bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformation of ECOWAS as a Security Apparatus and Its Implications in Ghana's Political Orientation, 1990-2000
Authors: Dapaah-Agyemang Joshua
Published: March 2003

The transformation of the Economic Community of West African States (ECOWAS) from an economic integration scheme to a political security scheme and its implications in Ghana political orientation was born out of the unanticipated changes of the post-1990s sub-regional civil conflicts and especially after the end of the Cold War. This history gave Ghana an opportunity within ECOWAS' transformation from economic integration to politico-security organization. In this connection, Ghana's foreign policy took a new turn affecting the whole West African sub-region that was precipitated partly by the Liberian civil war. My principal argument is that despite Ghana's adoption of purposeful isolationism in the early 1980s, the followed trends of events of Ghana foreign policy, at least on the sub-regional level, is a reflection of internal and external factors such as the transformation of ECOWAS security apparatus due to conflicts in some member states, but not the leadership style of the government. Therefore, in order to apprehend the reason behind the dynamics of Ghana's foreign policy change and adjustment, in particular Ghana's strategies and perception of its interest in the sub-regional level, one has to consider a number of crucial factors such as the political and economic milieu in which ECOWAS is engaged, vis-à-vis Ghana's government actions.
The Untold and Alternate Story of the Indian Subcontinent's War of Independence
Authors: Ali Tahseen
Published: March 2003

When the Indian subcontinent became independent in August 1947, it marked the end of the foreign occupation of the largest country in the world. Renowned for his part in that long struggle for independence was the famous Mahatma ('Great-souled one') Mohandas K. Gandhi, the proponent of non-violence, and his western-educated disciple, Jawaharlal Nehru. Gandhi was said to have charmed the British with his strength and simplicity and compel them into withdrawing from the subcontinent. Yet against the background of Gandhi's famous struggle whispers of another movement were heard, complete with its own leaders and its own vision on the fight for freedom. This paper takes a closer look at that struggle, and its efficacy in the quest for Indian independence. What were its goals and its guiding principles? How did it compare with Gandhi's struggle? This is the untold and alternate story of the Indian subcontinent's war of independence, and the men and women whose sacrifices created an immortal saga of patriotism.
The Lomé Convention from Inception to the Dynamics of the Post-Cold War, 1957-1990s
Authors: Montana Ismael Musah
Published: March 2003

From the early 1960s through the late 1980s, Lomé Convention, the chief achievement of Euro-African Caribbean and Pacific (ACP) countries' entente, has been an interdependent form of partnership that has offered ACP states a privileged position in the European Economic Commission's Market. Although considered a cornerstone and model for Europe's North-South economic cooperation, changes that occurred in the aftermath of the Cold War had drastic effects on the nature of this historic partnership. In the period between 1989 and 1995, profound changes occurred in international relations following the end of the Cold War, followed by the subsequent liberalization of East European states' economies, the creation of the Euro-Mediterranean partnership, and the restructuring of Europe's internal as well as external policies, in part, affected the ACP's privileged position in the European Union. The concept of Cold War context used in this article will be narrower (economic implications) rather than that commonly employed in the study of superpower rivalry. The framework employed throughout the paper is a conceptual and critical survey of the Lomé Convention's history, from its inception to the changing dynamics of the post Cold-War world. The paper critically examines the divergence of interpretations of the relevance and obsolescence of the Convention in the post-Cold War context. "The World is changing. It has changed for the ACP States; it will change for the Community; it is changing all around us."
A promoter mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causes X-linked sideroblastic anemia
Authors: Bekri Soumeya , May Alison , Cotter Philip D. , Al-Sabah Ala I. , Guo Xiaojun , Masters Gillian S. , Bishop David F.
Published: April 2003

X-linked sideroblastic anemia (XLSA) is caused by mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). XLSA was diagnosed in a 32-year-old woman with a mild phenotype and moderately late onset. Pyridoxine therapy had no effect in the proband, but in her affected son engendered a modest increase in hemoglobin concentration and a 4-fold reduction in ferritin iron. Molecular analysis identified a C to G transversion at nucleotide -206 from the transcription start site, as defined by primer extension, in the proximal promoter region of ALAS2. No other mutations were found in the promoter region, the flanking intronic sequences, the exons, or the 3′ genomic region. The same mutation was found in her affected son but not in any other of her unaffected relatives. The mutation resulted in a 94% loss of activity relative to the wild-type sequence for a luciferase reporter construct containing the proximal 293 nucleotides (nt's) of the ALAS2 promoter when transfected into human erythroid K562 cells. Confirming the mutation's deleterious effect, the ALAS2 mRNA level in the proband's erythroid precursors was reduced 87%. The mutation occurred in or near 3 different putative transcription factor binding sites of unknown erythroid importance. The dramatic decreases in reporter activity and mRNA level suggest that the region of the mutation may bind a novel and important erythroid regulatory element.
Presence of the BCR-ABL mutation Glu255Lys prior to STI571 (imatinib) treatment in patients with Ph+ acute lymphoblastic leukemia
Authors: Hofmann Wolf-Karsten , Komor Martina , Wassmann Barbara , Jones Letetia C. , Gschaidmeier Harald , Hoelzer Dieter , Koeffler H. Phillip , Ottmann Oliver G.
Published: April 2003

