bluuebunny/crossref_metadata_2025 huggingface dataset:
Reciprocal effects of C/EBPα and PKCδ on JunB expression and monocytic differentiation depend upon the C/EBPα basic region
Authors: Liu Huaitian , Keefer Jeffrey R. , Wang Qian-fei , Friedman Alan D.
Published: May 2003

Monocytic differentiation of 32DPKCδ cells in response to activation of protein kinase C δ (PKCδ) by phorbol 12-myristate 13-acetate (PMA) was inhibited by exogenous CCAAT/enhancer binding protein α–estradiol receptor (C/EBPα-ER), which impeded morphologic maturation and induction of macrosialin mRNA. Inhibition of monopoiesis was also evident in 32DPKCδ subclones expressing C/EBPαLeu12Val-ER, which cannot dimerize or bind DNA because of mutation of the leucine zipper, C/EBPαGZ-ER, in which the leucine zipper has been replaced by the GCN4 zipper, or C/EBPαΔ3-8-ER, lacking the C/EBPα transactivation domains. In contrast, C/EBPαBR3-ER, containing a mutant basic region, did not inhibit monocytic differentiation. C/EBPα-ER strongly inhibited endogenous AP-1 DNA-binding. Supershift analysis revealed that the major AP-1 complex contains JunB. Activation of C/EBPα-ER specifically reduced endogenous JunB RNA and protein and exogenous JunB levels without affecting endogenous or exogenous c-Jun. The stability of PMA-induced JunB was not affected. Thus, C/EBPα-ER suppresses both JunB transcription and posttranscriptional protein generation or induction. PU.1 levels and activity were increased. The Leu12Val, GZ, and Δ3-8 mutants also inhibited JunB expression, whereas the BR3 mutant was ineffective, indicating that inhibition of JunB expression and monocytic differentiation by C/EBPα-ER depends upon an interaction mediated by its basic region. Exogenous JunB restored AP-1 DNA-binding but did not prevent inhibition of macrosialin expression by C/EBPα-ER, indicating that JunB is not the only target relevant to inhibition of monopoiesis by C/EBPα.
Nephrotic syndrome with crescent formation and massive IgA deposition following allogeneic bone marrow transplantation for natural killer cell leukemia/lymphoma
Authors: Kimura Shinya , Horie Akeyo , Hiki Yoshiyuki , Yamamoto Chie , Suzuki Satoru , Kuroda Junya , Deguchi Masayo , Kato Gen-ichi , Karasuno Takahiro , Hiraoka Akira , Yoshikawa Toshikazu , Maekawa Taira
Published: May 2003

We describe herein a case of nephrotic syndrome (NS) following allogeneic bone marrow transplantation (allo-BMT) for natural killer cell leukemia/lymphoma. Histologic studies defined the diagnosis as crescentic glomerulonephritis with massive immunoglobulin A (IgA) deposition, which has never been reported in NS cases following allo-BMT. Most of the massive infiltrated cells in the interstice were CD3+CD4−CD8+ T cells derived from the donor. We observed mesangial deposition of Haemophilus parainfluenza outer membrane (OMHP) antigen and decreased glycosylation of the IgA1 hinge in the recipient's samples is consistent with the recently reported pathogenesis of IgA nephropathy. Further, the titer of IgA antibody against the donor serum was as high as other IgA nephropathy cases. These findings suggest that NS and crescentic glomerulonephritis in this case occurred as one of the forms of chronic graft-versus-host disease (GVHD), and that IgA deposition was associated with H parainfluenza and decreased glycosylation of the IgA1 hinge.
Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model
Authors: Pawlinski Rafal , Pedersen Brian , Kehrle Bettina , Aird William C. , Frank Rolf D. , Guha Mausumee , Mackman Nigel
Published: May 2003

In septic shock, tissue factor (TF) activates blood coagulation, and cytokines and chemokines orchestrate an inflammatory response. In this study, the role of Egr-1 in lipopolysaccharide (LPS) induction of TF and inflammatory mediators in vivo was evaluated using Egr-1+/+ and Egr-1−/− mice. Administration of LPS transiently increased the steady-state levels of Egr-1 mRNA in the kidneys and lungs of Egr-1+/+ mice with maximal induction at one hour. Egr-1 was expressed in epithelial cells in the kidneys and lungs in untreated and LPS-treated mice. LPS induction of monocyte chemoattractant protein mRNA in the kidneys and lungs of Egr-1−/− mice was not affected at 3 hours, but its expression was significantly reduced at 8 hours compared with the expression observed in Egr-1+/+ mice. Similarly, LPS induction of TF mRNA expression in the kidneys and lungs at 8 hours was reduced in Egr-1−/− mice. However, Egr-1 deficiency did not affect plasma levels of tumor necrosis factor α in endotoxemic mice. Moreover, Egr-1+/+ and Egr-1−/− mice exhibited similar survival times in a model of acute endotoxemia. These data indicate that Egr-1 does not contribute to the early inflammatory response in the kidneys and lungs or the early systemic inflammatory response in endotoxemic mice. However, Egr-1 does contribute to the sustained expression of inflammatory mediators and to the maximal expression of TF at 8 hours in the kidneys and lungs.
G-protein–coupled receptor signaling in Syk-deficient neutrophils and mast cells
Authors: Mócsai Attila , Zhang Hong , Jakus Zoltán , Kitaura Jiro , Kawakami Toshiaki , Lowell Clifford A.
Published: May 2003

