinct dose-dependent effects of plasmin and TPA on coagulation and hemorrhage
Authors: Stewart Daphne , Kong Mansze , Novokhatny Valery , Jesmok Gary , Marder Victor J.
Published: April 2003

All thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a direct-acting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages.
Downstream effectors of oncogenic ras in multiple myeloma cells
Authors: Hu Liping , Shi Yijiang , Hsu Jung-hsin , Gera Joseph , Van Ness Brian , Lichtenstein Alan
Published: April 2003

Ectopic expression of mutated K-ras or N-ras in the interleukin 6 (IL-6)–dependent ANBL6 multiple myeloma cell line induces cytokine-independent growth. To investigate the signaling pathways activated by oncogenic ras that may stimulate IL-6–independent growth, we compared ANBL6 cells stably transfected with mutated K or N-ras genes with wild-type ras–expressing control cells identically transfected with an empty vector. Upon depletion of IL-6, both mutated ras–containing myeloma lines demonstrated constitutive activation of mitogen-activated extracellular kinase 2(MEK)/extracellular signal–regulated kinase (ERK), phosphatidylinositol-3 kinase (PI3-kinase)/AKT, mammalian target of rapamycin (mTOR)/p70S6-kinase, and nuclear factor kappaB (NF-kB) pathways. In contrast, signal transducer and activator of transcription–3 (STAT-3) was not constitutively tyrosine phosphorylated in mutant ras–expressing cells. We used several maneuvers in attempts to selectively target these constitutively active pathways. The mTOR inhibitors rapamycin and CCI-779, the PI3-kinase inhibitor LY294002, and the MEK inhibitor PD98059 all significantly curtailed growth of mutant ras–containing cells. Farnesyl transferase inhibitors, used to target ras itself, had modest effects only against mutant N-ras–containing cells. Growth of mutant N-ras–containing myeloma cells was also inhibited by acute expression of the IKBsuperrepressor gene, which abrogated NF-kB activation. These results indicate that several pathways contributing to stimulation of cytokine-independent growth are activated downstream of oncogenic ras in myeloma cells. They also suggest that therapeutic strategies that target these pathways may be particularly efficacious in patients whose myeloma clones contain ras mutations.
Cotreatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) enhances imatinib-induced apoptosis of Bcr-Abl–positive human acute leukemia cells
Authors: Nimmanapalli Ramadevi , Fuino Lianne , Stobaugh Corinne , Richon Victoria , Bhalla Kapil
Published: April 2003

Here we demonstrate that treatment with SAHA (suberoylanilide hydroxamic acid), a known inhibitor of histone deacetylases (HDACs), alone induced p21 and/or p27 expressions but decreased the mRNA and protein levels of Bcr-Abl, which was associated with apoptosis of Bcr-Abl–expressing K562 and LAMA-84 cells. Cotreatment with SAHA and imatinib (Gleevec) caused more down-regulation of the levels and auto-tyrosine phosphorylation of Bcr-Abl and apoptosis of these cell types, as compared with treatment with either agent alone (P < .05). This finding was also associated with a greater decline in the levels of phospho-AKT and Bcl-xL. Significantly, treatment with SAHA also down-regulated Bcr-Abl levels and induced apoptosis of CD34+ leukemia blast progenitor cells derived from patients who had developed progressive blast crisis (BC) of chronic myelocytic leukemia (CML) while receiving therapy with imatinib. Taken together, these findings indicate that cotreatment with SAHA enhances the cytotoxic effects of imatinib and may have activity against imatinib-refractory CML-BC.
Cobalamin deficiency with and without neurologic abnormalities: differences in homocysteine and methionine metabolism
Authors: Carmel Ralph , Melnyk Stepan , James S. Jill
Published: April 2003

