bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical relevance of T‐cell receptor delta gene rearrangements in childhood B‐precursor cell acute lymphoblastic leukaemia
Authors: DÍaz M. A. , Garcia‐Sanchez F. , Vicario J. L. , Ramirez M. , Balas A. , Madero L.
Published: November 2003

The large majority of childhood B‐precursor cell acute lymphoblastic leukaemia cases present IgH and TCRδ gene rearrangements. These rearrangements have been widely used as specific markers for monitoring minimal residual disease. However, their prognostic value still remains unclear. In order to determine whether IgH and TCRδ gene rearrangements have any influence on relapse and event‐free survival (EFS), we analysed the clinical impact of these genetic characteristics in 51 B‐precursor acute lymphoblastic leukaemia patients. 46/51 patients (90.2%) showed IgH gene rearrangements by Southern blot and/or polymerase chain reaction (PCR) analysis. No statistically significant associations were found between IgH gene rearrangement pattern and age, sex, WBC count, immunophenotype, risk factor, relapse or EFS. 27/41 patients (66%) showed Vδ2Dδ3 recombination by Southern blot and/or PCR analysis. At a median follow‐up of 53 months the estimated 5‐year EFS probability was 78 ± 3% for the whole group. The EFS probability among patients with a Vδ2Dδ3 recombination pattern in the TCRδ locus was 90 ± 3%, whereas for patients without Vδ2Dδ3 recombination was 39 ± 13% (P < 0.005).

IgH rearrangement patterns do not appear to influence relapse or EFS probability. However, TCRδ gene rearrangement patterns have a relevant impact on the relapse rate and the EFS probability. Patients with Vδ2Dδ3 recombination have better clinical outcome than patients without this recombination, independent of any other prognostic factors.
Role of the CD40 and CD95 (APO‐1/Fas) antigens in the apoptosis of human B‐cell malignancies
Authors: WANG DAKUN , FREEMAN GORDON J. , LEVINE HERBERT , RITZ JEROME , ROBERTSON MICHAEL J.
Published: November 2003

Ligation of CD40 inhibits apoptosis and stimulates proliferation of normal B cells, whereas ligation of CD95 (APO‐1/Fas) induces apoptosis of activated lymphocytes. Aberrant signalling through the CD40 and CD95 antigens could thus participate in the pathogenesis of lymphoid malignancies. The expression and function of CD40 and CD95 on neoplastic B cells from patients with acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL) and non‐Hodgkin’s lymphoma (NHL) were examined. CD40 was expressed by all 30 B‐cell tumours, whereas CD95 was detected on neoplastic B cells in only one of 10 cases of ALL, two of 10 cases of CLL, and three of 10 cases of NHL. Incubation with an agonistic CD95 monoclonal antibody (MoAb) did not augment apoptosis in any of the unstimulated B‐cell neoplasms. CD40 triggering did not consistently inhibit spontaneous apoptosis, but ultimately stimulated the growth of neoplastic B cells in most cases. Furthermore, CD40 activation led to up‐regulation of the CD95 antigen in all 30 B‐cell neoplasms. Ligation of CD95 on CD40‐activated tumour cells augmented apoptosis in five of 10 ALL, three of 10 CLL, and nine of 10 NHL cases. The degree of apoptosis induced by CD95 triggering was greater for NHL cells than for ALL cells or CLL cells. Bcl‐2 expression by ALL and NHL cells was substantially decreased after in vitro culture, whereas Bcl‐2 expression by CLL cells was not significantly changed. However, there was no correlation between the level of Bcl‐2 expression and sensitivity to CD95‐mediated apoptosis. Thus, factors other than levels of CD95 and Bcl‐2 determine susceptibility of malignant B cells to apoptosis after CD95 triggering. CD40‐activated lymphoma cells appear to be very sensitive to CD95‐mediated apoptosis, suggesting potential strategies for treatment of NHL. Elucidation of the mechanisms underlying resistance of ALL and CLL cells to CD95 triggering may facilitate the development of novel therapeutic approaches to these diseases as well.
Rheological analysis of the formation of rosettes by red blood cells parasitized by Plasmodium falciparum
Authors: Chu Yen , Haigh Teresa , Nash Gerard B.
Published: November 2003