The Syk tyrosine kinase is essential for immunoreceptor and multiple integrin functions as well as being implicated in signaling from G-protein–coupled receptors (GPCR) in cell lines, transfection systems, and pharmacologic studies. In contrast, using Syk-deficient primary cells, we show here that Syk does not play a major functional role in chemoattractant/chemokine signaling in neutrophils and mast cells. syk−/− neutrophils showed normal respiratory burst and degranulation in response to the bacterial peptide formyl-Met-Leu-Phe (fMLP). The migration of neutrophils toward fMLP was similarly not affected by the syk−/−mutation. fMLP initiated normal Ca2+-signal, activation of the extracellular signal-related kinase (ERK) and p38 mitogen–activated protein (MAP) kinase cascades, and polymerization of cellular actin in the absence of Syk.syk−/− and wild-type neutrophils also responded similarly to LTB4, C5a, and the chemokines macrophage inflammatory protein-1 (MIP-1)α or MIP-2, both in functional assays and in intracellular signaling mechanisms. Furthermore, bone marrow–derived syk−/− mast cells showed normal activation of the Akt, ERK, and p38 MAP kinase pathways when stimulated by the GPCR ligand adenosine. We conclude that, in contrast to previous reports, Syk does not play a major role in GPCR signaling.
Effects of combined chelation treatment with pyridoxal isonicotinoyl hydrazone analogs and deferoxamine in hypertransfused rats and in iron-loaded rat heart cells
Authors: Link Gabriela , Ponka Prem , Konijn Abraham M. , Breuer William , Cabantchik Z. Ioav , Hershko Chaim
Published: May 2003

Although iron chelation therapy with deferoxamine (DFO) results in improved life expectancy of patients with thalassemia, compliance with parenteral DFO treatment is unsatisfactory, underlining the need for alternative drugs and innovative ways of drug administration. We examined the chelating potential of pyridoxal isonicotinoyl hydrazone (PIH) analogs, alone or in combination with DFO, using hypertansfused rats with labeled hepatocellular iron stores and cultured iron-loaded rat heart cells. Our in vivo studies using 2 representative PIH analogs, 108-o and 109-o, have shown that PIH analogs given orally are 2.6 to 2.8 times more effective in mobilizing hepatocellular iron in rats, on a weight-per-weight basis, than parenteral DFO administered intraperitoneally. The combined effect of DFO and 108-o on hepatocellular iron excretion was additive, and response at a dose range of 25 to 200 mg/kg was linear. In vitro studies in heart cells showed that DFO was more effective in heart cell iron mobilization than all PIH analogs studied. Response to joint chelation with DFO and PIH analogs was similar to an increase in the equivalent molar dose of DFO alone, rather than the sum of the separate effects of the PIH analog and DFO. This finding was most likely the result of iron transfer from PIH analogs to DFO, a conclusion supported directly by iron-shuttle experiments using fluorescent DFO. These findings provide a rationale for the combined, simultaneous use of iron-chelating drugs and may have useful, practical implications for designing novel strategies of iron chelation therapy.
Granulocyte colony-stimulating factor and the risk of secondary myeloid malignancy after etoposide treatment
Authors: Relling Mary V. , Boyett James M. , Blanco Javier G. , Raimondi Susana , Behm Frederick G. , Sandlund John T. , Rivera Gaston K. , Kun Larry E. , Evans William E. , Pui Ching-Hon
Published: May 2003

Event-free survival for children with acute lymphoblastic leukemia (ALL) now exceeds 80% in the most effective trials. Failures are due to relapse, toxicity, and second cancers such as therapy-related myeloid leukemia or myelodysplasia (t-ML). Topoisomerase II inhibitors and alkylators can induce t-ML; additional risk factors for t-ML remain poorly defined. The occurrence of t-ML among children who had received granulocyte colony-stimulating factor (G-CSF) following ALL remission induction therapy prompted us to examine this and other putative risk factors for t-ML in 412 children treated on 2 consecutive ALL protocols from 1991 to 1998. All children received etoposide and anthracyclines, 99 of whom received G-CSF; 284 also received cyclophosphamide, 58 of whom also received cranial irradiation. There were 20 children who developed t-ML at a median of 2.3 years (range, 1.0-6.0 years), including 16 cases of acute myeloid leukemia, 3 myelodysplasia, and 1 chronic myeloid leukemia. Stratifying by protocol, the cumulative incidence functions differed (P = .017) according to the use of G-CSF and irradiation: 6-year cumulative incidence (standard error) of t-ML of 12.3% (5.3%) among the 44 children who received irradiation without G-CSF, 11.0% (3.5%) among the 85 children who received G-CSF but no irradiation, 7.1% (7.2%) among the 14 children who received irradiation plus G-CSF, and 2.7% (1.3%) among the 269 children who received neither irradiation nor G-CSF. Even when children receiving irradiation were excluded, the incidence was still higher in those receiving G-CSF (P = .019). In the setting of intensive antileukemic therapy, short-term use of G-CSF may increase the risk of t-ML.
Cytokine-mediated regulation of iron transport in human monocytic cells
Authors: Ludwiczek Susanne , Aigner Elmar , Theurl Igor , Weiss Günter
Published: May 2003