The unknown biochemical basis for neurologic dysfunction in cobalamin deficiency and the frequent divergence between neurologic and hematologic manifestations led us to study homocysteine metabolism in 22 patients with pernicious anemia. Serum levels of total homocysteine (tHcy), methionine, S-adenosylmethionine (AdoMet), cysteine, cysteinylglycine (cys-gly), and glutathione (GSH) were measured. Only levels of tHcy and cysteine were increased and only GSH was decreased in cobalamin deficiency as a whole, compared with 17 control subjects. AdoMet correlated only with methionine levels (P = .015) and cysteine only with cys-gly (P = .007) in healthy subjects, but in cobalamin-deficient patients AdoMet correlated instead with cysteine, cys-gly, and folate levels only (P = .008,P = .03, and P = .03, respectively). Significant differences appeared in clinically subgrouped cobalamin-deficient patients. The 11 patients with neurologic defects had higher mean levels of folate (27.9 versus 15.4 nM), AdoMet (117.2 versus 78.6 nM), cysteine (462 versus 325 μM), and cys-gly (85.0 versus 54.7 μM) than the 11 neurologically unaffected patients. Cobalamin therapy restored all metabolic changes to normal. The results indicate that changes in several metabolic pathways differ in patients with and without neurologic dysfunction. Cysteine levels were the most significant predictors of neurologic dysfunction, but it is unclear if they are direct or indirect indicators of neurotoxicity. The higher AdoMet levels in neurologically affected patients may result from inhibition of glycine N-methyltransferase by those patients' higher folate levels. The origin of the folate differences is unclear and possibly varied. Low AdoMet and GSH levels were independent predictors of anemia.
IQGAP2 functions as a GTP-dependent effector protein in thrombin-induced platelet cytoskeletal reorganization
Authors: Schmidt Valentina A. , Scudder Lesley , Devoe Craig E. , Bernards André , Cupit Lisa D. , Bahou Wadie F.
Published: April 2003

Human blood platelets are anucleate cells whose response to extracellular stimuli results in actin cytoskeleton rearrangements, thereby providing the critical initial step in the regulation of hemostasis. The serine protease α-thrombin, known to activate platelets by cleavage of a family of protease-activated receptors (PARs), is the most potent physiologic activator of human platelets, though downstream effector proteins uniquely linked to platelet cytoskeletal actin polymerization remain largely uncharacterized. The gene encoding the putative rac1/cdc42 effector protein IQGAP2 was identified within the PAR gene cluster at 5q13, flanked telomeric byPAR1 and encompassing PAR3. Immunofluorescence microscopy demonstrated IQGAP2 expression in filopodial extensions of activated platelets and colocalized with F-actin in lamellipodia and filopodia of IQGAP2-transfected COS1 cells. Platelet activation by α-thrombin, but not saturating concentrations of fibrillar collagen or adenosine 5′-diphosphate, uniquely assemble an IQGAP2/arp2/3–actin cytoplasmic complex, an association regulated by guanosine triphosphate rac1 ([GTP]rac1) but not by [GTP]cdc42. Likewise, only thrombin-activated platelets resulted in rapid translocation of IQGAP2 to the platelet cytoskeleton. These observations identify a physiologic scaffolding function for IQGAP2 and establish the presence of a functional genomic unit in humans uniquely evolved to regulate thrombin-induced platelet cytoskeletal actin reorganization.
Stimulation of osteoprotegerin production is responsible for osteosclerosis in mice overexpressing TPO
Authors: Chagraoui Hédia , Tulliez Micheline , Smayra Tarek , Komura Emiko , Giraudier Stéphane , Yun Theodore , Lassau Nathalie , Vainchenker William , Wendling Françoise
Published: April 2003

Myelofibrosis and osteosclerosis are prominent features arising in mice overexpressing thrombopoietin (TPO). The pivotal role of transforming growth factor β1 (TGF-β1) in the pathogenesis of myelofibrosis has been documented, but the mechanisms mediating osteosclerosis remain unclear. Here, we used mice deficient in osteoprotegerin (OPG), a secreted inhibitor of bone resorption, to determine whether osteosclerosis occurs through a deregulation of osteoclastogenesis. Marrow cells from opg-deficient mice (opg−/−) or wild-type (WT) littermates were infected with a retrovirus encoding TPO and engrafted into anopg−/− or WT background for long-term reconstitution. The 4 combinations of graft/host (WT/WT,opg−/−/opg−/−,opg−/−/WT, and WT/opg−/−) were studied. Elevation of TPO and TGF-β1 levels in plasma was similar in the 4 experimental groups and all the mice developed a similar myeloproliferative syndrome associated with severe myelofibrosis. Osteosclerosis developed in WT hosts engrafted with WT or opg−/− hematopoietic cells and was associated with increased OPG levels in plasma and decreased osteoclastogenesis. In contrast,opg−/− hosts exhibited an osteoporotic phenotype and a growth of bone trabeculae was rarely seen. These findings suggest that osteosclerosis in mice with TPO overexpression occurs predominantly via an up-regulation of OPG in host stromal cells leading to disruption of osteoclastogenesis.
Combined carrier status of prothrombin 20210A and factor XIII-A Leu34 alleles as a strong risk factor for myocardial infarction: evidence of a gene-gene interaction
Authors: Butt Christopher , Zheng Hong , Randell Edward , Robb Desmond , Parfrey Patrick , Xie Ya-Gang
Published: April 2003