The tyrosine kinase inhibitor STI571 (imatinib) binds competitively to the adenosine triphosphate (ATP) binding site of the ABL kinase, thereby inhibiting auto- and substrate phosphorylation of the oncogenic protein BCR-ABL and preventing the activation of downstream signaling pathways. Comparative studies on leukemic cell samples obtained from chronic myelogenous leukemia (CML) and Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) patients before and after treatment with STI571 reported point mutations in resistant samples after a short time of therapy. The aim of this study was to determine whether patients with Ph+ ALL in whom resistance developed as a consequence of the Glu255Lys mutation already harbored this subclone prior to STI571 treatment. First, the migration pattern of cDNAs from 30 bone marrow samples from patients with Ph+ ALL was analyzed by polymerase chain reaction–single strand conformation polymorphism (PCR-SSCP). Thereafter, detailed mutational analysis using genomic DNA was performed on initial STI571-naive bone marrow samples of 4 individuals with Ph+ ALL, for whom the mutation Glu255Lys in association with STI571 treatment had been shown. A 166-bp PCR fragment spanning from nucleotide (nt) 862 to nt 1027 was cloned, and 108 clones per sample were analyzed by direct sequencing. This more sensitive technique revealed the presence of the Glu255Lys mutation in 2 initial samples, one clone each. We identified for the first time the mutation Glu255Lys in STI571-naive leukemic samples of Ph+ ALL patients. The findings suggest that the mutation exists in a very small subpopulation of leukemic cells at the beginning of STI571 therapy.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces antiapoptotic and proapoptotic signals in acute myeloid leukemia
Authors: Faderl Stefan , Harris David , Van Quin , Kantarjian Hagop M. , Talpaz Moshe , Estrov Zeev
Published: April 2003

High levels of cytokines are associated with a poor prognosis in acute myeloid leukemia (AML). However, cytokines may induce, on one hand, survival factor expression and cell proliferation and, on the other hand, expression of inhibitory signals such as up-regulation of suppressors of cytokine signaling (SOCS) and induce apoptotic cell death. Because blasts from patients with AML express high procaspase protein levels, we asked whether granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances procaspase protein production in AML cells. In the GM-CSF–responsive OCIM2 AML cell line, GM-CSF induced signal transducer and activator of transcription 5 (Stat 5) phosphorylation, up-regulated cyclin D2, and stimulated cell cycle progression. Concurrently, GM-CSF stimulated expression of SOCS-2 and -3 and of procaspases 2 and 3 and induced caspase 3 activation, poly(ADP[adenosine 5′-diphosphate]-ribose) polymerase (PARP) cleavage, and apoptotic cell death. The Janus kinase (Jak)–Stat inhibitor AG490 abrogated GM-CSF–induced expression of procaspase 3 and activation of caspase 3. Under the same conditions GM-CSF up-regulated production of BAX as well as Bcl-2, Bcl-XL, survivin, and XIAP. GM-CSF also increased procaspase 3 protein levels in OCI/AML3 and Mo7e cells, suggesting that this phenomenon is not restricted to a single leukemia cell line. Our data suggest that GM-CSF exerts a dual effect: it stimulates cell division but contemporaneously up-regulates Jak-Stat–dependent proapoptotic proteins. Up-regulation of procaspase levels in AML is thus a beacon for an ongoing growth-stimulatory signal.
Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation
Authors: Allsopp Richard C. , Morin Gregg B. , DePinho Ronald , Harley Calvin B. , Weissman Irving L.
Published: April 2003

Telomere shortening ultimately limits the replicative life span of cultured human somatic cells. Telomeres also shorten during replicative aging in vivo in hematopoietic cells, including early hematopoietic progenitors and hematopoietic stem cells (HSCs), from humans and mice, despite readily detectable levels of telomerase in these cells. To assess the relevance of telomerase to the long-term replicative capacity of HSCs in vivo, we serially transplanted HSCs from wild-type and telomerase-deficient mice until exhaustion and monitored telomere length in HSCs during this process. Telomerase-deficient HSCs could be serially transplanted for only 2 rounds, whereas wild-type HSCs could be serially transplanted for at least 4 rounds. Furthermore, the rate of telomere shortening was increased approximately 2-fold during serial transplantation of telomerase-deficient HSCs. These findings suggest that one role for telomerase in the HSC is to partially counter the rate of telomere shortening during division of HSCs, thereby preventing premature loss of telomere function and providing added replicative capacity.
Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation
Authors: Mielcarek Marco , Martin Paul J. , Leisenring Wendy , Flowers Mary E. D. , Maloney David G. , Sandmaier Brenda M. , Maris Michael B. , Storb Rainer
Published: April 2003