Under chronic inflammatory conditions cytokines induce a diversion of iron traffic, leading to hypoferremia and retention of the metal within the reticuloendothelial system. However, the regulatory pathways underlying these disturbances of iron homeostasis are poorly understood. We investigated transferrin receptor (TfR)–dependent and –independent iron transport mechanisms in cytokine-stimulated human monocytic cell lines THP-1 and U937. Combined treatment of cells with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) reduced TfR mRNA levels, surface expression, and iron uptake, and these effects were reversed by interleukin-10 (IL-10), thus stimulating TfR-mediated iron acquisition. IFN-γ and LPS dose-dependently increased the cellular expression of divalent metal transporter-1, a transmembrane transporter of ferrous iron, and stimulated the uptake of nontransferrin bound iron (NTBI) into cells. At the same time, IFN-γ and LPS down-regulated the expression of ferroportin mRNA, a putative iron exporter, and decreased iron release from monocytes. Preincubation with IL-10 partly counteracted these effects. Our results demonstrate that the proinflammatory stimuli IFN-γ and LPS increase the uptake of NTBI via stimulation of divalent metal transporter-1 expression and cause retention of the metal within monocytes by down-regulating ferroportin synthesis. Opposite, the anti-inflammatory cytokine IL-10 stimulates TfR-mediated iron uptake into activated monocytes. The regulation of iron transport by cytokines is a key mechanism in the pathogenesis of anemia of chronic disease and a promising target for therapeutic intervention.
Aberrant DNA methylation of p57KIP2 identifies a cell-cycle regulatory pathway with prognostic impact in adult acute lymphocytic leukemia
Authors: Shen LanLan , Toyota Minoru , Kondo Yutaka , Obata Toshiro , Daniel Sophia , Pierce Sherry , Imai Kohzoh , Kantarjian Hagop M. , Issa Jean-Pierre J. , Garcia-Manero Guillermo
Published: May 2003

P57KIP2 is a cyclin-dependent kinase inhibitor silenced in a variety of human malignancies. DNA methylation of a region surrounding the transcription start site of p57KIP2 was found in acute lymphocytic leukemia (ALL)–derived cell lines. Methylation of this region correlated with gene silencing, and treatment of methylated/silenced cell lines with 5-aza-2′-deoxycytidine resulted in gene re-expression. P57KIP2 was methylated in 31 (50%) of 63 patients with newly diagnosed ALL, and in 11 (52%) of 21 patients with relapsed ALL. In 5 of them (25%), methylation was acquired at relapse. No association was observed between methylation of p57KIP2 alone and clinical-biologic characteristics studied, including overall survival (OS) or disease-free survival. Methylation of multiple genes in a cell-cycle regulatory pathway composed of p73, p15, and p57KIP2 occurred in 22% of Philadelphia chromosome (Ph)–negative patients. Ph-negative patients with methylation of 2 or 3 genes of this pathway had a significantly worse median OS compared with those with methylation of 0 or 1 gene (50 vs 467 weeks, respectively;P = .02). Our results indicate that p57KIP2 is frequently methylated in adult patients with ALL, and that inactivation of a pathway composed of p73, p15, and p57KIP2 predicts for poor prognosis in Ph-negative patients.
Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents
Authors: Zhou Yan , Hileman Elizabeth O. , Plunkett William , Keating Michael J. , Huang Peng
Published: May 2003

2-Methoxyestradiol (2-ME), a new anticancer agent currently in clinical trials, has been demonstrated to inhibit superoxide dismutase (SOD) and to induce apoptosis in leukemia cells through a free radical–mediated mechanism. Because the accumulation of superoxide (O2−) by inhibition of SOD depends on the cellular generation of O2−, we hypothesized that the endogenous production of superoxide may be a critical factor that affects the antileukemia activity of 2-ME. In the present study, we investigated the relationship between cellular O2− contents and the cytotoxic activity of 2-ME in primary leukemia cells from 50 patients with chronic lymphocytic leukemia (CLL). Quantitation of O2− revealed that the basal cellular O2− contents are heterogeneous among patients with CLL. The O2− levels were significantly higher in CLL cells from patients with prior chemotherapy. CLL cells with higher basal O2− contents were more sensitive to 2-ME in vitro than those with lower O2− contents. There was a significant correlation between the 2-ME–induced O2−increase and the loss of cell viability. Importantly, addition of arsenic trioxide, a compound capable of causing reactive oxygen species (ROS) generation, significantly enhanced the activity of 2-ME, even in the CLL cells that were resistant to 2-ME alone. These results suggest that the cellular generation of O2− plays an important role in the cytotoxic action of 2-ME and that it is possible to use exogenous ROS-producing agents such as arsenic trioxide in combination with 2-ME to enhance the antileukemia activity and to overcome drug resistance. Such a combination strategy may have potential clinical applications.
Hyperhomocysteinemia accelerates atherosclerosis in cystathionine β-synthase and apolipoprotein E double knock-out mice with and without dietary perturbation
Authors: Wang Hong , Jiang XiaoHua , Yang Fan , Gaubatz John W. , Ma Lang , Magera Mark J. , Yang XiaoFeng , Berger Peter B. , Durante William , Pownall Henry J. , Schafer Andrew I.
Published: May 2003

Although hyperhomocysteinemia is an independent risk factor for cardiovascular disease, a direct role for homocysteine (Hcy) in this disease remains to be shown. Whereas diet-induced hyperhomocysteinemia promotes atherosclerosis in animal models, the effects of Hcy on atherogenesis in the absence of dietary perturbations is not known. We have generated double knock-out mice with targeted deletions of the genes for apolipoprotein E (apoE) and cystathionine β-synthase (CBS), which converts Hcy to cystathionine. ApoE−/−/CBS−/− mice developed aortic lesions even in the absence of dietary manipulation; lesion area and lesion cholesteryl ester (CE) and triglyceride (TG) contents increased with animal age and plasma Hcy levels. Plasma total cholesterol was significantly increased, whereas high density lipoprotein (HDL) cholesterol and TG concentrations of apoE−/−/CBS−/− mice were decreased. Cholesterol esterification and activities of enzymes catalyzing CE or TG formation in the vessel wall and in peritoneal macrophages were not changed by hyperhomocysteinemia. However, uptake of human acetyl-LDL, but not native low density lipoprotein (LDL), by mouse peritoneal macrophages was higher in the presence of hyperhomocysteinemia. These results suggest that isolated hyperhomocysteinemia is atherogenic and alters hepatic and macrophage lipoprotein metabolism, in part, by enhancing uptake of modified LDL.
Induction of heme oxygenase-1 and stimulation of cGMP production by hemin in aortic tissues from hypertensive rats
Authors: Ndisang Joseph Fomusi , Wu Lingyun , Zhao Weimin , Wang Rui
Published: May 2003