Studies associating the prothrombin 20210G>A (FII 20210A), factor V Leiden (FVL), and factor XIII Leu34 (FXIII-A Leu34) alleles with myocardial infarction (MI) have yielded conflicting results. Complicated gene-gene interactions, small sample sizes, and heterogeneous genetic and environmental backgrounds may contribute to opposing findings. Simultaneous analysis of multiple gene variants in a large sample size from a genetically isolated population may overcome these weaknesses. Genotyping was performed in 500 MI patients and 500 control subjects from the genetically isolated Newfoundland population to determine the prevalence of the FII 20210A, FVL, and FXIII-A Leu34 variants and their association with MI. Gene-gene interactions were also analyzed. The prevalence of the FII 20210A allele was higher in MI patients (3.2%) than in control subjects (1.0%;P = .015). The FII 20210A allele was also 5.6-fold higher in MI patients younger than 51 years than in age-matched control subjects (P = .04). FVL showed 3.9-fold higher prevalence in young patients than in patients older than 50 years (P = .004) and 2.7-fold higher than in age-matched control subjects (P = .007). Furthermore, the prevalence of combined carriers of the FXIII-A L34 and FII 20210A alleles was 12-fold higher in MI patients than in control subjects (P = .002) and with 92% penetrance. There was disequilibrium of the FXIII-A Leu34 allele to MI patients carrying the FII 20210A allele as a genetic background. Based on our data, we determined that (1) the FII 20210A allele is a risk factor for MI, possibly important for early onset; (2) FVL may predispose for early-onset MI; (3) the FXIII-A Leu34 allele predisposes for MI in males only; however, (4) interaction between the FII 20210A and FXIII-A Leu34 alleles forms a synergistic coeffect that strongly predisposes for MI, placing combined carriers at high risk for MI.
Multiple myeloma tumor progression in the 5T2MM murine model is a multistage and dynamic process of differentiation, proliferation, invasion, and apoptosis
Authors: Asosingh Kewal , De Raeve Hendrik , Van Riet Ivan , Van Camp Benjamin , Vanderkerken Karin
Published: April 2003

At clinical presentation, multiple myeloma (MM) is already a well-established disease. The processes involved in earlier stages are, however, unknown. Here the 5T2MM murine model was used to analyze differentiation, proliferation, invasion, and apoptosis of MM cells during disease progression. Naive mice were injected with 5T2MM cells and from the onset of the experiment 3 mice were killed each week until the end stage. Myeloma cells were isolated from the bone marrow and selected by sequential gating of 5T2MM idiotype+ cells by flow cytometry. Microscopic analysis of these sorted 5T2MM idiotype+ cells confirmed their identity as true myeloma cells. Based on serum paraprotein concentration and bone marrow tumor load, 3 disease stages were distinguished: a quiescent stage, an intermediate stage, and an end stage, of slow, moderate, and accelerated tumor progression, respectively. In the quiescent stage, the majority of the myeloma cells were CD45+CD138−IL-6Rα+, corresponding to an immature, invasive, and apoptosis-resistant phenotype. In the end stage the majority of the myeloma cells had differentiated into CD45−CD138+IL-6Rα− cells, corresponding to a mature, less invasive, and apoptosis-sensitive phenotype. In the intermediate stage a gradual transition from the quiescent toward the end stage was observed. In line with these data, analysis of sorted 5T2MM cells demonstrated a significant decrease in invasive capacity and a significant increase in (dexamethasone-induced) apoptosis sensitivity and in proliferation during the disease progression. These data suggest that myeloma disease progression is a multistage and dynamic process of differentiation, proliferation, invasion, and apoptosis.
Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML)
Authors: Guan Yinghui , Gerhard Brigitte , Hogge Donna E.
Published: April 2003

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture–initiating cells (LTC-ICs) directly isolated from patients are actively cycling, quiescent progenitors are present in most samples. In the current study,3H-thymidine (3H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G0, G1, and S/G2+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the 3H-Tdr suicide results, with NOD/SL-ICs found almost exclusively among G0 cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly, after 72 hours in serum-free culture with or without Steel factor (SF), Flt-3 ligand (FL), and interleukin-3 (IL-3), most G0 AML cells entered active cell cycle (percentage of AML cells remaining in G0 at 72 hours, 1.2% to 37%, and 0% to 7.6% in cultures without and with growth factors [GFs], respectively) while G0 cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF, FL, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, 3 of 4 samples contained an internal tandem duplication of theFLT3 gene. In summary, quiescent leukemic cells, including NOD/SL-ICs, are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs.