bluuebunny/crossref_metadata_2025 huggingface dataset:
DEUX FAUSSES LETTRES DE JULIEN L'APOSTAT (LA LETTRE AUX JUIFS, EP. 51 [WRIGHT], ET LA LETTRE À ARSACIUS, EP. 84 [BIDEZ])
Authors: Van Nuffelen Peter
Published: October 2002

Pratiquement toute collection épistolaire ancienne contient de fausses lettres et le corpus de Julien l'Apostat (361-363) n'y fait pas exception. Deux catégories sont à distinguer. Les lettres inauthentiques sont des lettres originales d'un autre auteur qui ont été introduites plus tard dans la collection sous le nom de Julien. Les lettres de Julien à Jamblique furent, à cet égard, sans doute écrites par un inconnu dans les années 314-19. La lettre de Julien aux Argiens est de deux siècles plus ancienne qu'on ne le croyait en fonction de sa place dans le corpus de l'empereur. La deuxième catégorie comprend les faux purs et simples : un auteur postérieur a imité le style de Julien afin de faire passer un certain message comme étant de l'empereur. Nous étudierons deux cas de ce dernier. D'abord nous démontrerons que la fameuse lettre aux Juifs est fausse. L'authenticité en a été débattue depuis longtemps, sans qu'on ait vu qu'elle reflète la situation juridique d'après 429. Ensuite, alors que l'authenticité de la lettre au grand-prêtre Arsacius n'a jusqu'ici jamais été mise en question, nous croyons cependant être en mesure d'avancer suffisamment d'éléments pour au moins ouvrir le dossier. À notre avis elle trahit la main d'un chrétien de la première moitié du cinquième siècle.
Metastable Si-Fe phases in hydrothermal sediments of Atlantis II Deep, Red Sea
Authors: Taitel-Goldman N. , Singer A.
Published: October 2002

Amorphous silicate sediments comprise most of the upper part of the sedimentary column of the Atlantis II Deep, whereas more crystalline phases are found in the lower part. The objective of this study was to focus on the newly-formed, metastable, hydrothermal phases, that eventually transform into clays or Fe oxides.

Short-range ordered Si-Fe phases (Si/Fe = 0.2 0.6) rounded to elliptical in shape, and amorphous Si-Fe platy phases (Si/Fe = 0.09 pure Si) comprise some of the amorphous sediments in Atlantis II Deep. The rounded particles have distinct electron-dense (thicker, relatively ordered) margins and a less crystalline inner core. They have rhombohedral symmetry with unit-cell parameters of a= 0.504 nm and c= 1.08 nm. They presumably transform into crystalline clay minerals in statunascendi(at the formative stage) in the sediments. Synthesis in NaCl brines designed to simulate crystallization of the rounded particles, was performed successfully. Similar rounded phases (Si/Fe = 0.26) were synthesized under saline, neutral, hydrothermal conditions (40°C, pH 7, 2 M NaCl) with initial Si/Fe = 1.5. We suggest that the metastable rounded particles precipitate from the hot brine created by the encounter between Red Sea Deep Water and the hydrothermal brine underneath. Within a few thousands years they disintegrate and transform into clay minerals, probably nontronite.
Distinct contributions of CD4+ and CD8+naive and memory T-cell subsets to overall T-cell–receptor repertoire complexity following transplantation of T-cell–depleted CD34-selected hematopoietic progenitor cells from unrelated donors
Authors: Eyrich Matthias , Croner Tanja , Leiler Christine , Lang Peter , Bader Peter , Klingebiel Thomas , Niethammer Dietrich , Schlegel Paul G.
Published: October 2002

Normalization of restricted T-cell–receptor (TCR) repertoire is critical following T-cell–depleted (TCD) stem cell transplantation. We present a prospective study analyzing respective contributions of naive and memory T-cell subsets within the CD4+ and CD8+ compartments to the evolution of overall TCR-repertoire complexity following transplantation of CD34-selected peripheral blood progenitor cells from unrelated donors. During the first year after transplantation, sorted CD4/45RA, CD4/45R0, CD8/45RA, and CD8/45R0 subsets were analyzed at 3-month intervals for TCR-repertoire complexity by CDR3 size spectratyping. Skew in TCR-repertoire was observed only in early memory-type T cells. CD4+ and CD8+ subsets differed in clonal distribution of CDR3 sizes, with rapid Gaussian normalization of bands in CD4/45R0+ T cells. Naive T cells displayed normal repertoire complexity and contributed significantly to skew correction. Our data provide direct evidence for an important role of de novo maturation of naive T cells in normalization of an initially restricted TCR-repertoire following transplantation of CD34-selected, TCD-depleted peripheral blood progenitors from unrelated donors.
Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein
Authors: Iwata Mineo , Graf Lynn , Awaya Norihiro , Torok-Storb Beverly
Published: October 2002

DNA spotted microarrays were used to compare gene expression profiles from 2 functionally distinct human marrow stromal cell lines: HS-27a, which supports cobblestone area formation by early hematopoietic progenitors, and HS-5, which secretes multiple cytokines that support the proliferation of committed progenitors. One unexpected result was the high level of interleukin-7 receptor (IL-7R) gene expression in HS-27a stromal cells. Northern blot analysis confirmed the IL-7R RNA expression, and Western blots for the IL-7R protein detected both a full-length (90-kd) IL-7R and a smaller 30-kd fragment in both HS-27a cells and primary stromal cell cultures, whereas only the 90-kd receptor protein was detected in peripheral blood mononuclear cells. Biotinylated IL-7 was shown to bind to HS-27a cells under physiologic conditions, and this binding was inhibited by blocking anti–IL-7 antibodies. Tyrosine phosphorylation of several proteins (55 kd, 30 kd, and 24 kd) in HS-27a cells was rapidly increased after incubation with recombinant IL-7. One of the phosphorylated proteins proved to be the 30-kd IL-7R fragment. Exposure of HS-27a cells to IL-7 resulted in a 10-fold increase in secretion of IL-6 into culture supernatants but no increase in the cytokines stromal cell–derived factor 1, macrophage inflammatory protein 1α, or IL-1β. The up-regulation of IL-6 secretion is associated with a rapid but transient increase in detectable levels of IL-6 messenger RNA. These data suggest that IL-7 may function to regulate the milieu of the microenvironment by modulating IL-6 secretion by the IL-7R–expressing stromal elements.
Molecular and functional characterization of a natural homozygous Arg67His mutation in the prothrombin gene of a patient with a severe procoagulant defect contrasting with a mild hemorrhagic phenotype
Authors: Akhavan Sepideh , De Cristofaro Raimondo , Peyvandi Flora , Lavoretano Silvia , Landolfi Raffaele , Mannucci Pier M.
Published: October 2002

In a patient who presented with a severe coagulation deficiency in plasma contrasting with a very mild hemorrhagic diathesis a homozygous Arg67His mutation was identified in the prothrombin gene. Wild-type (factor IIa [FIIa]-WT) and mutant Arg67His thrombin (FIIa-MT67) had similar amidolytic activity. By contrast, the kcat/Km value of fibrinopeptide A hydrolysis by FIIa-WT and FIIa-MT67 was equal to 2.1 × 107M−1s−1 and 9 × 105M−1s−1. Decreased activation of protein C (PC) correlated with the 33-fold decreased binding affinity for thrombomodulin (TM; Kd = 65.3 nM vs 2.1 nM, in FIIa-MT67 and in FIIa-WT, respectively). In contrast, hydrolysis of PC in the absence of TM was normal. The Arg67His mutation had a dramatic effect on the cleavage of protease-activated G protein–coupled receptor 1 (PAR-1) 38-60 peptide (kcat/Km = 4 × 107M−1s−1 to 1.2 × 106M−1s−1). FIIa-MT67 showed a weaker platelet activating capacity, attributed to a defective PAR-1 interaction, whereas the interaction with glycoprotein Ib was normal. A drastic decrease (up to 500-fold) of the second-order rate constant pertaining to heparin cofactor II (HCII) interaction, especially in the presence of dermatan sulfate, was found for the FIIa-MT67 compared with FIIa-WT, suggesting a severe impairment of thrombin inhibition by HCII in vivo. Finally, the Arg67His mutation was associated with a 5-fold decrease of prothrombin activation by the factor Xa-factor Va complex, perhaps through impairment of the prothrombin-factor Va interaction. These experiments show that the Arg67His substitution affects drastically both the procoagulant and the anticoagulant functions of thrombin as well as its inhibition by HCII. The mild hemorrhagic phenotype might be explained by abnormalities that ultimately counterbalance each other.
ATM mutations are associated with inactivation of theARF-TP53 tumor suppressor pathway in diffuse large B-cell lymphoma
Authors: Grønbæk Kirsten , Worm Jesper , Ralfkiaer Elisabeth , Ahrenkiel Vibeke , Hokland Peter , Guldberg Per
Published: October 2002

The ATM serine-threonine kinase plays a central role in the cellular response to DNA damage. Germ-line mutations in theATM gene cause ataxia-telangiectasia (A-T), a multisystem disorder associated with predisposition to lymphoma and acute leukemia. Moreover, somatic ATM mutations have been identified in T-cell prolymphocytic leukemia, mantle cell lymphoma, and B-cell chronic lymphocytic leukemia. In this study, the entire ATMcoding sequence was examined in genomic DNA from 120 lymphoid neoplasms. Novel mutations and mutations implicated in cancer and/or A-T were found in 9 of 45 diffuse large B-cell lymphomas (DLBCLs), 2 of 24 follicular lymphomas, and 1 of 27 adult acute lymphoblastic leukemias, whereas no such mutations were detected among 24 peripheral T-cell lymphomas. The mutational spectrum consisted of 2 nonsense mutations, 1 mutation affecting RNA splicing, and 10 missense variants. Most of these mutations were associated with loss or mutation of the paired ATM allele, consistent with biallelic inactivation of ATM. Of the 9 DLBCLs with ATM mutations, 7 also carried TP53 mutations and/or deletions of theINK4a/ARF locus (P = .003). TheATM 735C>T substitution previously considered a rare normal variant was found to be 5.6 times more frequent in individuals with DLBCL than in random individuals (P = .026), suggesting that it may predispose to B-cell lymphoma. Our data suggest that ATM mutations contribute to the development of DLBCL, and that ATM and the ARF-p53 tumor suppressor pathway may cooperate in the pathogenesis of this malignancy.
Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques
Authors: Nathwani Amit C. , Davidoff Andrew M. , Hanawa Hideki , Hu Yunyu , Hoffer Fredric A. , Nikanorov Alexander , Slaughter Clive , Ng Catherine Y. C. , Zhou Junfang , Lozier Jay N. , Mandrell Timothy D. , Vanin Elio F. , Nienhuis Arthur W.
Published: October 2002

The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 × 1012 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B.
The role of posttransplantation maintenance chemotherapy in improving the outcome of autotransplantation in adult acute lymphoblastic leukemia
Authors: Powles Ray , Sirohi Bhawna , Treleaven Jennifer , Kulkarni Samar , Tait Diana , Singhal Seema , Mehta Jayesh
Published: October 2002

Extending the principle of conventional acute lymphoblastic leukemia (ALL) therapy to transplantation, 77 adult patients receiving autografts in first remission after melphalan with or without total body irradiation were scheduled to receive 6-mercaptopurine (6MP), methotrexate (MTX), and vincristine-prednisone (VP) for 2 years after transplantation to reduce relapse. Seventy-one percent of patients received 6MP, 57% received MTX, and 38% received VP. Thirty patients had a relapse at 1.5 to 80 months (median, 12.5 months), 15 in the first year and 7 beyond 3 years. The cumulative incidence of relapse at 10 years was 42% (95% CI, 31%-55%). The 10-year probabilities of disease-free survival (DFS) and overall (OS) survival were 50% (95% CI, 38%-62%) and 53% (95% CI, 41%-65%), respectively. Age older than 30 years, more than 4 weeks to attain remission, and high-risk karyotypes, for example, t(9;22) or t(4;11), were adverse features contributing to the identification of 3 prognostic risk groups with 0, 1, and 2 adverse features, respectively: standard (47%), intermediate (36%), and high (17%). The 10-year cumulative incidences of relapse (20%, 48%, 85%; P < .0001) and probabilities of DFS (72%, 41%, 10%; P = .0003) were significantly different among these groups. In Cox analysis of the 71 patients alive and well 120 days after transplantation, those receiving 2 or 3 maintenance chemotherapy agents had significantly lower relapse rates and superior DFS compared with those receiving 0 or 1 agent. Our data suggest that maintenance chemotherapy improves the outcome of patients with ALL undergoing autografting. However, it is unlikely that autograft-based strategies are optimal for the high-risk group of patients who should be considered for alternative-donor allograft procedures.
Activation-induced cell death of aggressive histology lymphomas by CD40 stimulation: induction of bax
Authors: Szocinski Jamie L. , Khaled Annette R. , Hixon Julie , Halverson Douglas , Funakoshi Satoshi , Fanslow William C. , Boyd Ann , Taub Dennis D. , Durum Scott K. , Siegall Clay B. , Longo Dan L. , Murphy William J.
Published: October 2002

CD40 is present on both normal and neoplastic B-lineage cells. CD40 stimulation of normal B cells has been shown to promote normal growth and differentiation, whereas aggressive histology B lymphomas are growth inhibited. The inhibition of neoplastic B-cell growth is believed to occur via activation-induced cell death in which stimuli that typically promote the growth of normal cells prevent the growth of their neoplastic counterparts. We show here that CD40 stimulation using either a soluble recombinant human CD40 ligand (srhCD40L) or anti-CD40 monoclonal antibody resulted in apoptosis of human Burkitt lymphoma cell lines. Additional studies examining the mechanism of CD40-mediated death revealed an increase in bax messenger RNA with a subsequent increase in Bax protein in the mitochondria of the treated cells. In vitro exposure of the cells to bax antisense oligonucleotides resulted in a significant decline in Bax protein levels and partial protection from CD40-mediated death, indicating that induction of Bax was at least one mechanism underlying this inhibitory effect of CD40 stimulation on lymphomas. When immunodeficient mice bearing Burkitt lymphoma were treated with srhCD40L, significant increases in survival were observed indicating a direct antitumor effect as a result of CD40 stimulation in vivo. Overall, these results demonstrate that CD40 ligation of aggressive histology B-lymphoma cells results in inhibition both in vitro and in vivo and thus may be of potential clinical use in their treatment.
Dendritic cells are functionally defective in multiple myeloma: the role of interleukin-6
Authors: Ratta Marina , Fagnoni Francesco , Curti Antonio , Vescovini Rosanna , Sansoni Paolo , Oliviero Barbara , Fogli Miriam , Ferri Elisa , Della Cuna Gioacchino Robustelli , Tura Sante , Baccarani Michele , Lemoli Roberto M.
Published: October 2002

We studied concentration, phenotype, and function of peripheral blood (PB) dendritic cells (DCs) from patients with multiple myeloma (MM). The absolute number of circulating precursors of myeloid and plasmacytoid DCs was significantly lower in MM patients than in healthy subjects. After maturation, PBDCs from MM patients showed significantly lower expression of HLA-DR, CD40, and CD80 antigens and impaired induction of allogeneic T-cell proliferation compared with controls. Remarkably, they were not capable of presenting the patient-specific tumor idiotype to autologous T cells. Conversely, DCs generated in vitro from CD14+ monocytes from the same patients, and PBDCs freshly isolated from healthy donors efficiently stimulated allogeneic and autologous T cells. To clarify the mechanism of PBDC deficiency in MM, we investigated the effects of the main plasma cell growth factor, interleukin-6 (IL-6), on the development of DCs from CD34+ cells. IL-6 inhibited the colony growth of CD34+ DC progenitors and switched the commitment of CD34+ cells from DCs to CD14+CD1a−CD86−CD80− CD40±HLA-DR ± monocytic cells exerting potent phagocytic activity but no antigen-presentation capacity. This effect was reversed by anti–IL-6 antibodies. Growing CD34+ cells in the presence of autologous serum (without IL-6) also suppressed the development of functional DCs. This study demonstrates that PBDCs from MM patients are functionally defective, partially because of IL-6–mediated inhibition of development. This brings into question the advisability of using PBDCs as antigen carriers for immunotherapy trials in MM. The results also suggest a novel mechanism whereby myeloma cells escape immune recognition.
Response of hairy cells to IFN-α involves induction of apoptosis through autocrine TNF-α and protection by adhesion
Authors: Baker Peter K. , Pettitt Andrew R. , Slupsky Joseph R. , Chen Hai J. , Glenn Mark A. , Zuzel Mirko , Cawley John C.
Published: October 2002

Although hairy cell leukemia is uniquely sensitive to interferon-α (IFN-α), the biologic basis for this phenomenon remains unclear. Here we examine the effects of IFN-α on cultured hairy cells (HCs), taking into account the possible modifying influence of cell adhesion. We make the novel observation that therapeutic concentrations of IFN-α kill nonadherent HCs by inducing apoptosis. In keeping with the persistence of HCs in tissues during therapy, such killing was inhibited by integrin-mediated adhesion to vitronectin or fibronectin. Exposure of HCs to IFN-α resulted in a marked increase in tumor necrosis factor-α (TNF-α) secretion. Furthermore, blocking antibodies to TNF-RI or TNF-RII protected HCs from IFN-α–induced apoptosis, demonstrating that such killing was mediated by TNF-α. In the absence of IFN-α, exogenous TNF-α did not induce HC apoptosis, showing that IFN-α sensitized HCs to the proapoptotic effect of autocrine TNF-α. This sensitization to TNF-α–induced killing was attributable to suppression of IAP (inhibitors of apoptosis) production known to be regulated by the cytoprotective nuclear factor–κB–dependent arm of TNF-α signaling. Moreover, engagement of the receptors for fibronectin or vitronectin prevented this IFN-α–induced down-regulation of IAPs. Understanding of the signals involved in the combined effects of IFN-α and TNF-α and abrogation of those induced by integrin engagement offers the possibility of sensitizing other malignant cells to IFN-α–induced killing and thereby extending the therapeutic use of this cytokine.
Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study
Authors: Keating Michael J. , Flinn Ian , Jain Vinay , Binet Jacques-Louis , Hillmen Peter , Byrd John , Albitar Maher , Brettman Lee , Santabarbara Pedro , Wacker Bret , Rai Kanti R.
Published: October 2002

This study investigated the efficacy, safety, and clinical benefit of alemtuzumab (Campath-1H) for patients with relapsed or refractory B-cell chronic lymphocytic leukemia exposed to alkylating agents and having failed fludarabine therapy. Ninety-three patients received alemtuzumab in 21 centers worldwide, with the aim to obtain an overall response rate of at least 20%. Dosage was increased gradually (target 30 mg, 3 times weekly, for a maximum of 12 weeks). Infection prophylaxis was mandatory, beginning on day 8, and continuing for a minimum of 2 months after treatment. Responses were assessed at weeks 4, 8, and 12, and patients were followed for 34 months. Overall objective response in the intent-to-treat population (n = 93) was 33% (CR 2%, PR 31%). Median time to response was 1.5 months (range, 0.4-3.7 months). Median time to progression was 4.7 months overall, 9.5 months for responders. At data cut-off, 27 patients (29%) were alive; overall median survival was 16 months (95% CI: 11.8-21.9) and 32 months for responders. Nineteen responders survived more than 21 months. Clinical benefit was observed both in responders and in patients with stable disease. The most common adverse events were related to infusion, generally grade 1 or 2 in severity, occurring mainly in the first week. Grade 3 or 4 infections were reported in 25 patients (26.9%). However, only 3 (9.7%) of 31 patients who responded to alemtuzumab treatment developed grade 3 or 4 infections on the study. Alemtuzumab induced significant responses in these patients with clinical benefit in the majority and with acceptable toxicity in a high-risk group.
Analysis of fibrinogen γ-chain truncations shows the C-terminus, particularly γIle387, is essential for assembly and secretion of this multichain protein
Authors: Okumura Nobuo , Terasawa Fumiko , Tanaka Hitoshi , Hirota Masako , Ota Hiroyoshi , Kitano Kiyoshi , Kiyosawa Kendo , Lord Susan T.
Published: October 2002

To examine the role of the fibrinogen γ chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated γ chains, terminating between residues γ379 and the C-terminus, γ411. The variant fibrinogens were synthesized from altered γ-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with γ chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the γ chain, from 1.4 μg/mL for normal fibrinogen to 0.39 μg/mL for γ 387 fibrinogen. Immunoassays of cell lysates showed that all variant γ chains were synthesized, although the levels varied significantly. For variants longer than 386 residues, levels decreased with length but remained near normal. In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced. Quantitative reverse transcription–polymerase chain reaction demonstrated that the γ-chain messenger RNA level was independent from chain length. Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules. For shorter variants, these intermediates were not evident. We conclude that residues near the C-terminus of the γ chain are essential for fibrinogen assembly, and more specifically, that γ387 is critical. We propose that the loss of residue γ387 destabilized the structure of γ chain, preventing assembly of αγ and βγ dimers, essential intermediates in the assembly of normal fibrinogen.
Polymorphisms in the thymidylate synthase and serine hydroxymethyltransferase genes and risk of adult acute lymphocytic leukemia
Authors: Skibola Christine F. , Smith Martyn T. , Hubbard Alan , Shane Barry , Roberts Abby C. , Law Graham R. , Rollinson Sara , Roman Eve , Cartwright Raymond A. , Morgan Gareth J.
Published: October 2002

We previously reported that 2 polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene at positions C677T and A1298C were associated with lower risk of adult acute lymphocytic leukemia (ALL). In the present study, we have examined whether polymorphisms in other folate-metabolizing genes play a role in ALL susceptibility. Polymorphisms in methionine synthase (MS A2756G), cytosolic serine hydroxymethyltransferase (SHMT1 C1420T), and a double (2R2R) or triple (3R3R) 28-bp tandem repeat in the promoter region of thymidylate synthase (TS) were studied and found to modulate ALL risk. In a univariate analysis, SHMT1 1420CT individuals exhibited a 2.1-fold decrease in ALL risk (odds ratio [OR] = 0.48; 95% confidence interval [CI], 0.25-0.91), whereas the 1420TT genotype conferred a 3.3-fold reduction in risk (OR = 0.31; 95% CI, 0.10-0.90). Similarly, TS 2R3R individuals exhibited a 2.8-fold reduction in ALL risk (OR = 0.36; 95% CI: 0.16-0.83), while the TS 3R3R genotype conferred an even greater level of protection (OR = 0.25; 95% CI, 0.08-0.78). However, no significant associations were evident for the MS 2756AG polymorphism (OR = 0.79; 95% CI, 0.38-1.7). In addition, potential interactions between theSHMT1 and TS or MS genes were observed. TS 3R3R individuals who were SHMT1 1420CT/TT had a 13.9-fold decreased ALL risk (OR = 0.072; 95% CI, 0.0067-0.77). Further, MS 2756AG individuals who were SHMT1 1420CT/TT had a 5.6-fold reduction in ALL risk (OR = 0.18; 95% CI, 0.05-0.63). This study suggests an important role for uracil misincorporation and resultant chromosomal damage in the pathogenesis of ALL, and that genetic interactions involving low penetrance polymorphisms in folate-metabolizing genes may increase ALL risk.
Sensitive detection of human cytomegalovirus peptide–specific cytotoxic T-lymphocyte responses by interferon-γ–enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation
Authors: Hebart Holger , Daginik Senay , Stevanovic Stefan , Grigoleit Ulrich , Dobler Andrea , Baur Manuela , Rauser Georg , Sinzger Christian , Jahn Gerhard , Loeffler Juergen , Kanz Lothar , Rammensee Hans-Georg , Einsele Hermann
Published: October 2002

Reconstitution of human cytomegalovirus (HCMV)–specific cytotoxic T lymphocytes (CTLs), predominantly directed against pp65, provides protective immunity for the development of HCMV disease after allogeneic stem cell transplantation (SCT). To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-γ release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8+ T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide–specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen–specific CD4+ T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35->180 days). Peak counts of peptide-specific CD8+ T cells ranged from 0.14 to 60.6 cells/μL; those of protein-specific CD4+ T cells ranged from 0.64 to 18.97 cells/μL. Reconstitution of HCMV-peptide–specific CD8+ T cells and protein-specific CD4+ T cells was associated with clearance of HCMV infection (r2 = 0.89, P < .0001 and r2 = 0.61, P = .0045, respectively). HCMV infection recurred after documentation of HCMV-specific T-cell reconstitution (n = 4) when immunosuppression was intensified. Patients in whom late-onset HCMV disease developed lacked HCMV-protein–specific T cells at 3 months after SCT. In conclusion, prospective monitoring of HCMV-specific CD4+ and CD8+ T-cell reconstitution can be performed rapidly by using flow cytometry after specific stimulation with HCMV peptides and proteins and might help to further improve clinical management of HCMV infection after allogeneic SCT.
A new adenoviral helper–dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo
Authors: Ehrhardt Anja , Kay Mark A.
Published: October 2002

We have developed a new helper-dependent (HD) adenoviral vector FTC that contains 3 cis-acting sequences as stuffer DNA: a human fragment of alphoid repeat DNA, matrix-attachment regions (MARs), and the hepatocyte control region enhancer. To determine the most robust human coagulation factor IX (hFIX) expression cassette in an adenovirus, we first tested different hFIX expression sequences with or without flanking MARs in first-generation adenoviral vectors. After intravenous infusion of the vector, serum levels of up to 100 000 ng/mL hFIX (normal level, 5000 ng/mL) were obtained at nontoxic doses. In order to make a direct comparison, a first-generation and a gene-deleted vector with identical hFIX expression cassettes were constructed. Both first-generation and HD adenovirus–treated animals demonstrated a threshold effect in a dose-response study. With the administration of 2 × 109transducing particles of either vector, supraphysiological serum levels of hFIX were obtained, with the highest expression (41 000 ng/mL) occurring during the first 2 months after injection. The serum factor IX concentrations, while remaining in the therapeutic range, slowly declined by 95% over a period of 1 year. At this dose, interleukin-6 and tumor necrosis factor–α serum concentrations were elevated in animals that received the first-generation but not the HD vector. This study compares the properties of a gene-deleted and first-generation adenovirus with equivalent expression cassettes and suggests that thecis-DNA elements contained in the vector and expression cassette have important effects on gene expression in vivo.
Hematopoietic progenitor/stem cells immortalized byLhx2 generate functional hematopoietic cells in vivo
Authors: Pinto do Ó Perpétua , Richter Karin , Carlsson Leif
Published: October 2002

Hematopoietic stem cells (HSCs) are unique in their capacity to maintain blood formation following transplantation into immunocompromised hosts. Expansion of HSCs in vitro is therefore important for many clinical applications but has met with limited success because the mechanisms regulating the self-renewal process are poorly defined. We have previously shown that expression of the LIM-homeobox gene Lhx2 in hematopoietic progenitor cells derived from embryonic stem cells differentiated in vitro generates immortalized multipotent hematopoietic progenitor cell lines. However, HSCs of early embryonic origin, including those derived from differentiated embryonic stem cells, are inefficient in engrafting adult recipients upon transplantation. To address whetherLhx2 can immortalize hematopoietic progenitor/stem cells that can engraft adult recipients, we expressed Lhx2 in hematopoietic progenitor/stem cells derived from adult bone marrow. This approach allowed for the generation of immortalized growth factor–dependent hematopoietic progenitor/stem cell lines that can generate erythroid, myeloid, and lymphoid cells upon transplantation into lethally irradiated mice. When transplanted into stem cell–deficient mice, these cell lines can generate a significant proportion of circulating erythrocytes in primary, secondary, and tertiary recipients for at least 18 months. Thus, Lhx2immortalizes multipotent hematopoietic progenitor/stem cells that can generate functional progeny following transplantation into lethally irradiated hosts and can long-term repopulate stem cell–deficient hosts.
Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to early-acting cytokines
Authors: Henckaerts Els , Geiger Hartmut , Langer Jessica C. , Rebollo Patricia , Van Zant Gary , Snoeck Hans-Willem
Published: October 2002

Quantitative trait analysis may shed light on mechanisms regulating hematopoiesis in vivo. Strain-dependent variation existed among C57BL/6 (B6), DBA/2, and BXD recombinant inbred mice in the responsiveness of primitive progenitor cells to the early-acting cytokines kit ligand, flt3 ligand, and thrombopoietin. A significant quantitative trait locus was found on chromosome 2 that could not be confirmed in congenic mice, however, probably because of epistasis. Because it has been shown that alleles of unknown X-linked genes confer a selective advantage to hematopoietic stem cells in vivo in humans and in cats, we also analyzed reciprocal male D2B6F1 and B6D2F1 mice, revealing an X-linked locus regulating the responsiveness of progenitor and stem cells to early-acting factors. Among DBA/2, B6, and BXD recombinant inbred mice, correlating genetic variation was found in the absolute number and frequency of Lin−Sca1++kit+ cells, which are highly enriched in hematopoietic progenitor and stem cells, and in the number of Lin−Sca1++kit− cells, a population whose biologic significance is unknown, suggesting that both populations are functionally related. Suggestive quantitative trait loci (QTLs) for the number of Lin−Sca1++ cells on chromosomes 2, 4, and 7 were confirmed in successive rounds of mapping. The locus on chromosome 2 was confirmed in congenic mice. We thus demonstrated genetic variation in the response to cytokines critical for hematopoiesis in vivo and in the pool size of cells belonging to a phenotype used to isolate essentially pure primitive progenitor and stem cells, and we identified loci that may be relevant to the regulation of hematopoiesis in steady state.
Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor–dependent procoagulant activity
Authors: Sabatier Florence , Roux Veronique , Anfosso Francine , Camoin Laurence , Sampol José , Dignat-George Françoise
Published: October 2002

In the present study we investigated whether endothelial microparticles (EMPs) can bind to monocytic THP-1 cells and modulate their procoagulant properties. Using flow cytometry, we demonstrated that EMPs express adhesive receptors similar to those expressed by activated endothelial cells. Expression of endothelial antigens by THP-1 cells incubated with EMP was shown by immunoperoxidase staining and flow cytometry using antibodies directed against E-selectin, VCAM-1, and endoglin. EMP binding to THP-1 cells was time- and concentration- dependent, reached a plateau at 15 minutes, and had an EMP-to-monocyte ratio of 50:1. EMP binding was not affected by low temperature and was not followed by the restoration of phosphatidylserine asymmetry, suggesting that adhesion was not followed by fusion. A 4-hour incubation of THP-1 cells with EMP led to an increase in procoagulant activity as measured by clotting assay. Concomitantly, THP-1 exhibited increased levels of tissue factor (TF) antigen and TF mRNA compared to control cells. The ability of EMP to induce THP-1 procoagulant activity was significantly reduced when THP-1 cells were incubated with EMP in the presence of blocking antibodies against ICAM-1 and β2 integrins. These results demonstrate that EMPs interact with THP-1 cells in vitro and stimulate TF-mediated procoagulant activity that is partially dependent on the interaction of ICAM-1 on EMP and its counterreceptor, β2 integrins, on THP-1 cells. Induction of procoagulant activity was also demonstrated using human monocytes, suggesting a novel mechanism by which EMP may participate in the dissemination and amplification of procoagulant cellular responses.
Characterization of caspase-8L: a novel isoform of caspase-8 that behaves as an inhibitor of the caspase cascade
Authors: Himeji Daisuke , Horiuchi Takahiko , Tsukamoto Hiroshi , Hayashi Kenshi , Watanabe Takeshi , Harada Mine
Published: October 2002

Caspase-8 (Fas-associating protein with death domain–like interleukin-1β– converting enzyme [FLICE]/MACH/Mch5) belongs to a family of cysteine proteases presumed to be the apex of the apoptotic signaling pathways. We recently reported the presence of a novel isoform of caspase-8, named caspase-8L, generated by the alternative splicing of human caspase-8 gene, from human peripheral blood lymphocytes by reverse transcriptase–polymerase chain reaction. We herein report a functional analysis of caspase-8L in the Fas-mediated apoptotic pathway. Caspase-8L is missing the catalytic site of caspase-8 but retains 2 N-terminal repeats of the death-effector domain. The caspase-8L messenger RNA was detected in various tissues but not in any cell lines examined. In human peripheral blood lymphocytes, caspase-8L was strongly suggested to be expressed at the protein level. In MCF-7 cells, caspase-8L transfection itself did not affect cell viability but instead inhibited the apotosis induced by the cotransfection of caspase-8 in a dominant negative manner. Moreover, Fas-mediated apoptosis was inhibited in caspase-8L–transfected Jurkat cells, which were associated with a reduction in the caspase-8 catalytic activity. In vitro binding assays demonstrated that caspase-8L bound to FADD (Fas-associating protein with death domain) and caspase-8a and blocked the binding of caspase-8 to FADD. In in vivo binding assays, transfected caspase-8L bound to endogeneous FADD. Thus, caspase-8L acts as an inhibitor of caspase-8 by interfering with the binding of caspase-8 to FADD and is involved in the regulation of Fas-mediated apoptosis.
Measurement of spleen volume by ultrasound scanning in patients with thrombocytosis: a prospective study
Authors: Picardi Marco , Martinelli Vincenzo , Ciancia Rosanna , Soscia Ernesto , Morante Roberto , Sodano Antonio , Fortunato Giuliana , Rotoli Bruno
Published: October 2002

Spleen size was assessed in 73 patients with thrombocytosis and in 15 healthy subjects, comparing palpation with ultrasonography (US) measurement of longitudinal diameter and volume. Intraobserver and interobserver variability for volume on US, checked in 12 patients, was very low. Correlation between spleen volume measured by US and that measured by computed tomography was excellent. Splenomegaly was detected by palpation in 25% of patients, by US assessment of longitudinal diameter in 33%, and by US assessment of volume in 52%. After diagnostic work-up, 54 patients had a diagnosis of essential thrombocythemia (ET), 4 of idiopathic myelofibrosis (IMF), and 15 of secondary thrombocytosis (ST). Spleen volume in patients with ST was in the normal range (138 ± 47 mL) and was significantly lower than that in patients with ET or IMF (370 ± 210 mL;P < .001). Thus, US-measured volume was the most sensitive method for identifying nonpalpable splenomegaly in patients with primary myeloproliferative diseases, and it may help in distinguishing these diseases from reactive disorders.
Induction of T-cell tolerance to an MHC class I alloantigen by gene therapy
Authors: Bagley Jessamyn , Tian Chaorui , Sachs David H. , Iacomini John
Published: October 2002

Induction of immunologic tolerance to alloantigens is a major goal in the field of transplantation. Here, we demonstrate that efficient transduction and expression of a retrovirally transduced major histocompatibility complex (MHC) class I gene(H-2Kb) in bone marrow (BM)–derived cells, resulting in a permanent state of hematopoietic molecular chimerism, induces stable tolerance to the transduced gene product. Reconstitution of lethally irradiated syngeneic recipients with BM transduced with virus encoding H-2Kb resulted in life-long expression of the retroviral gene product on the surface of BM-derived hematopoietic lineages including Sca-1+, lineage negative, hematopoietic progenitors. T cells from mice receiving MHC-transduced BM were unable to kill targets expressing H-2Kbbut were able to respond to third-party controls. Mice reconstituted with H-2Kb-transduced BM exhibited long-term acceptance of H-2Kb mismatched skin grafts but were able to rapidly reject third-party control grafts. Thus, gene therapy approaches may be used to induce T-cell tolerance.
Tissue factor is the receptor for plasminogen type 1 on 1-LN human prostate cancer cells
Authors: Gonzalez-Gronow Mario , Gawdi Govind , Pizzo Salvatore V.
Published: October 2002

Tissue factor (TF), the initiator of the extrinsic pathway of coagulation, binds plasminogen (Pg) with high affinity through an interaction between kringles 1-3 of Pg and the extracellular domain of TF. We investigated the binding of Pg type 1 (Pg 1) and Pg type 2 (Pg 2) to highly invasive, TF-expressing, 1-LN human prostate tumor cells and to TF isolated from 1-LN cell membranes. Pg 1, containing both N-linked and O-linked oligosaccharide chains, bound to isolated TF with high affinity, whereas Pg 2, containing only one O-linked oligosaccharide chain, did not bind to TF. Although Pg 1 and Pg 2 bind to 1-LN cells, only anti-TF antibodies inhibited the binding of Pg 1, suggesting that TF functions as the receptor for Pg 1 on 1-LN cells. Binding of Pg 1 to isolated TF was inhibited by 6-aminohexanoic acid and α-methylmannoside, suggesting that Pg 1 l-lysine binding sites and the biantennary, mannose-containing N-linked oligosaccharide chain are involved in this interaction. Binding of Pg 1 to 1-LN cells promoted activation by receptor-bound urinary-type Pg activator (u-PA) and initiated a Ca++ signaling cascade. In previous studies we demonstrated that the Pg 2 O-linked carbohydrate chain is essential for its binding to CD26 on 1-LN cells. The current studies suggest that Pg oligosaccharide chains regulate the binding of Pg 1 and Pg 2 to separate receptors on the cell surface.
Does treatment with intermittent infusions of intravenous anti-D allow a proportion of adults with recently diagnosed immune thrombocytopenic purpura to avoid splenectomy?
Authors: Cooper Nichola , Woloski B. Michael R. , Fodero Erin M. , Novoa Maria , Leber Melissa , Beer Juerg H. , Bussel James B.
Published: October 2002

This study explored whether repeated infusions of intravenous anti-D could allow adults with recently diagnosed immune thrombocytopenic purpura (ITP) who had failed an initial steroid course to postpone and ultimately avoid splenectomy. Twenty-eight Rh+, nonsplenectomized adults with ITP diagnosed within 1 to 11 months and platelet counts 30 × 109/L (30 000/μL) or below were enrolled. Anti-D was infused whenever the platelet count decreased to 30 × 109/L (30 000/μL) or below. “Response” was defined as a platelet increase of more than 20 × 109/L (20 000/μL) to more than 30 × 109/L (30 000/μL) within 7 days of treatment. Patients were a median 3.5 months from ITP diagnosis at enrollment and had received a median of 2 previous therapies, including prednisone in 26 of 28 cases. They were followed for a median 26 months. A total of 93% responded to their initial infusion of anti-D, and 68% repeatedly responded with counts maintained above 30 × 109/L (30 000/μL) using anti-D alone. Currently, 12 (43%) of 28 patients have been off all treatment for more than 6 months without undergoing splenectomy, 6 maintaining counts above 100 × 109/L (100 000/μL). Seven continue on treatment, 8 underwent splenectomy, and 1 was lost to follow-up at 10 months. One patient discontinued anti-D because of toxicity. Patients with platelet counts at least 14 × 109/L (14 000/μL) at enrollment were more likely to discontinue treatment (P < .05). Anti-D was an effective maintenance treatment for two thirds of Rh+, nonsplenectomized adults with ITP who had failed an initial steroid course. Intermittent infusions of intravenous anti-D allowed more than 40% of these adults to avoid splenectomy and to achieve stable platelet counts off all therapy, even after many months of treatment. Platelet count at study entry was the primary predictor of outcome.
Shear-dependent morphology of von Willebrand factor bound to immobilized collagen
Authors: Novák Levente , Deckmyn Hans , Damjanovich Sándor , Hársfalvi Jolán
Published: October 2002

We have developed an immunogold von Willebrand factor (VWF) detection method that permits almost complete coverage of individual VWF molecules, and by this unequivocal localization and morphologic analysis of collagen-bound VWF by atomic force microscopy (AFM). Perfusion of gel filtration–purified VWF in parallel plate perfusion chambers over glass coverslips coated with calf skin collagen, followed by AFM imaging in air, enabled us to assess possible morphologic differences between VWF bound at low (0.07 N/m2 = 0.7 dynes/cm2) and high (4.55 N/m2 = 45.5 dynes/cm2) shear stresses. No significant differences in VWF morphology were found, the molecules were oriented almost randomly, and there were no clear signs of VWF “uncoiling” either at a high or at a low shear regime. After perfusing 1 μg/mL VWF for 5 minutes, surface coverage at high shear was almost twice the one seen at low shear, and some larger and more irregularly shaped VWF molecules could be seen at high shear. This difference disappeared, however, at 15 minutes of perfusion and was probably caused by diffusion kinetics. Moreover, the presence of 68 × 109/L washed fixed platelets in the perfusate did not have any visible effect on VWF morphology at high versus low shear stress. These findings suggest that shear stress does not influence significantly the overall molecular morphology of VWF during its binding to collagen-coated surface and are consistent with a constitutively expressed affinity of collagen-bound VWF for glycoprotein Ib.
Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor–1
Authors: Fink Trine , Kazlauskas Arunas , Poellinger Lorenz , Ebbesen Peter , Zachar Vladimir
Published: October 2002

Plasminogen activator inhibitor–1 (PAI-1) plays a key role in control of coagulation and tissue remodeling and has been shown to be regulated by a number of cell stimuli, among those hypoxia. In this study we characterize the hypoxia-mediated induction of PAI-1 in human hepatoma cell line HepG2. We found that PAI-1 is tightly regulated in a narrow oxygen gradient. After incubation at oxygen concentrations of 1% to 2%, a 60-fold increase in PAI-1 messenger RNA levels was observed, whereas mild hypoxic conditions of more than 3.5% did not appear to induce transcription. Moreover, increased levels of PAI-1 protein were observed after incubation at low oxygen tensions. Through sequence analysis, several putative hypoxia-response elements (HREs 1-5) were identified in the human PAI-I promoter. Reporter gene assays showed that the HRE-2 (−194 to −187) was necessary and sufficient for the hypoxia-mediated response. By electrophoretic mobility assay we observed hypoxia-dependent binding of a protein complex to the HRE-2 motif. Further analysis demonstrated that HRE-2 was specifically recognized by the hypoxia-inducible transcription factor 1α–arylhydrocarbon nuclear translocator complex. Taken together, our data demonstrate that hypoxia-induced transcription is mediated through HIF-1 interaction with the HRE-2 site of the human PAI-1 promoter.