Heme oxygenase (HO) and carbon monoxide (CO) have been implicated in the modulation of various cardiovascular functions including blood pressure (BP) regulation. Up-regulating the HO/CO system lowers BP in young (8-week-old) but not in adult (20-week-old) spontaneously hypertensive rats (SHRs). The mechanisms for this selective effect are largely unknown. We investigated the effects of HO-1 inducer, hemin, on the HO/CO-soluble gyanylyl cyclase (sGC)/cGMP system in the aorta of prehypertensive (4-week-old) young and adult SHRs as well as age-matched Wistar-Kyoto rats (WKYs). Reduced expressions of HO-1, HO-2, and sGC proteins associated with depressed HO activity and cGMP levels were detected in young SHRs. These deficiencies were significantly reversed by hemin treatment. Macrophage infiltration of vascular tissues was more significant in adult SHRs than adult WKYs, but invisible in young SHRs and WKYs. Hemin treatment did not alter macrophage infiltration of vascular tissues in young SHRs. The same hemin administration resulted in a significant decrease in BP (from 148.6 ± 3.2 to 125.8 ± 2.6 mmHg, P < .01) in young SHRs, but not in prehypertensive or adult SHRs or WKYs of all ages. The HO inhibitor zinc protoporphyrin abrogated the hemin effect in young SHRs. Aortic tissues became desensitized to YC-1, an activator sGC, in adult SHRs. Thus, in young SHRs the expression and function of the HO/CO-sGC/cGMP system were suppressed, constituting a pathogenic mechanism for the development of hypertension. In adult SHRs, the HO/CO-sGC/cGMP system appeared normal, but desensitization of the sGC/cGMP pathway caused hypertension to prevail.
AML-1A and AML-1B regulation of MIP-1α expression in multiple myeloma
Authors: Choi Sun J. , Oba Tomoko , Callander Natalie S. , Jelinek Diane F. , Roodman G. David
Published: May 2003

Macrophage inflammatory protein-1α (MIP-1α) is produced in high concentration by multiple myeloma (MM) cells in about 70% of patients, and MIP-1α levels correlate with their disease activity. Patients who have high levels of MIP-1α have a poor prognosis. Furthermore, blocking MIP-1α expression in an in vivo model of human MM profoundly decreases both tumor burden and bone destruction, suggesting that MIP-1α is an important mediator of MM bone disease. Therefore, to analyze the regulation of MIP-1α production in MM, we cloned the human MIP-1α promoter and characterized the transcription factor (TF) motifs that control MIP-1α expression in MM cells. The proximal region of MIP-1α promoter was composed of 2 sets of identical transcription regulatory regions consisting of GATA-2+ AML-1+ C/EBPα motifs. Since 2 alternatively spliced variants of the acute myeloid leukemia-1 (AML-1) class of TFs can bind the AML-1 region, AML-1A and AML-1B, the relationship between the expression levels of AML-1A or AML-1B in MM cells and their capacity to express MIP-1α was examined. AML-1A mRNA was relatively overexpressed compared with AML-1B in MM cell lines that produced high levels of MIP-1α (> 1 ng/mL per 106 cells per 72 hours), but AML-1A was not increased in MM cell lines that expressed less than 200 pg/mL MIP-1α. More importantly, the ratio of AML-1A to AML-1B mRNA levels was also increased in 3 of 3 highly purified myeloma cells from patients with MM who expressed increased amounts of MIP-1α. The ratio of AML-1A to AML-1B mRNA in patients with MM was 8-fold higher than in healthy controls. Transduction of AML-1B into the MM-derived MM.1S and ARH-77 cells totally blocked MIP-1α production, while AML-1A did not further increase the already high levels of MIP-1α produced by these cells. Taken together, these data demonstrate that in patients with MM who produce increased concentrations of MIP-1α, the relative level of AML-1B is significantly decreased compared with healthy controls. The data suggest that strategies that enhance AML-1B expression or decrease AML-1A in MM cells may be beneficial therapeutically.
Excess hemoglobin digestion and the osmotic stability ofPlasmodium falciparum–infected red blood cells
Authors: Lew Virgilio L. , Tiffert Teresa , Ginsburg Hagai
Published: May 2003

During their asexual reproduction cycle (about 48 hours) in human red cells, Plasmodium falciparum parasites consume most of the host cell hemoglobin, far more than they require for protein biosynthesis. They also induce a large increase in the permeability of the host cell plasma membrane to allow for an increased traffic of nutrients and waste products. Why do the parasites digest hemoglobin in such excess? And how can infected red cells retain their integrity for the duration of the asexual cycle when comparably permeabilized uninfected cells hemolyse earlier? To address these questions we encoded the multiplicity of factors known to influence host cell volume in a mathematical model of the homeostasis of a parasitized red cell. The predicted volume changes were subjected to thorough experimental tests by monitoring the stage-related changes in the osmotic fragility of infected red cell populations. The results supported the model predictions of biphasic volume changes comprising transient shrinkage of infected cells with young trophozoites followed by continuous volume increase to about 10% lower than the critical hemolytic volume of approximately 150 fL by the end of the asexual cycle. Analysis of these results and of additional model predictions demonstrated that the osmotic stability of infected red cells can be preserved only by a large reduction in impermeant solute concentration within the host cell compartment. Thus, excess hemoglobin consumption represents an essential evolutionary strategy to prevent the premature hemolysis of the highly permeabilized infected red cell.
Interleukin-7 improves CD4 T-cell reconstitution after autologous CD34 cell transplantation in monkeys
Authors: Storek Jan , Gillespy Thurman , Lu Hailing , Joseph Ansamma , Dawson Monja A. , Gough Michael , Morris Julia , Hackman Robert C. , Horn Peter A. , Sale George E. , Andrews Robert G. , Maloney David G. , Kiem Hans-Peter
Published: May 2003

In mice, interleukin-7 (IL-7) hastens T-cell reconstitution and might cause autoimmune diseases, lymphoma, and osteoporosis. We assessed the effect of IL-7 on T-cell reconstitution and toxicity in baboons that underwent total body irradiation followed by autologous transplantation of marrow CD34 cells. Three baboons received placebo and 3 baboons received recombinant human IL-7 (rhIL-7, 75 μg/kg twice a day subcutaneously) between 6 and 10 weeks after transplantation. The mean increase in blood absolute CD4 T-cell counts was 0.9-fold in the placebo-treated animals versus 9.0-fold in those treated with IL-7 (P = .02). The increase observed in the IL-7–treated animals appeared attributable to peripheral expansion rather than de novo generation. The IL-7–treated animals had greater mean increases in the volumes of the spleen (2.0-fold with placebo versus 4.5-fold with IL-7, P = .02) and lymph nodes (1.8-fold with placebo versus 4.1-fold with IL-7,P = .10) but not the thymus (3.4-fold with placebo versus 1.1-fold with IL-7, P = .18). Side effects of IL-7 included thrombocytopenia and possibly neutropenia and hemolytic anemia. One IL-7–treated animal failed to thrive due to a disease resembling graft-versus-host disease. No animals developed lymphoma. Bone density was not decreased. In conclusion, IL-7 raises CD4 T-cell counts in irradiated primates. It remains to be determined whether this is associated with clinical benefit.
Gene expression profiling reveals a highly specialized genetic program of plasma cells
Authors: Underhill Gregory H. , George David , Bremer Eric G. , Kansas Geoffrey S.
Published: May 2003

The formation of terminally differentiated plasma cells represents the critical final step in B-cell differentiation. In this study, utilizing oligonucleotide microarray analysis, we describe the highly specialized genetic profile exhibited by terminally differentiated plasma cells. A total of 1476 known genes were differentially expressed by plasma cells compared with B cells. Plasma cells displayed an up-regulation, induction, or a selective retention of a unique constellation of transcription factors, including members of the AP-1, nuclear factor–κB (NF-κB), nuclear factor of activated T cells (NFAT), and octamer binding factor families. Interestingly, plasma cells also displayed a down-regulation of several RNA polymerase I– related factors, consistent with terminal differentiation, and exhibited a down-regulation of the TATA box binding protein. Furthermore, plasma cells displayed alterations in multiple components of the Wnt and Notch signaling pathways and showed a unique pattern of apoptosis and proliferation-associated genes. Unexpectedly, plasma cells displayed an up-regulation of 2 factors normally associated with microenvironmental positioning of neuronal cells, reelin and neuropilin-1. These results supply insight into the developmental genetics of plasma cell differentiation and provide a foundation for further analysis of plasma cell biology.
Immunoglobulin light chain variable (V) region genes influence clinical presentation and outcome in light chain–associated amyloidosis (AL)
Authors: Abraham Roshini S. , Geyer Susan M. , Price-Troska Tammy L. , Allmer Cristine , Kyle Robert A. , Gertz Morie A. , Fonseca Rafael
Published: May 2003

Light chain–associated amyloidosis (AL) is a plasma cell dyscrasia in which the secreted monoclonal immunoglobulin (Ig) light chains form amyloid fibrils. There is considerable heterogeneity in clinical presentation, and prognosis of the disease relates to the severity of organ dysfunction induced by amyloid deposits. The mechanisms by which the amyloid fibrils are deposited as well as the predilection for specific organ sites have not been clearly elucidated. This study characterizes the repertoire of immunoglobulin light chain variable genes used by the clonal B cell in AL amyloid patients, and the association of light chain variable region (VL) genes with clinical presentation and outcome is assessed in 58 (32 λ and 26 κ) patients. A preferential use of VL germ-line genes was noted for both AL κ and λ patients. There was a significant correlation between the use of the Vλ VI germ-line donor, 6a, and renal involvement as well as the Vλ III gene, 3r, with soft-tissue AL. The use of a biased VL gene repertoire also correlated with clinical outcome, revealing important trends for predicting prognosis. The use of Vλ II germ-line genes was associated with cardiac amyloidosis and affected survival adversely. The presence of multiple myeloma also correlated with a poor prognosis. The presence of renal disease, on the other hand, was associated with improved survival. Therefore, identification of the clonal VL gene in AL has important implications in determining clinical outcome.
Utility of FDG-PET scanning in lymphoma by WHO classification
Authors: Elstrom Rebecca , Guan Liang , Baker Gary , Nakhoda Khozaim , Vergilio Jo-Anne , Zhuang Hongming , Pitsilos Stephanie , Bagg Adam , Downs Lisa , Mehrotra Amit , Kim Scott , Alavi Abass , Schuster Stephen J.
Published: May 2003

We retrospectively evaluated 18fluoro-2-deoxyglucose positron emission tomography (FDG-PET) scans in 172 patients with lymphoma and correlated results with pathologic diagnosis using the World Health Organization (WHO) classification system. In total, FDG-PET detected disease in at least one site in 161 patients (94%) and failed to detect disease in 11 patients (6%). The most frequent lymphoma diagnoses were diffuse large B-cell lymphoma (LBCL; n = 51), Hodgkin lymphoma (HL; n = 47), follicular lymphoma (FL; n = 42), marginal zone lymphoma (MZL; n = 12), mantle cell lymphoma (MCL; n = 7), and peripheral T-cell lymphoma (PTCL; n = 5). FDG-PET detected disease in 100% of patients with LBCL and MCL and in 98% of patients with HL and FL. In contrast, FDG-PET detected disease in only 67% of MZL and 40% of PTCL. Comparison with bone marrow biopsies showed that FDG-PET was not reliable for detection of bone marrow involvement in any lymphoma subtype.
Prognostic significance of cytogenetic clonal evolution in patients with chronic myelogenous leukemia on imatinib mesylate therapy
Authors: Cortes Jorge E. , Talpaz Moshe , Giles Francis , O'Brien Susan , Rios Mary Beth , Shan Jianqin , Garcia-Manero Guillermo , Faderl Stefan , Thomas Deborah A. , Wierda William , Ferrajoli Allessandra , Jeha Sima , Kantarjian Hagop M.
Published: May 2003

Cytogenetic clonal evolution (CE) is a known poor prognostic factor in Philadelphia chromosome–positive chronic myelogenous leukemia (Ph-positive CML). However, its prognostic relevance in the era of imatinib therapy is unknown. We investigated the independent prognostic relevance of CE in 498 patients with Ph-positive CML treated with imatinib for chronic or accelerated phases. One hundred twenty-one patients had CE alone (n = 70) or with other accelerated phase criteria (n = 51). Patients were compared in 4 categories: chronic phase (n = 295), CE only (n = 70), accelerated phase without CE (n = 82), and accelerated phase with CE (n = 51). Statistical methods used established methodologies for univariate and multivariate analyses. In chronic and accelerated phases of CML, CE was not associated with significant differences in major or complete cytogenetic response rates, but it was an independent poor prognostic factor for survival by multivariate analyses in both chronic (P = .005) and accelerated phase (P = .03). Multivariate analyses conducted at the 3-month landmark (including the 3-month cytogenetic response) identified the lack of cytogenetic response at 3 months to be a stronger independent poor prognostic factor for survival than CE for both chronic (major cytogenetic response versus other) and accelerated phase (any cytogenetic response versus other). We conclude that cytogenetic CE is not an important factor for achieving major or complete cytogenetic response with imatinib mesylate therapy, but it is an independent poor prognostic factor for survival in both chronic and accelerated phases of CML. The 3-month cytogenetic response to imatinib mesylate refined the prognostic relevance of such studies in patients on imatinib mesylate therapy.
Patterns of somatic mutations in VH genes reveal pathways of clonal transformation from MGUS to multiple myeloma
Authors: Zojer Niklas , Ludwig Heinz , Fiegl Michael , Stevenson Freda K. , Sahota Surinder S.
Published: May 2003

Monoclonal gammopathy of undetermined significance (MGUS) can transform to multiple myeloma (MM). In myeloma, mutated VHgenes with sequence homogeneity reveal a postfollicular origin. Previously, some MGUS cases showed mutated VH genes with intraclonal variation, indicating an earlier stage of arrest. We investigated progression from 2 of 2 MGUS to MM, in which VH genes confirmed clonal evolution. In one MGUS case, intraclonal heterogeneity was evident, and transformation to myeloma occurred rapidly with apparent homogeneity in the emergent clone. However, residual MGUS-derived sequences were detectable at this time. Heterogeneity in MGUS does not associate with benign disease, but it indicates an origin from a tumorigenic cell, most likely surface immunoglobulin+, undergoing somatic mutation. The remaining case displayed intraclonal homogeneity at the MGUS stage, conceivably resulting from a self-cloning outgrowth from MGUS with heterogeneity. Transformation can occur at either MGUS stage, but it involves a single cell in which somatic mutation is then silent.
ICAM-directed vascular immunotargeting of antithrombotic agents to the endothelial luminal surface
Authors: Murciano Juan-Carlos , Muro Silvia , Koniaris Lauren , Christofidou-Solomidou Melpo , Harshaw David W. , Albelda Steven M. , Granger D. Neil , Cines Douglas B. , Muzykantov Vladimir R.
Published: May 2003

Drug targeting to a highly expressed, noninternalizable determinant up-regulated on the perturbed endothelium may help to manage inflammation and thrombosis. We tested whether inter-cellular adhesion molecule-1 (ICAM-1) targeting is suitable to deliver antithrombotic drugs to the pulmonary vascular lumen. ICAM-1 antibodies bind to the surface of endothelial cells in culture, in perfused lungs, and in vivo. Proinflammatory cytokines enhance anti-ICAM binding to the endothelium without inducing internalization. 125I-labeled anti-ICAM and a reporter enzyme (β-Gal) conjugated to anti-ICAM bind to endothelium and accumulate in the lungs after intravenous administration in rats and mice. Anti-ICAM is seen to localize predominantly on the luminal surface of the pulmonary endothelium by electron microscopy. We studied the pharmacological effect of ICAM-directed targeting of tissue-type plasminogen activator (tPA). Anti-ICAM/tPA, but not control IgG/tPA, conjugate accumulates in the rat lungs, where it exerts plasminogen activator activity and dissolves fibrin microemboli. Therefore, ICAM may serve as a target for drug delivery to endothelium, for example, for pulmonary thromboprophylaxis. Enhanced drug delivery to sites of inflammation and the potential anti-inflammatory effect of blocking ICAM-1 may enhance the benefit of this targeting strategy.
Efficacy and safety of rituximab in type II mixed cryoglobulinemia
Authors: Zaja Francesco , De Vita Salvatore , Mazzaro Cesare , Sacco Stefania , Damiani Daniela , De Marchi Ginevra , Michelutti Angela , Baccarani Michele , Fanin Renato , Ferraccioli Gianfranco
Published: May 2003

The best treatment of type II mixed cryoglobulinemia (MC) has still to be defined. Antiviral treatment for the frequent underlying infectious trigger hepatitis C virus (HCV) may be ineffective, contraindicated, or not tolerated in a fraction of cases, whereas current immunosuppressive treatments may lead to relevant complications. Selective B-cell blockade with rituximab was used in this study, based on favorable results in preliminary experience. Fifteen consecutive patients with type II MC (HCV-related in 12 of 15) were treated with rituximab, 375 mg/m2 intravenously weekly for 4 weeks. Only medium- to low-dose steroids were allowed, if already administered at the time of recruitment. All patients had active disease, poorly controlled or difficult to manage with previous treatments, including corticosteroids in all. Efficacy and safety of rituximab therapy were evaluated in the following 6 months. The overall follow-up after rituximab treatment ranged from 9 to 31 months. Rituximab proved effective on skin vasculitis manifestations (ulcers, purpura, or urticaria), subjective symptoms of peripheral neuropathy, low-grade B-cell lymphoma, arthralgias, and fever. Nephritis of recent onset went into remission in one case. Laboratory features, that is, significantly decreased serum rheumatoid factor and cryoglobulins and increased C4, were consistent with the clinical efficacy. Treatment was well tolerated, with no infectious complications. Thrombosis of retinal artery or self-limiting panniculitis occurred in one patient each. Rituximab may represent a safe and effective alternative to standard immunosuppression in type II MC. Controlled studies are needed to better define drug indications and the cost-efficacy profile in the different systemic manifestations.
Antigen-primed CD8+ T cells can mediate resistance, preventing allogeneic marrow engraftment in the simultaneous absence of perforin-, CD95L-, TNFR1-, and TRAIL-dependent killing
Authors: Komatsu Masanobu , Mammolenti Michele , Jones Monica , Jurecic Roland , Sayers Thomas J. , Levy Robert B.
Published: May 2003

Engraftment failure following allogeneic bone marrow (BM) transplantation is of clinical concern particularly involving T-cell–depleted inoculum and transplantations for aplastic anemia. Immune resistance by lymphoid and natural killer (NK) populations with “barrier” function is well established. Major histocompatibility complex (MHC)–identical marrow allografts were examined to investigate effector pathways in non-NK–mediated resistance. Barrier function was examined in cytotoxic normal and deficient B6 (H-2b) recipients primed to donor minor histocompatibility antigen (MiHA) prior to BM transplantation. Host resistance was sensitively evaluated by colony-forming unit (CFU) assays to directly assess for donor progenitor cell (PC) and peripheral chimerism. B6 host CD8+ T cells but not CD4+ or NK1.1+ cells effected rejection of primitive (CFU-HPP [high-proliferative potential]) and lineage-committed (CFU-IL3/GM [interleukin 3/granulocyte macrophage]) allogeneic donor progenitors. To address complementation by the cytotoxic pathways existing in singly deficient (perforin or FasL) recipients, cytotoxically double (perforin plus FasL) deficient (cdd) recipients were used. Resistance in B6-cdd recipients was comparable to that of wild-type B6 recipients and was also dependent on CD8+ T cells. A “triple” cytotoxic deficient model, involving transplantation of TNFR1−/− (tumor necrosis factor receptor 1) progenitor grafts did not diminish the ability of B6-cdd recipients to reject allografts. Finally, injection of anti-TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) monoclonal antibody (mAb) in B6-cdd recipients also failed to inhibit rejection of TNFR1−/− marrow grafts. In total, these studies demonstrate that CD8+ host T cells can effectively resist MHC-matched MiHA-mismatched donor PCs via alternative effector pathway(s) independent of perforin-, FasL-, TNFR-1–, and TRAIL-dependent cytotoxicity. Therefore, inhibition of these effector pathways in sensitized recipients is unlikely to result in stem cell engraftment following PC allografts.
Continuous absence of metaphase-defined cytogenetic abnormalities, especially of chromosome 13 and hypodiploidy, ensures long-term survival in multiple myeloma treated with Total Therapy I: interpretation in the context of global gene expression
Authors: Shaughnessy John , Jacobson Joth , Sawyer Jeff , McCoy Jason , Fassas Athanasios , Zhan Fenghuang , Bumm Klaus , Epstein Joshua , Anaissie Elias , Jagannath Sundar , Vesole David , Siegel David , Desikan Raman , Munshi Nikhil , Badros Ashraf , Tian Erming , Zangari Maurizio , Tricot Guido , Crowley John , Barlogie Bart
Published: May 2003

Metaphase cytogenetic abnormalities (CAs), especially of chromosome 13 (CA 13), confer a grave prognosis in multiple myeloma even with tandem autotransplantations as applied in Total Therapy I, which enrolled 231 patients between 1989 and 1994. With a median follow-up of almost 9 years, the prognostic implications of all individual CAs, detected prior to treatment and at relapse, were investigated. Among all CAs and standard prognostic factors examined prior to therapy, only hypodiploidy and CA 13 (hypo–13 CA), alone or in combination, were associated with shortest event-free survival and overall survival (OS). The shortest postrelapse OS was observed with hypo–13 CA, which was newly detected in 18 of all 28 patients presenting with this abnormality at relapse. Superior prognosis was associated with the absence of any CA at both diagnosis and relapse (10-year OS, 40%). The lack of independent prognostic implications of other CAs points to a uniquely aggressive behavior of hypo–13 CA (present in 16% of patients at diagnosis). With the use of microarray data in 146 patients enrolled in Total Therapy II, overexpression of cell cycle genes distinguished CA from no CA, especially in cases of del(13) detected by interphase fluorescence in situ hybridization (FISH). FISH 13, resulting in a haploinsufficiency of RB1 and other genes mapping to chromosome 13, as well as activation of IGF1R, appears to have an amplifying effect on cell cycle gene expression, thus providing a molecular explanation for the dire outcome of patients with CA 13 compared with those with other CAs.
Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270
Authors: Ding Zhongren , Kim Soochong , Dorsam Robert T. , Jin Jianguo , Kunapuli Satya P.
Published: May 2003

Human platelets express 2 G protein–coupled nucleotide receptors: the platelet adenosine diphosphate (ADP) receptor coupled to stimulation of phospholipase C (P2Y1) via heterotrimeric guanosine 5-triphosphate (GTP)–binding protein Gq, and the platelet ADP receptor coupled to inhibition of adenylyl cyclase (P2Y12) via heterotrimeric GTP-binding protein Gi. Although these 2 receptors are encoded on the same chromosome and have similar pharmacologic profiles, they have different reactivities toward thiol reagents. The thiol agent p-chloromercuribenzene sulfonic acid (pCMBS) and the active metabolites from antiplatelet drugs, clopidogrel and CS-747, inactivate the P2Y12 receptor and are predicted to interact with the extracellular cysteine residues on the P2Y12 receptor. In this study we identified the reactive cysteine residues on the human P2Y12 receptor by site-directed mutagenesis using pCMBS as the thiol reagent. Cys97Ser and Cys175Ser mutants of the P2Y12 receptor did not express when transfected into Chinese hamster ovary (CHO-K1) cells, indicating the essential nature of a disulfide bridge between these residues. The Cys17Ser, Cys270Ser, and Cys17Ser/Cys270Ser double mutants had similar median effective concentration (EC50) values for ADP and 2-methylthio–ADP (2-MeSADP) when compared with the wild-type P2Y12. Similarly, the median inhibitory concentration (IC50) values for BzATP (2′,3′-O-(4- benzoylbenzoyl) adenosine 5′-triphosphate), an antagonist of the P2Y12 receptor, also did not differ dramatically among these mutants and the wild-type P2Y12receptor. pCMBS inactivated the wild-type P2Y12 receptor in a concentration-dependent manner, whereas it had no effect on the P2Y1 receptor. Finally, pCMBS partially affected the Gi coupling of Cys17Ser or Cys270Ser receptor mutants, but had no effect on Cys17Ser/Cys270Ser P2Y12receptor–mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y1 receptor, which has 2 essential disulfide bridges linking its extracellular domains, the P2Y12 receptor has 2 free cysteines in its extracellular domains (Cys17 and Cys270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS-747 form disulfide bridges with both Cys17 and Cys270 in the P2Y12 receptor, and thereby inactivate the receptor.
Repopulating defect of mismatch repair–deficient hematopoietic stem cells
Authors: Reese Jane S. , Liu Lili , Gerson Stanton L.
Published: May 2003

Mismatch repair deficiency is associated with carcinogenesis, increased spontaneous and induced mutagenesis, and resistance to methylating agents. In humans, leukemias and lymphomas arise in the background of mismatch repair deficiency, raising the possibility that hematopoiesis is abnormal as well. To address hematopoiesis in MSH2–/– mice, we collected marrow and performed serial transplantations of these cells, alone or mixed with wild-type cells, into lethally irradiated healthy mice. Transplant recipients were observed or treated with the methylating agent, temozolomide (TMZ). Methylating agent tolerance was evident by the competitive survival advantage of MSH2–/– marrow progenitors compared with wild-type cells after each TMZ exposure. However, serial repopulation by MSH2–/– cells was deficient compared with wild-type cells. In recipients of mixed populations, the MSH 2–/– cells were lost from the marrow, and mice receiving MSH2–/– cells plus TMZ could not be reconstituted in the third passage, whereas all wild-type cell recipients survived. No differences in telomere length, cell cycle distribution, or homing were observed, but an increase in microsatellite instability was seen in the MSH2–/– early progenitor colony-forming unit (CFU) and Sca+Kit+lin–-derived clones. Thus, mismatch repair deficiency is associated with a hematopoietic repopulation defect and stem cell exhaustion because of accumulation of genomic instability.
Neonatal or hepatocyte growth factor–potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B
Authors: Xu Lingfei , Gao Cuihua , Sands Mark S. , Cai Shi-Rong , Nichols Timothy C. , Bellinger Dwight A. , Raymer Robin A. , McCorquodale Stephanie , Ponder Katherine Parker
Published: May 2003

Hemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus–based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 μg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.