It is unknown whether the severity, timing, and quality of graft-versus-host disease (GVHD) may be different after nonmyeloablative as compared with myeloablative hematopoietic stem cell transplantation (HSCT). Therefore, GVHD incidence, morbidity of skin, liver, and gut, requirements for immunosuppressive therapy, and survival were retrospectively analyzed in 44 patients who underwent nonablative HSCT and 52 who underwent ablative HSCT (median ages, 56 and 54 years, respectively). The nonablative transplantation regimen consisted of low-dose total body irradiation (TBI), preceded in some patients by fludarabine administration and followed in all patients by immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP). Those who underwent myeloablative HSCT were prepared with different TBI- and non-TBI–containing regimens and received CSP plus methotrexate or MMF for GVHD prophylaxis. The cumulative incidence of grades II-IV acute GVHD was lower after nonablative transplantation (64% vs 85%; P = .001), but there were no differences in the cumulative incidence of chronic GVHD requiring treatment (73% vs 71%; P = .96). Nonablative transplantation was associated with the delayed initiation of steroid treatment for GVHD (0.95 months vs 3.0 months; P < .001) and with the use of fewer systemic immunosuppressants in the first 3 months after transplantation (P ≤ .04). This corresponded to more prevalent skin and more severe gut morbidity 6 to 12 months after nonablative transplantation. Our results show that nonablative HSCT is associated with a syndrome of acute GVHD occurring after day 100 in many patients. This “late-onset acute GVHD” should be taken into consideration in the design of prospective studies comparing GVHD resulting from the two types of transplantation procedures.
Early changes in gene expression profiles of hepatic GVHD uncovered by oligonucleotide microarrays
Authors: Ichiba Tamotsu , Teshima Takanori , Kuick Rork , Misek David E. , Liu Chen , Takada Yuichiro , Maeda Yoshinobu , Reddy Pavan , Williams Debra L. , Hanash Samir M. , Ferrara James L. M.
Published: April 2003

The liver, skin, and gastrointestinal tract are major target organs of acute graft-versus-host disease (GVHD), the major complication of allogeneic bone marrow transplantation (BMT). In order to gain a better understanding of acute GVHD in the liver, we compared the gene expression profiles of livers after experimental allogeneic and syngeneic BMT using oligonucleotide microarray. At 35 days after allogeneic BMT when hepatic GVHD was histologically evident, genes related to cellular effectors and acute-phase proteins were up-regulated, whereas genes largely related to metabolism and endocrine function were down-regulated. At day 7 after BMT before the development of histologic changes in the liver, interferon γ (IFN-γ)–inducible genes, major histocompatibility (MHC) class II molecules, and genes related to leukocyte trafficking had been up-regulated. Immunohistochemistry demonstrated that expression of IFN-γ protein itself was increased in the spleen but not in hepatic tissue. These results suggest that the increased expression of genes associated with the attraction and activation of donor T cells induced by IFN-γ early after BMT is important in the initiation of hepatic GVHD in this model and provide new potential molecular targets for early detection and intervention of acute GVHD.
Oxidative stress is a critical mediator of the angiotensin II signal in human neutrophils: involvement of mitogen-activated protein kinase, calcineurin, and the transcription factor NF-κB
Authors: El Bekay Rajaa , Álvarez Moisés , Monteseirín Javier , Álba Gonzalo , Chacón Pedro , Vega Antonio , Martín-Nieto José , Jiménez Juan , Pintado Elízabeth , Bedoya Francisco J. , Sobrino Francisco
Published: April 2003

Neutrophils are mobilized to the vascular wall during vessel inflammation. Published data are conflicting on phagocytic nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activation during the hypertensive state, and the capacity of angiotensin II (Ang II) to modulate the intracellular redox status has not been analyzed in neutrophils. We here describe that Ang II highly stimulates endogenous and extracellular O2- production in these cells, consistent with the translocation to the cell membrane of the cytosolic components of NADPH oxidase, p47phox, and p67phox. The Ang II–dependent O2- production was suppressed by specific inhibitors of AT1 receptors, of the p38MAPK and ERK1/2 pathways, and of flavin oxidases. Furthermore, Ang II induced a robust phosphorylation of p38MAPK, ERK1/2, and JNK1/2 (particularly JNK2), which was hindered by inhibitors of NADPH oxidase, tyrosine kinases, and ROS scavengers. Ang II increased cytosolic Ca2+ levels—released mainly from calcium stores—enhanced the synthesis de novo and activity of calcineurin, and stimulated the DNA-binding activity of the transcription factor NF-κB in cultured human neutrophils. Present data demonstrate for the first time a stimulatory role of Ang II in the activation of phagocytic cells, underscore the relevant role of ROS as mediators in this process, and uncover a variety of signaling pathways by which Ang II operates in human neutrophils.
Ontogeny and innate properties of neonatal dendritic cells
Authors: Sun Cheng-Ming , Fiette Laurence , Tanguy Myriam , Leclerc Claude , Lo-Man Richard
Published: April 2003

