bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for differences in the mechanisms by which antibodies against CD44 promote adhesion of erythroid and granulopoietic progenitors to marrow stromal cells
Authors: Oostendorp Robsert A. J. , Spitzer Elisabeth , Brandl Martina , Eaves Connie J. , DÖrmer Peter
Published: March 2003

Adhesive interactions between haemopoietic progenitor cells and stromal elements involve a number of different molecules, some of which may be progenitor‐lineage‐ and stage‐specific. CD44 is one such molecule, although little is known about the mechanism(s) by which it is involved. In this study, several anti‐CD44 monoclonal antibodies (mAb) increased the adherence of clonogenic cells, without affecting the total number of types of progenitors recoverable from the adhesion cultures. All of these mAb recognized epitopes on the globular head of CD44. In contrast, two mAb that recognized other regions of CD44 reduced progenitor adhesion to stroma. The mechanism by which one of the anti‐CD44 mAb (L178) enhanced progenitor adhesion did not involve CD44‐crosslinking, and was independent of VLA‐4‐, VLA‐5‐ or LFA‐1‐mediated interactions, Ca or Mg cations, or accessory cells. In addition, CD44 expression on both progenitors and stromal cells contributed to L178‐enhanced progenitor adhesion. Baseline adherence of erythroid progenitors to stroma required tyrosine kinase activity, whereas that of granulopoietic progenitors did not. However, the increase in adhesion did require tyrosine kinase activation. Additional experiments suggested that enhanced adhesion of CFU‐GM to stroma may also be adenylate cyclase‐dependent. Taken together, the present studies indicate both similarities and differences in the mechanisms of CD44‐mediated adhesion of erythroid and granulopoietic progenitors to stromal cells.
Tumourigenicity of MTG8, a leukaemia‐related gene, in concert with v‐Ha‐ras gene in BALB/3T3 cells
Authors: Sueoka Eisaburo , Sueoka Naoko , Okabe Sachiko , Komori Atsumasa , Suganuma Masami , Kozu Tomoko , Fujiki Hirota
Published: March 2003

The MTG8 (ETO) gene has been identified as the translocation partner of AML1 (PEBP2αB or CBFα2) gene in the AML1/MTG8 (ETO) fused gene caused by t(8;21) translocation in human acute myeloid leukaemia, M2 type. Although AML1/MTG8 chimaeric protein is known to inhibit the functioning of AML1 protein, the precise function of MTG8 gene itself is not known yet. We studied the significance of MTG8 gene in the oncogenicity of AML1/MTG8 fused gene, by introducing full‐length MTG8 cDNA into both BALB/3T3 cells containing v‐Ha‐ras gene (Bhas 42 cells) and BALB/3T3 cells without v‐Ha‐ras gene.

Irrespective of the overexpression of MTG8 gene in both groups of cells, Bhas‐MTG8 clones which contained v‐Ha‐ras gene and expressed the MTG8 gene at a level more than twice that of parental Bhas 42 cells induced cell transformation, whereas BALB‐MTG8 clones without v‐Ha‐ras gene did not. Furthermore, injection of the transformed Bhas‐MTG8 clones into the subcutaneous tissue of nude mice induced tumours, whereas that of BALB‐MTG8 clones did not. These results suggest that MTG8 gene product, in cooperation with viral Ras protein, resulted in tumour formation. We provide the first evidence that MTG8 gene by itself has a carcinogenic property within the AML1/MTG8 (ETO) fused gene.
Trisomy 13 and myeloid malignancy — characteristic blast cell morphology: a United Kingdom Cancer Cytogenetics Group survey
Authors: Mehta  , Bain  , Fitchett  , Shah  , Secker‐Walker
Published: March 2003

We retrospectively report data on 28 patients with haematological malignancy and trisomy 13 (25 cases) or tetrasomy 13 (three cases) as the primary acquired cytogenetic change. Peripheral blood and/or bone marrow morphology was reviewed in 25/28 cases and the final diagnosis was as follows: AML M0 (11), AML M1 (6), AML M2 (2), AML M4 (2), AML M5b (1), AML M6 (1), RAEB‐t (3), RAEB (1), RA (1). All three cases with tetrasomy 13 had AML M0. Characteristic small hand‐mirror blasts with cytoplasmic blebs and tails and scanty small granules were seen in 13/25 cases and 18/25 cases had small blasts which could easily be mistaken for lymphoblasts. Trilineage dysplasia was present in 8/28 cases. Median patient survival was 3 months. We conclude that trisomy 13 is particularly associated with acute myeloid leukaemia with minimal differentiation (AML M0), often has distinctive morphological features, and has a poor prognosis.
Plasminogen activator in acute myeloid leukaemic marrows: u‐PA in contrast to t‐PA in normal marrow
Authors: Mcwilliam Nicola , Robbie Linda , Booth Nuala , Bennett Bruce
Published: March 2003

Leukaemic and normal bone marrow samples were compared in terms of their content of the fibrinolytic agents, tissue plasminogen activator (t‐PA) and urokinase‐type plasminogen activator (u‐PA) and their inhibitors, plasminogen activator inhibitors 1 and 2 (PAI‐1 and PAI‐2). Normal marrow contained t‐PA as the principal plasminogen activator, whereas in leukaemic marrow samples u‐PA was the predominant activator. Both normal and leukaemic marrows contained PAI‐1 in similar amounts, but whereas normal marrow contained significant amounts of PAI‐2 the leukaemic marrows contained very little. Plasminogen activators were present in uncomplexed, active forms and plasmin–α2‐antiplasmin complexes were generated locally more prominently in leukaemic marrows. u‐PA associated with blast cells may contribute to the severe forms of haemorrhage sometimes occurring in myeloid types of leukaemia.
Antithrombotic activity of a fucosylated chondroitin sulphate from echinoderm: sulphated fucose branches on the polysaccharide account for its antithrombotic action
Authors: Mourão Paulo A. S. , Guimarães Marco A. M. , Mulloy Barbara , Thomas Stephen , Gray Elaine
Published: March 2003

The antithrombotic activity of a fucosylated, chondroitin‐sulphate‐like polysaccharide extracted from the body wall of sea cucumber, and of chemically modified derivatives of the same polysaccharide, have been assessed using a stasis thrombosis model in rabbits. Intravenous administration of the native polysaccharide reduced thrombosis in a dose‐dependent manner and, at a dose of 1.5 mg/kg (60 IU/kg) body weight, completely prevented thrombosis after 10 min stasis. Removal of the sulphated fucose branches of the polysaccharide abolished antithrombotic effectiveness.

After intravenous injection of an antithrombotic dose of radioactively labelled polysaccharide, a correlation was observed between removal of radioactivity from the plasma and decrease in ex vivo APTT values, demonstrating that antithrombotic effectiveness depends on the level of circulating polysaccharide rather than on an indirect effect of the polysaccharide on the vascular endothelium.

Reduction of the glucuronic acid carboxyl groups in the polysaccharide did not affect its in vitro and in vivo activities. Both partial defucosylation and desulphation of the polysaccharide abolished all its anticoagulant or antithrombotic action.
Similar characteristics of the CDR3 of VH1‐69/DP‐10 rearrangements in normal human peripheral blood and chronic lymphocytic leukaemia B cells
Authors: Brezinschek Hans‐Peter , Brezinschek Ruth I , DÖrner Thomas , Lipsky Peter E
Published: March 2003

The variable heavy chain (VH) gene segment VH1‐69/DP‐10 has been shown to be over‐represented in B‐cell chronic lymphocytic leukaemia (CLL). Because of certain similar characteristics of their complementarity determining region 3 (CDR3), including preferential utilization of JH6 elements and an extended length, it has been suggested that antigenic stimulation might be involved in leukaemogenesis. Utilizing single‐cell PCR to amplify and sequence genomic DNA from individual normal human peripheral blood B cells, we have obtained 7/421 productively and 1/69 nonproductively rearranged VH genes that used VH1‐69/DP‐10. All productive rearrangements were unmutated, used JH6 and had an average CDR3 length similar to that previously found in VH1‐69/DP‐10‐expressing CLL cells. These results suggest that CLL may arise from B cells commonly found in the peripheral B‐cell repertoire and do not represent expansion of a unique subset of specific antigen‐reactive B cells.
Anti‐B4‐blocked ricin: a phase II trial of 7 day continuous infusion in patients with multiple myeloma
Authors: Grossbard Michael L. , Fidias Panos , Kinsella Jeanne , O'toole John , Lambert John M. , Blattler Walter A. , Esseltine Dixie , Braman Ginny , Nadler Lee M. , Anderson Kenneth C.
Published: March 2003

This phase II trial was undertaken to determine the toxicities, response rate, pharmacokinetics and frequency of human anti‐mouse antibody (HAMA) and anti‐ricin antibody (HARA) when the B‐cell restricted immunotoxin anti‐B4‐bR was administered to patients with previously treated multiple myeloma (MM).

Five patients with MM were scheduled to receive a 7 d continuous infusion of anti‐B4‐bR. The initial four patients received therapy at 40 μg/kg lean body weight (LBW)/d. Two patients received a 7 d infusion, one patient received 6 d, and another patient 5 d of therapy. The fifth patient was treated for 7 d at a lower dose of 30 μg/kg LBW/d because of the side‐effects observed in the initial patients.

Pharmacokinetic studies demonstrated a peak serum level >2.6 n M in three of the patients. Side‐effects of therapy included hepatic transaminase elevations, myalgias, thrombocytopenia, nausea, vomiting, decrease in performance status, and capillary leak syndrome. One patient developed HAMA and two patients HARA. One patient developed neurologic toxicity with akinetic mutism, and died following therapy. No patient demonstrated a significant decline in M‐component during therapy.

We concluded that anti‐B4‐bR can be administered by continuous infusion to patients with multiple myeloma, although immunotoxin levels >3 n M were associated with increased incidence of toxicity and required dose adjustment. Future trials using anti‐B4‐bR in MM will be needed to determine the optimal dose and administration schedule in this patient population, and to determine whether there is evidence of biologic activity.
K‐252a‐induced polyploidization and differentiation of a human megakaryocytic cell line, Meg‐J: transient elevation and subsequent suppression of cyclin B1 and cdc2 expression in the process of polyploidization
Authors: Iwabe Koji , Teramura Masanao , Yoshinaga Kentaro , Kobayashi Shoko , Hoshikawa Yutaka , Maeda Tatsuya , Hatakeyama Masanori , Mizoguchi Hideaki
Published: March 2003

Megakaryocytes are unique haemopoietic cells which undergo DNA replication, giving rise to polyploid cells. However, little is known about the mechanism of megakaryocytic polyploidization. To address this issue, we used the human megakaryocytic cell line Meg‐J. In the presence of K‐252a (an indolocarbasole derivative), Meg‐J cells stopped proliferation and exhibited additional megakaryocytic features, including morphological changes, polyploidization, and increases in the levels of surface expression of platelet glycoprotein (GP) IIb/IIIa and GPIb. Thrombopoietin (TPO) promoted the K‐252a‐induced polyploidization and megakaryocytic differentiation. In the process of K‐252a‐induced polyploidization, levels of expression of both cdc2 and cyclin B1 were elevated transiently and subsequently decreased. This suggested that the polyploidization process in Meg‐J cells was at least in part associated with a transient elevation and subsequent decrease in the expression of cdc2/cyclin B1 complex, a critical kinase involved in G2/M cell cycle transition.
Kaposi's sarcoma‐associated herpesvirus (KSHV) in bone marrow biopsies of patients with Waldenstrom's macroglobulinaemia
Authors: Brousset Pierre , Theriault Catherine , Roda Daniel , Attal Michel , Delsol Georges
Published: March 2003

Kaposi's sarcoma‐associated herpesvirus (KSHV) is suspected to play a role in the aetiology of multiple myeloma. Because of similarities in the pathophysiology of multiple myeloma and Waldenstrom's macroglobulinaemia (WM), we investigated DNA samples from 20 bone marrow biopsies with WM for the detection of KSHV by PCR (KS330/ORF26). We performed two rounds of amplification and found that only 1/20 of the DNA samples obtained from biopsies had a detectable KSHV sequence. The positive patient was also infected by the human immunodeficiency virus (HIV). Our data provide evidence that KSHV cannot be implicated in the pathogenesis of WM.
Recombinant human natural autoantibodies against GPIIb/IIIa inhibit binding of autoantibodies from patients with AITP
Authors: Escher  , Müller  , Vogel  , Miescher  , Stadler  , Berchtold
Published: March 2003

Autoimmune thrombocytopenic purpura (AITP) is caused by autoantibodies predominantly against platelet membrane glycoproteins (GP) IIb/IIIa and GPIb/IX. Naturally occurring autoantibodies have been described against a variety of autoantigens; it has been suggested that perturbation of their regulation may be associated with autoimmune diseases. Using a combinatorial Fab phagemid library from an individual immunized with human RhD+ red blood cells, we evaluated the presence of natural anti‐GPIIb/IIIa autoantibodies as well as their relation to AITP‐associated anti‐GPIIb/IIIa autoantibodies. Selection on native GPIIb/IIIa and characterization of positive clones by inhibition studies against murine monoclonal anti‐GPIIb/IIIa antibodies and by DNA analysis revealed the presence of two distinct recombinant anti‐GPIIb/IIIa autoantibodies, which partially inhibited binding of affinity‐purified platelet‐associated autoantibodies from 8/12 AITP patients. Our results demonstrated that GPIIb/IIIa‐specific Fab directed against conformational epitopes within the GPIIb/IIIa complex may be cloned from the genome of an individual immunized with RhD+ red blood cells, who was not affected by AITP. The partial inhibition of binding of platelet‐associated autoantibodies from AITP patients to GPIIb/IIIa by the recombinant anti‐GPIIb/IIIa phage clones suggests recognition of closely related antigenic epitopes. These phage clones may represent down‐regulated, potentially pathological autoantibodies and could be used as new tools for investigation of AITP.
Two M‐components in a single cell lineage in a patient with a dual isotype secretory B‐cell tumour
Authors: Sakashita Chizuko , Saito Takako , Kurosu Tetsuya , Yoshinaga Haruhiko , Kumagai Takasi , Yamamoto Koh , Miki Tohru , Koyama Takatoshi , Miura Osamu , Nemoto Tetsuo , Asakawa Hideo , Hirosawa Shinsaku
Published: March 2003

We describe a case of Waldenström's macroglobulinaemia with two M‐components (IgM and IgG) with the same λ light chain. Southern blot analysis of bone marrow cells showed rearrangements of immunoglobulin heavy and λ light chain genes.

Sequencing of the complementarity determining region 3 of the two γ and μ transcripts showed 100% homology. Immunofluorescence study showed that most cells stained for both IgG and IgM. These findings indicated that a single population of cells was expressing two isotypic variants of IgG and IgM, as the genes responsible for production of both components had the same origin.
Bb1‐3, a transgenic hybrid cell line with erythroid and megakaryocytic differentiation potential that expresses high levels of human γ‐globin and human β‐globin
Authors: Thomson Andrew M. , Roberts Nigel A. , Wood William G.
Published: March 2003

We have characterized a murine hybrid cell line, Bb1‐3, generated by the fusion of mouse primary erythroblasts with MEL cells. It proliferated in serum‐free medium and displayed a low level of spontaneous erythroid and megakaryocyte differentiation. Terminal erythroid differentiation could be induced with HMBA and DMSO and was enhanced by serum. Treatment with phorbol esters resulted in a high proportion of megakaryocytes and the expression of megakaryocytic specific lineage markers. Bb1‐3 cells contain a human β‐globin transgene that was expressed at levels of 20–50% of the endogenous mouse globin genes. Initially, expression was largely limited to the β‐globin gene but after adaptation to serum free growth, equal expression of both the human γ‐ and human β‐globin genes was observed. This cell line provides further evidence that the differentiation potential of mouse erythroleukaemia cells is not restricted to the erythroid lineage and should be useful to study the mechanisms underlying both developmental globin gene regulation and the terminal differentiation of bipotential erythroid/megakaryocytic progenitor cells.
Successful management of concurrent congenital dyserythropoietic anaemia and autoimmune haemolytic anaemia with splenectomy
Authors: Schmidt Mary Lou , Joshi Sarita , Dechristopher Phillip J. , Mihalov Michael , Sosler Steven D.
Published: March 2003

This first known case of concurrent congenital dyserythropoietic anaemia (CDA) and autoimmune haemolytic anaemia (AIHA) which occurred in a hispanic male and spanned 6 years from the age of 2. Light and electron microscopy of bone marrow erythroblasts and immunophenotyping confirmed CDA; serum/eluate warm autoantibodies and positive direct antiglobulin tests (DAT) associated with severe, episodic anaemias established AIHA. Cytogenetic analysis of bone marrow cells and peripheral blood lymphocytes ascertained sex chromosome aneuploidy (48 XY,+ Y,+ Y). Recurrent, life‐threatening episodes of transfusion‐dependent anaemia refractory to steroids and intravenous immune globulin, were put into stable remission at age 8 years when splenectomy successfully managed both disorders.
Polymorphism in CD33 and CD34 genes: a source of minor histocompatibility antigens on haemopoietic progenitor cells?
Authors: Raptis  , Clave  , Mavroudis  , Molldrem  , Van Rhee  , Barrett
Published: March 2003

Following bone marrow stem cell transplantation allo‐responses against haemopoietic progenitor cells (HPC), causing graft rejection and graft‐versus‐leukaemia effects, can be induced by donor T cells recognizing peptides derived from polymorphic endogenous proteins present in HPC. Since CD33 and CD34 are both expressed on HPC, we looked for genetic polymorphisms that might be the source of minor histocompatibility antigens (mHA) on such cells. Bone marrow from 14 donors and their HLA‐identical recipients undergoing BMT for haematological malignancies were studied. Using non‐radioactive single‐strand conformation polymorphism analysis (cold SSCP) of complementary DNA encoding CD33 and CD34, three DNA polymorphisms, two in CD33 and one in CD34 were found and sequenced. Two were in non‐coding regions, but in CD33, ATA or ATG at codon 183 resulted in an Ile or Met in the protein sequence. Nonapeptides derived from both alleles were predicted to bind to HLA A68.1. Thus two alleles of CD33 protein exist that could be mHA. With an alternate allele frequency of < 10%, allo‐responses against CD33 would be uncommon after marrow transplantation. However, donors homozygous for this allele could be used to generate cytotoxic T cells against the frequent CD33 allele, for adoptive therapy of leukaemia.
Blastic variant of mantle cell lymphoma shows a heterogenous pattern of somatic mutations of the rearranged immunoglobulin heavy chain variable genes
Authors: Pittaluga Stefania , Tierens Anne , Pinyol Magda , Campo Elias , Delabie Jan , DE Wolf‐Peeters Chris
Published: March 2003

Mantle cell lymphoma is a distinct clinicopathological entity associated with t(11;14) and cyclin D1 overexpression. The majority of cases show uniform morphological and phenotypic features characterized by a monotonous proliferation of small‐to‐medium‐sized irregular B cells that express CD5 and bright surface immunoglobulin IgM and IgD. By sequence analysis of the rearranged immunoglobulin heavy chain variable genes (VH), it has been shown that these lymphoma cells carry little if no somatic mutations, as described for the fetal CD5+ cells or B1 cells. Besides mantle cell lymphoma with classic histological features, a morphological variant of mantle cell lymphoma with blastic features and a more aggressive clinical course has been described. To investigate whether this variant is closely related, by the cell of origin, to typical cases, we analysed the presence and the pattern of somatic mutations of the VH genes in a series of nine cases diagnosed as such. Our cases of blastic mantle cell lymphomas rearrange most frequently VH4 and VH3 family genes. In three cases there was a complete homology to published germline genes, and a near complete homology was documented in another three. In contrast, the remaining three cases showed somatic mutations in their rearranged VH genes. Mutation analysis revealed evidence for antigen selection in one of these three cases. Taken together, these data are similar to those of normal adult‐type B1 cells and those described for chronic lymphocytic leukaemia (CLL) but slightly different to those reported for classic mantle cell lymphoma. It is likely that blastic mantle cell lymphoma as well as CLL originates from adult‐type B1 cells. More cases will need to be studied to determine whether classic mantle cell lymphoma is different from the blastic subtype and if it arises from fetal‐type B1 cells.
Distribution of prostaglandin IP and EP receptor subtypes and isoforms in platelets and human umbilical artery smooth muscle cells
Authors: Paul  , Ashby  , Sheth
Published: March 2003

Prostaglandins act through specific receptors to stimulate cyclic AMP formation which inhibits platelet activation and relaxes vascular smooth muscle. We have used RT‐PCR combined with Southern blot analysis to determine the subtypes of prostaglandin receptor on platelets. Platelets expressed the EP4 rather than the EP2 prostaglandin EP receptor subtype, whereas vascular smooth muscle cells predominantly expressed the EP2 receptor. The IP receptor, which binds prostacyclin and couples to stimulation of adenylyl cyclase, and three isoforms of the inhibitory EP3 receptor were equally expressed in platelets, HEL cells and umbilical artery smooth muscle cells. The EP3‐II isoform showed variation in level of expression among the three cell types. As a positive control for the presence of platelet RNA, PCR was performed using primers specific for the alpha chain of the platelet membrane glycoprotein Ib. As a negative control for the absence of T and B cell contamination in the platelet RNA, PCR was performed using primers specific for the cell specific cluster determinants CD2 (a T‐cell marker) and CD20 (a B‐cell marker). The finding that platelets express both stimulatory and inhibitory prostaglandin receptors provides confirmation of a homeostatic model of regulation of platelet adenylyl cyclase previously proposed.
Effect of thrombopoietin on proliferation of blasts from CD7‐positive acute myelogenous leukaemia
Authors: Tokunaga Yasunobu , Miyamoto Toshihiro , Okamura Takashi , Eto Tetsuya , Gondo Hisashi , Kamimura Tomohiko , Okamura Seiichi , Niho Yoshiyuki
Published: March 2003

We investigated the effect of thrombopoietin (TPO) on the growth of leukaemic blasts from 30 acute myelogenous leukaemia (AML) patients according to the surface expression of CD7 and CD34: 10 patients were CD7 positive (CD7+), nine were CD7 negative/CD34+(CD7−/CD34+) and the remaining 11 were CD7−/CD34−. Significant growth response of leukaemic blasts to TPO was observed in 10/10 CD7+, 5/9 CD7−/CD34+ and 2/11 CD7−/CD34− AML cases using 3H‐thymidine incorporation. Synergistic stimulatory effects of TPO with stem cell factor (SCF), interleukin‐3 (IL‐3), granulocyte colony‐stimulating factor and granulocyte‐macrophage colony‐stimulating factor were observed in both TPO‐responding cases (9/17) and TPO‐non‐responding cases (8/13). In a leukaemic blast colony assay, significant growth response to TPO was observed in 5/6 CD7+ and 4/17 CD7− AML cases examined. However, the effect of TPO on the growth of CD7+ leukaemic blasts was not so potent as that of IL‐3 and SCF, both of which support the proliferation of primitive haemopoietic progenitors. Expression of c‐mpl (TPO receptor) was significantly higher in CD7+ AML cases than in CD7− cases, suggesting a relationship between expression of c‐mpl and proliferative response to TPO. These data indicate that CD7+ leukaemic blasts express functional TPO receptors and proliferate in response to TPO. These observations also imply that CD7 expression on AML blasts may indicate involvement of leukaemic progenitors at an early stage of multipotent haemopoietic stem cells.
A comparative analysis of lipid‐complexed and liposomal amphotericin B preparations in haematological oncology
Authors: Clark  , Mckendrick  , Tansey  , Franklin  , Chopra
Published: March 2003

No comparative clinical information on the properties of lipid‐associated amphotericin preparations is presently available. In this single‐centre retrospective analysis over a 5‐year period the indications, efficacy and toxicity of true liposomal amphotericin (AmBisome®) were compared with a lipid complexed preparation (Abelcet®). In a novel approach APACHE III scores were used in addition to neutrophil counts, disease status and additional immunosuppression to accurately assess the severity of illness in both groups and enable valid comparison. Overall, AmBisome at a median dose of 1.9 mg/kg/d was found to have similar clinical outcome to Abelcet at a median dose of 4.8 mg/kg/d. Nephrotoxicity and electrolyte abnormalities were similar in both groups. Rigors and febrile episodes were more common with Abelcet. Prospective randomized comparative trials are required to clarify the optimum dosages and therapeutic and economic issues associated with these agents.
Status of Plasmodium falciparum towards catalase
Authors: Clarebout  , Slomianny  , Delcourt  , Leu  , Masset  , Camus  , Dive
Published: March 2003

The role of endogenous and internalized catalase in the protection of Plasmodium against oxidant stress was studied. Catalase activities were measured in isolated Plasmodium falciparum at different stages of intererythrocytic development. Activities measured at late schizont stages were compared to parasite markers (glutamate dehydrogenase, SOD) and to red blood cell markers (haemoglobin, Cu/Zn‐SOD). The fate of the host cell catalase in the parasite digestive system was studied by immunoelectron microscopy using monoclonal antibodies. The internalized catalase appeared to be dissociated in the digestive system of the parasite and inactivated. To examine the protective role of the endogenous and internalized catalase in the parasite protection against oxidant stress, parasites were cultivated at two oxygen concentrations (5% and 20%) in inhibited catalase red blood cells. These experiments suggested that the catalases present both in red blood cell and parasite are not essential when parasites are cultivated under 5% oxygen, but are necessary to protect the parasite under 20% oxygen. Catalase may not be the main protective enzyme involved in the protection of P. falciparum in standard in vitro culture conditions, but may become critical under the higher oxygen tensions conditions encountered in vivo.
Identification of transcription factors expressed during ATRA‐induced neutrophil differentiation of HL60 cells
Authors: Mills  , Walsh  , Gilkes  , Woodgate  , Brown  , Burnett
Published: March 2003

A recent clinical therapeutic initiative has been the use of chemical agents which induce the leukaemic cells to overcome their block in differentiation. In order to understand this block the cascade of molecular events needs to be characterized. Haemopoietic differentiation is ultimately controlled at the level of gene transcription which is mediated by an array of transcription factors. Many transcription factors contain similar structural protein sequences, and we have used an RT‐PCR‐based approach to isolate sequences, from transcription factor gene families which share similar domains. Degenerate primers corresponding to the TFIIIA zinc‐finger consensus amino acid sequences and to the POU‐homeodomain and POU‐specific domain were used to amplify genes on the basis that they contained similarities in structural motifs shared within these families of transcription factors. A serum‐independent HL60 cell line was induced towards the neutrophil lineage by treatment with all‐trans retinoic acid (ATRA) for 24 h. CD38+ cells committed towards this lineage were enriched and a population of these cells treated with dihydroxyvitamin D3 to induce neutrophil maturation. RNA extracted from uninduced, ATRA‐induced CD38+ cells, and vitamin D3treated maturing cell cultures were amplified using the degenerate primers. PCR fragments were cloned, sequenced, clustered into homologous groups, and the group sequences searched on the GenBank database. The Oct 1 transcription factor, and a very close homologue, KIAA0144, was identified using the POU family primers. The zinc‐finger primers identified three zinc‐finger genes. The pattern of gene expression was suggested from the number of clones in each group at neutrophil commitment and maturation. The differential expression of the genes in the zinc finger and POU families will lead to a better understanding of the cascade of gene expression which occurs following ATRA‐induced differentiation.
Apparent reactivation of a red cell alloantibody in a healthy individual after G‐CSF administration
Authors: Norol  , Bonin  , Charpentier  , Bierling  , Beaujean  , Cartron  , Bories  , Kuentz
Published: March 2003

Granulocyte‐colony stimulating factor (G‐CSF) has been successfully administered to healthy subjects to mobilize peripheral blood stem cells (PBSC) for allogeneic transplantation. Adverse events are moderate. We report the first case of apparent reactivation of an alloantibody to a blood cell antigen (Jka) after G‐CSF administration to a healthy subject and its transmission to the PBSC transplant recipient; no concomitant reactivation of other alloantibodies was detected. This case raises questions on the effect of G‐CSF on the immune system and its safety in healthy individuals.
Mobilization and transplantation of Philadelphia chromosome‐negative peripheral blood progenitor cells in patients with CML
Authors: Waller Cornelius F. , Heinzinger Monika , Rosenstiel Antonia , Lange Winand
Published: March 2003

Mobilization and transplantation of peripheral blood progenitor cells (PBPCs) was investigated in patients with stage IA or stage IIA chronic myelogenous leukaemia (CML) using combination chemotherapy with idarubicin, cytosine arabinoside and etoposide (ICE) followed by simultaneous administration of rhG‐CSF and rhIL‐2 or rhG‐CSF alone. 17 patients (stage IA: 12; stage IIA: five) were mobilized. Chemotherapy, cytokine priming and collection of PBPCs were well tolerated. Using large‐volume aphereses, a median number of 10.6 × 107/kg b.w. of MNC were harvested, which yielded between 0.15 and 21.0 × 106 CD34+ cells/kg b.w. High numbers of CD34+ HLA‐DR− cells could be obtained. Autologous transplantation of mostly Ph− apheresis products was performed in 16/17 patients after high‐dose chemotherapy. 10 patients achieved a complete or major cytogenetic remission (stage IA: seven; stage IIA: three), but six patients did not respond cytogenetically. Median follow‐up after transplantation was 18+ months. Our data show that this very efficient treatment modality for PBPC mobilization in CML was safe and able to induce major and sustained cytogenetic responses in the majority of patients.
Anti‐endothelial cell antibodies in primary antiphospholipid syndrome and SLE: patterns of reactivity with membrane antigens on microvascular and umbilical venous cell membranes
Authors: Hill  , Phipps  , Hughes  , Greaves
Published: March 2003

Although there has been recent emphasis on autoantibodies to epitopes on beta‐2‐glycoprotein I and prothrombin in the pathogenesis of antiphospholipid syndrome (APS), antibodies other than those directed toward epitopes on phospholipid binding proteins are present. These include those reactive with antigens on platelet membrane glycoproteins, and with vascular endothelial cell membrane. As the pathogenesis of the thrombotic manifestations of APS remains unexplained, further characterization of these antibodies may be informative. We have confirmed anti‐endothelial cell binding to a range of cell membrane antigens in systemic lupus erythematosus (SLE) and primary APS. Furthermore, differences in both the pattern of antibody binding and band intensity between human umbilical vein (HUVEC) and human microvascular endothelial cells (HMEC‐1) were demonstrated. Of 17 primary APS sera, antibody binding to HUVEC cell membranes was found in nine and to HMEC‐1 membranes in seven. Binding at 72–79 kD was confined to HUVEC. In 32 SLE sera, binding to HUVEC and HMEC‐1 membranes was detected in 17 and 22 respectively, binding at 135–155 kD being confined to HMEC‐1. These results are consistent with the phenotypic variation in endothelial cells of different origins and confirm the frequent presence of autoantibodies reactive with vascular endothelium in both SLE and PAPS. Whether these antibodies could be involved in the pathogenesis of thrombosis, through induction of endothelial cell apoptosis or damage, remains to be determined.
In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice
Authors: Turner Curtis W , Archer David R , Wong John , Yeager Andrew M , Fleming William H
Published: March 2003

We have previously demonstrated that high levels of allogeneic, donor‐derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34+ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45+ human cells. CD45+ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3+ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16‐fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.
Long‐term administration of G‐CSF for aplastic anaemia is closely related to the early evolution of monosomy 7 MDS in adults
Authors: Kaito Ken , Kobayashi Masayuki , Katayama Toshio , Masuoka Hidekazu , Shimada Takaki , Nishiwaki Kaichi , Sekita Toru , Otsubo Hiroko , Ogasawara Yoji , Hosoya Tatsuo
Published: March 2003

There is an increasing incidence of the evolution of myelodysplastic syndrome (MDS) from aplastic anaemia (AA) with immunosuppressive treatment. In paediatric patients G‐CSF is also reported to increase MDS evolution, but this process is not precisely understood in children or in adults. Therefore risk factors of MDS evolution in adults are evaluated here. Of 72 patients, five developed MDS. In 47 patients without cyclosporine (CyA) or antithymocyte globulin (ATG) therapy, only one developed MDS with trisomy 8, 242 months after diagnosis. But of 25 patients treated with either CyA or ATG, four developed monosomy 7 MDS within 3 years. Of these 25 patients, 18 were treated with G‐CSF and the four patients (22.2%) who developed MDS were found in this group. The cumulative dose and the duration of G‐CSF administration were significantly elevated in patients who developed MDS when compared with those who did not, 822.3 ± 185.0 v 205.4 ± 25.5 μg/kg (P < 0.05) and 187.5 ± 52.5 v 72.0 ± 24.6 d (P < 0.002), respectively. However these two values for CyA did not differ significantly. Statistically, treatment with CyA, G‐CSF and combined G‐CSF and CyA were significantly related to MDS evolution. The administration of G‐CSF for more than a year was the most important factor (P = 0.00). These results suggested that a close relationship exists between G‐CSF and subsequent monosomy 7 MDS from AA in adults who receive immunosuppressive therapy. Long‐term administration of G‐CSF should be prohibited in order to prevent MDS evolution.
Splicing and missense mutations in the human FXIIIA gene causing FXIII deficiency: effects of these mutations on FXIIIA RNA processing and protein structure
Authors: Anwar Rashida , Gallivan Louise , Miloszewski Krzysztof J. A. , Markham Alexander F.
Published: March 2003

We have investigated the molecular basis of factor XIII (FXIII) deficiency in a family from the north‐west region of the U.K. and identified two sequence changes in the FXIII subunit A (FXIIIA) gene. We report a novel Asn to Lys mutation at codon 541, and a g → a mutation at the intron 5/exon 6 splice junction in the FXIIIA gene. The splicing mutation results in two abnormal FXIIIA transcripts. The Asn541 residue is important for stabilizing an external fold in the FXIIIA barrel 1 domain. The Asn541Lys mutation is expected to result in inappropriate folding and therefore an unstable FXIIIA molecule.
Effects of r‐metHuG‐CSF on polymorphonuclear leucocyte kinetics and function in patients on continuous ambulatory peritoneal dialysis
Authors: Turzanski  , Crouch  , Andrews  , Rose  , Finch  , Burden  , Holliday  , Fletcher
Published: March 2003

End‐stage renal failure (ESRF) patients undergoing continuous ambulatory peritoneal dialysis (CAPD) are immunocompromised and exhibit abnormal circulating polymorphonuclear leucocyte (PMN) function, including reduced phagocytosis and intracellular killing. Six uraemic patients on CAPD were each given 300 μg granulocyte‐colony stimulating factor (G‐CSF) every day for 5 d and PMN function tests were performed daily. By day 5 of the study CD11b expression was significantly decreased in response to N‐formylmethionylleucylphenylalanine (fMLP) and opsonized Staphylococcus epidermidis stimulation, and expression of L‐selectin (CD62L) was significantly decreased in response to opsonized Staphylococcus epidermidis stimulation. Further, superoxide anion production and FcγRI (CD64) expression were found to be significantly increased and FcγRIII (CD16) expression was lowered. Circulating white cell and PMN counts were significantly elevated in response to treatment. Administration of G‐CSF did not appear to have corrected the abnormalities in phagocytosis and intracellular killing. This study suggests that G‐CSF does no harm to ESRF patients and influences uraemic PMN function in a manner that is comparable to its effects on PMN in non‐uraemic subjects.
Cerebral candidiasis in a child 1 year after leukaemia
Authors: Tweddle Deborah A. , Graham J. Clive , Shankland Gillian S. , Kernahan Jennifer
Published: March 2003

We describe an unusual case of a late presentation of a fungal brain abscess in a non‐neutropenic child 1 year after completing chemotherapy for M5 acute myeloid leukaemia (AML). Biopsy of the mass identified candidal hyphae and the patient was treated with 5 mg/kg of liposomal amphotericin B for 6 weeks. The lesion resolved completely and the child remains well 2 years later. Invasive fungal infection should be included in the differential diagnosis of unexplained symptoms in patients who have previously received intensive chemotherapy.
Extensive phenotypic analysis of CD34 subsets in successive collections of mobilized peripheral blood progenitors
Authors: Perey Lucien , Peters Rowayda , Pampallona Sandro , Schneider Philippe , Gross Nicole , Leyvraz Serge
Published: March 2003

The transplantation of mobilized progenitor cells after high‐dose chemotherapy shortens haemopoietic engraftment. CD34 cell subsets were examined in 20 consecutive mobilized progenitor cell collections obtained from patients with solid tumours that had not been previously treated. The analysis of CD34 cells was based on the expression of intracellular antigens, surface antigens including CD38, and cell size using multi‐dimensional flow cytometry. We also correlated the numbers of stem cell subsets reinfused to haemopoietic recovery.

The majority of CD34+ cells expressed CD13 and CD33. A significant proportion was cytoplasmic myeloperoxidase (cMPO) positive. CD34+ MPO+ cells increased significantly in late collections. MPO expression was related to cell size. Cells expressing CD13 also increased in late collections in parallel to CFU‐GM count. Small subpopulations of CD34+ CD38+ were committed to B cells, T cells and erythroid cell lineages. A small population expressing the megakaryocytic antigen had a small size and were predominantly CD38−. A minor subpopulation expressed stem cells antigens. These were significantly higher in late collections (CD34+ Thy‐1+ and CD34+ CD33−). After mobilization, patients received three cycles of intensive chemotherapy followed by reinfusion of mobilized progenitors (5.45 × 106/kg CD34+ cells, range 3.4–11.88). The numbers of reinfused CD34 cells or the individual subsets did not influence recovery of leucocytes (9 d) or platelets (9 d).

In conclusion, the numbers of stem cells and their subsets differed between collections and, in unpretreated patients receiving intensive chemotherapy, there was no delayed engraftment when sufficient numbers of stem cells were reinfused. The recovery period was short and not correlated to any stem cell subsets.
Two novel mutations (Pro864His, Val867Glu) causing type 2A von Willebrand disease and affecting a single restriction site in exon 28
Authors: Bernardi  , Casonato  , Marchetti  , Gemmati  , Bizzaro  , Pontara  , Girolami
Published: March 2003

We detected two transversions in two unrelated Italian patients with type 2A von Willebrand disease (VWD): a C to A at nucleotide 8821 and a T to A at nucleotide 8830, resulting in the missense mutations Pro864His and Val867Glu respectively. Both mutations were in the heterozygous form and abolished the BstXI restriction site in exon 28 of the VWF gene. In both mutations plasma VWF multimer pattern improved by antiproteases. Moreover, DDAVP normalized plasma VWF multimers in the Pro864His patient, especially when protease inhibitors were present. These new mutations appear to be of the 2A VWD subtype due to the increased susceptibility to proteases.
Anti‐CD36 autoantibodies in thrombotic thrombocytopenic purpura and other thrombotic disorders: identification of an 85 kD form of CD36 as a target antigen
Authors: Schultz Duane R. , Arnold Patricia I. , Jy Wenche , Valant Peter A. , Gruber Julie , Ahn Yeon S. , Mao Fang W. , Mao Wei W. , Horstman Larry L.
Published: March 2003

The presence of anti‐CD36 antibodies in plasma of patients with thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), and heparin‐induced thrombocytopenia without/with thrombosis (HIT/HITT) has been examined by immunoblots, and a monoclonal antibody capture assay, the platelet‐associated IgG characterization assay (PAICA). Results with PAICA showed that 73% (8/11) of patients with TTP were positive, and 71% (10/14) by immunoblots. With ITP, 20% (6/30) were positive by PAICA and 19% (3/16) by immunoblots; HIT, 30% (3/10) were positive by PAICA and 60% (6/10) by immunoblot; HITT, 50% (2/4) by PAICA and 100% (4/4) by immunoblot. Purification of CD36 by fast protein liquid chromatography (FPLC) from Triton X‐100 extracts of normal platelet membranes resulted in the isolation of two different forms: the classic 88 kD form, and a second, lighter 85 kD form. Our data indicated that the patients' plasma autoantibodies reacted strongly with the 85 kD form. Conventional monoclonal and polyclonal antisera produced to the 88 kD form reacted strongly with the 88 kD form but weakly with the 85 kD form. These results confirm the possible importance of anti‐CD36 antibodies in the pathophysiology of TTP and other thrombocytopenias and demonstrate the presence of a previously unrecognized target antigen for these antibodies.
The impact of antenatal and perinatal variables on cord blood haemopoietic stem/progenitor cell yield available for transplantation
Authors: Shlebak  , Roberts  , Stevens  , Syzdlo  , Goldman  , Gordon
Published: March 2003

We investigated the impact of maternal and fetal variables on cord blood (CB) haemopoietic stem/progenitor cell content. These included maternal age, ethnic origin, parity, ABO and Rhesus D blood group, antenatal haemoglobin, alcohol and cigarette consumption at time of registration, mode of delivery, duration of the first and second stages of labour, gestational age, birth weight, cord pH and cord erythrocyte mean cell volume (MCV). Cord volumes and total nucleated cellularities (TNC) were recorded, the colony assay for granulocyte‐macrophage colony‐forming‐cells (CFU‐GM) was used to quantify the progenitor cells and the potential of CFU‐GM to produce secondary colonies on replating was used as a measure of progenitor cell quality. We found: (1) significantly greater ( P = 0.04) volumes were collected from babies who weighed  2.5 kg versus babies with a birth weight < 2.5 kg; (2) significantly greater numbers of mononuclear cells (MNC) from mothers who drank 0–3 units versus those who drank  4 units of alcohol weekly ( P = 0.03), and in babies with a cord pH  7.1 v > 7.1 ( P = 0.02); (3) Significantly greater numbers of cord CFU‐GM in mothers who drank 0–3 v 4 units weekly ( P = 0.004) and smokers of  10 v 0–9 cigarettes daily ( P = 0.02) and in spontaneous vaginal deliveries than assisted vaginal and caesarean deliveries ( P = 0.04), and (4) the potential of CFU‐GM to produce secondary colonies was significantly greater in CB derived from Caucasians than from non‐Caucasians ( P = 0.02); in assisted vaginal delivery v spontaneous vaginal ( P = 0.02) and in deliveries with prolonged first stage of labour v short first stage of labour ( P = 0.04). We conclude that antenatal and perinatal variables may influence the CB stem/progenitor cell yield available for transplantation.
In vitro antisickling activity of cromolyn sodium
Authors: Fall Ahmedou B. K. , Toppet Michèle , Ferster Alina , Fondu Pierre , Vanhaelen‐Fastré Renée , Vanhaelen Maurice
Published: March 2003

The improvement in sickle cell disease (SCD) children receiving cromolyn sodium therapy prompted us to investigate its antisickling activity in vitro. The number of sickle cells was determined in deoxygenated blood samples from 15 children with severe SCD. At the eight concentrations tested, cromolyn sodium exhibited a significantly higher activity than pentoxifylline, the standard compound. Therefore cromolyn sodium would appear to be an interesting candidate for SCD therapy and deserves further in vivo investigations.