We investigated whether a developmental immaturity of the dendritic cells (DCs) compartment could contribute to the high susceptibility to infections observed in newborns. DCs are among the first cells to colonize the spleen, but the ontogeny of DC subsets follows distinct steps. At birth, plasmacytoid DCs and CD4-CD8α- DCs are found in the spleen, whereas CD8α+ and CD4+ DCs are not present. Then, the CD8α+ DC compartment quickly develops and reaches an adult size by day 7, whereas the CD4+ DC compartment slowly increases to become predominant by the age of 3 weeks. The production of interleukin (IL)–12p70 by DCs is particularly efficient after birth, reflecting the stronger capacity of the neonatal CD8α- DCs to secrete IL-12 compared with its adult counterpart. Like-wise, neonatal DCs produced type I and II interferons. In vivo, following microbial stimulation, up-regulation of major histocompatibility complexes (MHCs) and of costimulatory molecules on DCs was induced clearly showing the activation of neonatal DCs in the neonatal environment. Therefore, despite a markedly different DC subset composition in early life compared with the adult DC compartment, neonatal DCs are fully competent in their innate immune functions.
Fibrinogen gamma-chain splice variant γ′ alters fibrin formation and structure
Authors: Cooper Amy V. , Standeven Kristina F. , Ariëns Robert A. S.
Published: March 2003

Fibrinogen γA/γ′ results from alternative splicing of mRNA. This variant, which constitutes approximately 8% to 15% of plasma fibrinogen, contains FXIII and thrombin binding sites. Our objective was to investigate whether γA/γ′ differs in fibrin formation and structure from the more common variant γA/γA. Both variants were separated and purified by anion-exchange chromatography. Fibrin formation and clot structure of the variants and unfractionated fibrinogen were investigated by turbidity and scanning electron microscopy (SEM). Thrombin cleavage of fibrinopeptides was analyzed by high-performance liquid chromatography (HPLC). Turbidity analysis showed significantly altered polymerization rates and overall fiber thickness in γA/γ′ clots compared with γA/γA and unfractionated fibrinogen. This finding was consistent with a range of thrombin concentrations. HPLC demonstrated reduced rates of fibrinopeptide B (FpB) release from γA/γ′ fibrinogen compared with γA/γA. Delayed FpB release was associated with delayed lateral aggregation of protofibrils and significant differences were found on SEM, with γA/γ′ clots consisting of smaller diameter fibers and increased numbers of branch points compared with both γA/γA and unfractionated fibrinogen. These results demonstrate that the γA/γ′ splice variant of fibrinogen directly alters fibrin formation and structure, which may help to explain the increased thrombotic risk associated with this variant.
Gene expression profiling of plasma cells and plasmablasts: toward a better understanding of the late stages of B-cell differentiation
Authors: Tarte Karin , Zhan Fenghuang , De Vos John , Klein Bernard , Shaughnessy John
Published: April 2003

Plasma cells (PCs), the end point of B-cell differentiation, are a heterogeneous cell compartment comprising several cell subsets from short-lived highly proliferative plasmablasts to long-lived nondividing fully mature PCs. Whereas the major transcription factors driving the differentiation of B cells to PCs were recently identified, the subtle genetic changes that underlie the transition from plasmablasts to mature PCs are poorly understood. We recently described an in vitro model making it possible to obtain a large number of cells with the morphologic, phenotypic, and functional characteristics of normal polyclonal plasmablastic cells (PPCs). Using Affymetrix microarrays we compared the gene expression profiles of these PPCs with those of mature PCs isolated from tonsils (TPCs) and bone marrow (BMPCs), and with those of B cells purified from peripheral blood (PBB cells) and tonsils (TBCs). Unsupervised principal component analysis clearly distinguished the 5 cell populations on the basis of their differentiation and proliferation status. Detailed statistical analysis allowed the identification of 85 PC genes and 40 B-cell genes, overexpressed, respectively, in the 3 PC subsets or in the 2 B-cell subsets. In addition, several signaling molecules and antiapoptotic proteins were found to be induced in BMPCs compared with PPCs and could be involved in the accumulation and prolonged survival of BMPCs in close contact with specialized stromal microenvironment. These data should help to better understand the molecular events that regulate commitment to a PC fate, mediate PC maintenance in survival niches, and could facilitate PC immortalization in plasma cell dyscrasias.
Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites
Authors: Chaussabel Damien , Semnani Roshanak Tolouei , McDowell Mary Ann , Sacks David , Sher Alan , Nutman Thomas B.
Published: April 2003

Monocyte-derived dendritic cells (DCs) and macrophages (Mϕs) generated in vitro from the same individual blood donors were exposed to 5 different pathogens, and gene expression profiles were assessed by microarray analysis. Responses to Mycobacterium tuberculosis and to phylogenetically distinct protozoan (Leishmania major, Leishmania donovani, Toxoplasma gondii) and helminth (Brugia malayi) parasites were examined, each of which produces chronic infections in humans yet vary considerably in the nature of the immune responses they trigger. In the absence of microbial stimulation, DCs and Mϕs constitutively expressed approximately 4000 genes, 96% of which were shared between the 2 cell types. In contrast, the genes altered transcriptionally in DCs and Mϕs following pathogen exposure were largely cell specific. Profiling of the gene expression data led to the identification of sets of tightly coregulated genes across all experimental conditions tested. A newly devised literature-based clustering algorithm enabled the identification of functionally and transcriptionally homogenous groups of genes. A comparison of the responses induced by the individual pathogens by means of this strategy revealed major differences in the functionally related gene profiles associated with each infectious agent. Although the intracellular pathogens induced responses clearly distinct from the extracellular B malayi, they each displayed a unique pattern of gene expression that would not necessarily be predicted on the basis of their phylogenetic relationship. The association of characteristic functional clusters with each infectious agent is consistent with the concept that antigen-presenting cells have prewired signaling patterns for use in the response to different pathogens.
The proapoptotic factor Nix is coexpressed with Bcl-xL during terminal erythroid differentiation
Authors: Aerbajinai Wulin , Giattina Mara , Lee Y. Terry , Raffeld Mark , Miller Jeffery L.
Published: April 2003

Transcriptional profiles of cultured primary human erythroid cells were examined to identify those genes involved in the control of erythroid growth during the terminal phase of maturation. Our in silico screening strategy indicated that a hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during erythropoiesis. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than reticulocytes. Polymerase chain reaction (PCR)–based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we propose that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation.
Sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor
Authors: Grundler Rebekka , Thiede Christian , Miething Cornelius , Steudel Christine , Peschel Christian , Duyster Justus
Published: April 2003

Activating mutations of FLT3 have been detected in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: internal tandem duplication (ITD) of sequences coding for the juxtamembrane domain and point mutations at codon 835 (Asp835) within the kinase domain. Both types of mutations constitutively activate the tyrosine kinase activity of FLT3 in experimental systems and result in factor-independent proliferation of Ba/F3 and 32D cells. Recently, novel mutations within the activation loop were identified in patients with AML: deletion of isoleucine 836 (Ile836del) and an exchange of isoleucine 836 to methionine plus an arginine insertion (Ile836Met+Arg). To examine whether the Ile836 mutations result in constitutive activation of the FLT3 receptor, we introduced both mutant FLT3 cDNAs transiently into HEK 293 cells. Both mutant FLT3 receptors were constitutively autophosphorylated in the absence of ligand and kinase activity led to constitutive activation of downstream signaling cascades as determined by activation of the STAT5 (signal transducer and activator of transcription 5) pathway. When stably expressed in the growth factor–dependent cell lines Ba/F3 and 32D, both deletion and insertion mutants led to factor-independent proliferation, indicating that both mutants have transforming capabilities. We then examined the sensitivity of the FLT3 ITD, FLT3 Asp835Tyr, and the novel FLT3 receptor mutants toward the kinase inhibitors AG1296, PKC412, and SU5614. We show that these FLT3 kinase inhibitors have distinct inhibitory potencies against different activating FLT3 receptor mutants. These results suggest that it may be useful to determine the exact kind of FLT3 mutation when applying receptor kinase inhibitors in clinical trials.
Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion
Authors: Watkins Nicholas A. , Du Lily M. , Scott J. Paul , Ouwehand Willem H. , Hillery Cheryl A.
Published: April 2003

The enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.
A novel missense mutation in the γ-glutamylcysteine synthetase catalytic subunit gene causes both decreased enzymatic activity and glutathione production
Authors: Hamilton David , Wu Jian Hui , Alaoui-Jamali Moulay , Batist Gerald
Published: April 2003

γ-Glutamylcysteine synthetase (γ-GCS) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis: the adenosine triphosphate (ATP)–dependent ligation of glutamate and cysteine. γ-GCS consists of a catalytic (γ-GCSH) and modifier (γ-GCSL) subunit. Hereditary deficiency of γ-GCS has been reported in a small number of patients and is associated with low erythrocyte levels of γ-GCS and GSH leading to hemolytic anemia. Here we report a novel γ-GCSH mutation, isolated from the cDNA of 2 related patients diagnosed with γ-GCS deficiency. Each was found to be homozygous for a C>T missense mutation at nucleotide 379, encoding for a predicted Arg127Cys amino acid change. Computerized structure modeling identified that the mutated amino acid lies within a cleft on the protein surface of γ-GCSH, and the border of this cleft was shown to contain Cys249, an evolutionarily conserved residue that has been proven to lie near the binding site of γ-GCSH. Transfection studies showed that the mutation is associated with decreased GSH production, and binding studies using purified recombinant protein showed that the mutant protein has markedly decreased enzymatic activity compared to wild type.
Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation
Authors: Arber Caroline , BitMansour Andrew , Sparer Timothy E. , Higgins John P. , Mocarski Edward S. , Weissman Irving L. , Shizuru Judith A. , Brown Janice M. Y.
Published: April 2003

Lymphoid deficiency after allogeneic hematopoietic cell transplantation (HCT) results in increased susceptibility to infection; however, transplantation of mature lymphocytes frequently results in a serious complication known as graft-versus-host disease (GVHD). Here we demonstrate in mice that both congenic as well as allogeneic transplantation of low numbers of highly purified common lymphoid progenitors (CLPs)—a rare population of lymphoid-lineage-committed bone marrow cells—accelerates immune reconstitution after lethal irradiation and rescue with hematopoietic stem cells (HSCs). After congenic transplantation, 3 × 103 CLPs protected against murine cytomegalovirus (MCMV) infection at a level roughly equivalent to 107 unfractionated lymph node cells. In the allogeneic model of matched unrelated donor HSC transplantation, cotransplantation of 3 × 103 CLPs protected thymus-bearing as well as thymectomized hosts from MCMV infection and attenuated disease severity. Immunohistochemistry in combination with antibody depletion of T and natural killer (NK) cells confirmed that CLP-derived as well as residual host lymphocytes contribute to antiviral protection. Importantly, transplantation of allogeneic CLPs provided a durable antiviral immunity without inducing GVHD. These data support the potential for composing grafts with committed progenitors to reduce susceptibility to viral infection following HCT.
Phase 2 study of rituximab in the treatment of cladribine-failed patients with hairy cell leukemia
Authors: Nieva Jorge , Bethel Kelly , Saven Alan
Published: April 2003

Hairy cell leukemia (HCL) is an indolent B-cell neoplasm, strongly expressing CD20. Despite initial very high response rates following cladribine, many patients (pts) ultimately relapse. Having relapsed after prior treatment with cladribine, 24 HCL pts (21 male, 3 female) with a median age of 53.5 years were treated with rituximab at 375 mg/m2 intravenously weekly for 4 weeks. Of the pts, 3 (13%) achieved complete remissions and 3 (13%), partial responses. Thus, 6 (25%) of 24 pts achieved a response following rituximab. At a median follow-up of 14.6 months, 2 responders have relapsed; median time to relapse was not yet reached. The only grade III or IV toxicities demonstrated were culture-negative febrile neutropenia, transient and reversible disseminated intravascular coagulation related to rituximab administration, and a diverticular abscess, each in single patients. Of 18 nonresponders, 9 pts subsequently received other treatments; 5 pts were retreated with cladribine, 3 underwent splenectomy, and 1 received pentostatin. Follow-up data are available on 7 of these 9 patients; all 7 patients achieved improvements in hematologic parameters. Rituximab, administered at this dose and schedule, has only modest single-agent activity in cladribine-failed HCL patients when compared with other agents active in this disease.
The minor histocompatibility antigen HA-3 arises from differential proteasome–mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein
Authors: Spierings Eric , Brickner Anthony G. , Caldwell Jennifer A. , Zegveld Suzanne , Tatsis Nia , Blokland Els , Pool Jos , Pierce Richard A. , Mollah Sahana , Shabanowitz Jeffrey , Eisenlohr Laurence C. , Van Veelen Peter , Ossendorp Ferry , Hunt Donald F. , Goulmy Els , Engelhard Victor H.
Published: April 2003

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)–identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr→Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.
Impaired thymic negative selection causes autoimmune graft-versus-host disease
Authors: Teshima Takanori , Reddy Pavan , Liu Chen , Williams Debra , Cooke Kenneth R. , Ferrara James L. M.
Published: April 2003

Animal models with impaired thymic negative selection do not always cause autoimmune diseases despite the development of an autoreactive T-cell repertoire. We investigated the requirements for the de velopment of systemic autoimmune disease by using bone marrow chimeras that lacked expression of major histocompatibility complex (MHC) class II on thymic antigen-presenting cells (APCs), leading to impaired negative selection. We found that impaired negative selection mediated by absence of MHC class II, but not MHC class I, permitted the development of systemic autoimmune disease that is indistinguishable from acute graft-versus-host disease (GVHD). Thymectomy prevented disease, confirming the causal association of the thymus with its development. Adoptive transfer of CD4+ T cells caused GVHD in secondary hosts only when they were irradiated, and cotransfer of peripheral CD4+ and CD8+ T cells from naive mice prevented the disease. These results demonstrate that impaired thymic negative selection can cause lethal autoimmune disease indistinguishable from acute GVHD in the context of a proinflammatory milieu when peripheral regulatory mechanisms are absent.
Successful treatment of murine β-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells
Authors: Persons Derek A. , Allay Esther R. , Sawai Nobukuni , Hargrove Phillip W. , Brent Thomas P. , Hanawa Hideki , Nienhuis Arthur W. , Sorrentino Brian P.
Published: April 2003

Successful gene therapy of β-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of β-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer. We used an oncoretroviral vector to transfer a methylguanine methyltransferase (MGMT) drug-resistance gene into normal bone marrow cells. These cells were transplanted into β-thalassemic mice given nonmyeloablative pretransplantation conditioning with temozolomide (TMZ) and O6-benzylguanine (BG). A majority of mice receiving 2 additional courses of TMZ/BG demonstrated in vivo selection of the drug-resistant cells and amelioration of anemia, compared with untreated control animals. These results were extended using a novel γ-globin/MGMT dual gene lentiviral vector. Following drug treatment, normal mice that received transduced cells had an average 67-fold increase in γ-globin expressing red cells. These studies demonstrate that MGMT-based in vivo selection may be useful to increase genetically corrected cells to therapeutic levels in patients with β-thalassemia.
The cognitive performance of patients with multiple sclerosis during periods of high and low fatigue
Authors: Parmenter Brett A , Denney Douglas R , Lynch Sharon G
Published: March 2003

The objective of this study was to examine whether multiple sclerosis (MS)-related fatigue affects patients’ cognitive performance. Thirty patients who had substantial fatigue in conjunction with MS and who reported marked diurnal variability in the severity of their fatigue were tested on two occasions: during a period of high fatigue and during a period of relatively low fatigue. The order of these sessions was counterbalanced across patients. During both sessions, patients completed a questionnaire concerning their present state of fatigue and a battery of neuropsychological tests of planning, selective attention, and paired associate learning. A lthough patients confirmed greater fatigue during the period of high fatigue and felt they had performed more poorly during this period, there were no differences in cognitive performance that could be attributed to fatigue. Instead, all subjects showed improvement from the first to the second session regardless of whether the latter entailed a period of high or low fatigue. In contrast to studies reporting fatigue-related declines in MS patients’ cognitive performance, no differences in performance were found when MS patients were tested during periods of high versus low fatigue. These contrasting results, stemming from differences in experimental design, are discussed in terms of their implications for assessing cognitive function in patients with MS.
Epilepsia partialis continua as a first symptom of multiple sclerosis: electrophysiological study of one case
Authors: Striano P , Striano S , Carrieri P B , Boccella P
Published: March 2003

Rationale: The prevalence of epilepsy in people with multiple sclerosis (MS) is higher than in the general population. Sometimes seizures are among the first symptoms and can be unusual in their semiology. In rare cases a long-lasting focal somatomotor status {i.e., epilepsia partialis continua (EPC)} has been reported. Case report: A 21-year-o ld male patient presented with a clinical picture of EPC as a first symptom of MS at age of 19. A neurophysiological study agreed with a cortical origin of myoclonic jerks. Conclusions: MS should be considered a rare but possible aetiology of EPC in adults.
Analyses of nursing home residents with multiple sclerosis and depression using the Minimum Data Set
Authors: Buchanan Robert J , Wang Suojin , Tai-Seale Ming , Ju Hyunsu
Published: March 2003

Depression is the most common psychiatric condition among people with multiple sclerosis (MS). A total of 14 009 people with MS at admission to a nursing facility were analyzed using the Minimum Data Set and 36% also had depression. This study developed profiles of nursing home residents with MS who also had depression and compared them with other residents with MS. MS residents with depression were significantly more likely to be female and younger than other MS residents, with significant racial differences as well. MS residents with depression were significantly more likely than other MS residents to have a history of mental health conditions, exhibit mood indicators, and have unsettled relationships. Both groups of MS residents had high levels of physical disability, although MS residents with depression tended to be slightly less disabled. MS residents with depression were more likely than other MS residents to experience daily pain and more likely to have the diseases common to all residents with MS. This research found that most MS residents with depression did not receive mental health services, demonstrating that nursing facilities must improve the mental healthcare provided to residents with MS with depression.
Can the Expanded Disability Status Scale be assessed by telephone?
Authors: Lechner-Scott J , Kappos L , Hofman M , Polman C H , Ronner H , Montalban X , Tintore M , Frontoni M , Buttinelli C , Amato M P , Bartolozzi M L , Versavel M , Dahlke F , Kapp J-F , Gibberd R
Published: March 2003

Information from patients who are unable to continue their visits to a study centre may be of major importance for the interpretatio n of results in multiple sclerosis (MS) clinical trials. To validate a questionnaire based on the Expanded Disability Status Scale (EDSS), patients in five different European centres were assessed independently by pairs of trained EDSS raters, first by telepho ne interview and a few days later by standardized neurological examination. Seventy women and 40 men with an average age of 43.7 years (range 19 -74 years) were included in the study. Mean EDSS score at the last visit was 4.5 (0 -9). EDSS assessment by telepho ne was highly correlated with the EDSS determined by physical examination (Pearson’s correlation coefficient -0.95). A n intraclass correlation coefficient (IC C) of 94.8% was found for the total sample; 77.6% and 86%, respectively, for patients with EDSSB-4.5 (n -46) and \-4.5 (n -64). Kappa values for full agreement were 0.48; for variation by -0.5 steps and -1.0 steps, 0.79 and 0.90, respectively. Best agreement could be found in higher EDSS scores, where assessment by telepho ne interview might be needed most. The telepho ne questionnaire is a valid tool to assess EDSS score in cases where the patient is unable to continue visiting a study centre or in long-term follow-up of trial participants.
Psychometric evaluation of the C hicago Multiscale Depression Inventory in multiple sclerosis patients
Authors: Chang C-H , Nyenhuis D L , Cella D , Luchetta T , Dineen K , Reder A T
Published: March 2003

Objective: The C hicago Multiscale Depression Inventory (CMDI), a comprehensive self-report measure of depression, has proven useful in the assessment of patients with chronic medical conditions. The objective of this study was to determine the validity of the C MDI in multiple sclerosis (MS) patients and explore the nature of depressive symptoms reported by people with MS. Method: Using a combined classical and modern psychometric appro ach, the factor structure of responses in MS patients was compared with that of a normative sample to confirm meaningful subtypes of depression in MS (mood, depressive cognition, and vegetative symptoms). Patient groups also were compared by disease severity to evaluate differences in depression associated with differences in disease severity. Results: The results supported the factor structures of the measure, which was internally consistent, reliable, and factorially valid. Some items function differently in MS patients when compared with depressed patients and normals, offering a further opportunity to understand the unique clinical aspects of depression in people with MS compared with those without a concurrent physical illness. Conclusions: Use of the C MDI to assess separate dimensions of depression may help to clarify the complex interrelationships among aspects of depression and health-related behavior.
Clinical presentation of primary progressive multiple sclerosis 10 years after the incidental finding of typical magnetic resonance imaging brain lesions
Authors: McDonnell G V , Cabrera-Gomez J , Calne D B , Li D KB , Oger J
Published: March 2003

Background: Subclinical multiple sclerosis (MS) has been identified incidentally at autopsy; apparently unaffected individuals with an affected twin have demonstrated magnetic resonance imaging (MRI) changes consistent with MS, and ‘MRI relapses’ are several times more common than clinical relapses. Case description: A 39-year-o ld, right-handed man underwent MRI and PET scanning in 1986 as a ‘normal’ control in a Parkinson’s disease study, where his father was the proband. MRI indicated multiple areas of abnormal signal intensity in a periventricular and grey -white matter junction distribution. Repeated clinical evaluations over the next 10 years were unchanged until 1996, when he complained of progressive weakness of the right foot and clumsiness in the right hand. MRI now indicated a further area of high signal intensity in the right posterior cord at the level of C 5/C 6. There was mild pyramidal distribution weakness in the right leg with an extensor plantar response on the same side. O ver the next five years there has been mild progression in weakness and fatigue and intermittent Lhermitte’s phenomenon. A t no stage has there been a history of relapse, cerebrospinal fluid examination was normal and evoked responses (visual and somatosensory) are normal. Conclusion: This case demonstrates the pheno menon of subclinical MS, unusually supported by prolonged clinical and MRI follow-up. The patient eventually became symptomatic nine years after MRI diagnosis and is following a primary progressive course. A lthough MRI is known to be sensitive in identifying subclinical ‘attacks’, the pattern illustrated here may actually be quite typical of primary progressive MS and is compatible with the later onset seen in this subgroup of patients.