Red blood cells infected by mature malarial parasites of the species Plasmodium falciparum can adhere to non‐parasitized red cells (rosetting) and also to endothelial cells (cytoadhesion). To investigate how the circulation might influence rosetting, we studied formation of rosettes in cell suspensions sheared in a cone‐and‐plate viscometer, and the ability of flowing non‐parasitized cells to bind to parasitized cells already adherent to a surface. After rosettes of strain R29 had been disrupted with fucoidan, they reformed slowly under stationary conditions but more rapidly in suspensions sheared at low stress (about 0.1–0.2 Pa). Strain Malayan Camp gave a lower rosetting frequency which actually increased at low shear. Increasing shear stress was associated with reduction in rosette formation, although rosetting occurred at >1 Pa, suggesting that rosettes could form in the systemic circulation. Rosetting inhibited adhesion of flowing parasitized cells to immobilized platelets (which express the cytoadhesion receptor CD36), as evidenced by increased adhesion after disruption of rosettes. The de‐rosetted adherent cells parasitized by R29 supported only a low level of rosetting when non‐parasitized cells were flowed over them at a wall shear of 0.1 Pa, with little increase if the stress was decreased to 0.05 Pa. Rosettes formed in the circulation might obstruct microvessels and inhibit cytoadhesion if they reached venules. However, if cytoadhesion occurred before rosetting, then adherent cells should not efficiently form rosettes.
Novel point mutation in the leucine‐rich motif of the platelet glycoprotein IX associated with Bernard‐Soulier syndrome
Authors: Suzuki Keijiroh , Hayashi Tomohiro , Yahagi Akito , Akiba Jiroh , Tajima Katsushi , Satoh Shinji , Sasaki Hideo
Published: November 2003

Bernard‐Soulier syndrome (BSS) is a rare inherited bleeding disorder which is caused by a qualitative or quantitative abnormality of the platelet glycoprotein (GP) Ib/IX/V complex. We examined a patient with BSS to find a molecular basis for the defect underlying this disease. The propositus was a 39‐year‐old Japanese female with life‐long bleeding diathesis. Sequence analysis of the GPIX gene revealed a T → C point mutation at nucleotide 1856 (EMBL, M80478), resulting in Phe55(TTT) → Ser(TCT) replacement. This substitution created a new MnlI restriction site in the mutant allele. Restriction analysis revealed that the propositus was homozygous for this sequence, and the same mutation was not detected in 57 unrelated Japanese subjects. Since this mutation is located in the leucine‐rich motif (LRM) of the GPIX polypeptide, the Phe55 → Ser substitution may result in an alteration of the LRM which leads to the impaired surface expression of GPIb/IX/V complex, a characteristic of BSS.
Aminoglycosides prevent and dissociate the aggregation of platelets in patients with EDTA‐dependent pseudothrombocytopenia
Authors: Sakurai Susumu , Shiojima Ichiro , Tanigawa Takeshi , Nakahara Kazuhiko
Published: November 2003

Although EDTA‐dependent pseudothrombocytopenia (EDTA‐PTCP) is of practical importance because failure to recognize this clinical entity may result in misdiagnosis and subsequent mismanagement of the patients, the pathophysiological nature of EDTA‐PTCP remains unknown. To develop an effective way to evaluate the platelet counts in patients with EDTA‐PTCP, we introduced aminoglycosides‐supplemented anticoagulating agents. When kanamycin was pre‐supplemented with EDTA for anticoagulating blood samples from EDTA‐PTCP patients there was no significant change in the platelet counts and the morphology of blood cells after 150 min of incubation at room temperature. Furthermore, when kanamycin was added to EDTA‐anticoagulated blood samples from EDTA‐PTCP patients within 30 min after blood withdrawal, rapid dissociation of platelets without apparent morphological changes of blood cells was observed, and complete blood cell counts as well as the histogram patterns were almost the same as those examined immediately after blood sampling. The dissociation of aggregated platelets was also detected when other antibiotics were used, although it was associated with some extent of morphological changes of blood cells. These findings indicate that the supplementation of aminoglycosides either before or after blood sampling is a useful method for the diagnosis EDTA‐PTCP and for the evaluation of platelet counts in patients with EDTA‐PTCP.
Alterations in Bcl‐2/Bax protein levels in platelets form part of an ionomycin‐induced process that resembles apoptosis
Authors: Vanags Daina M , Orrenius Sten , Aguilar‐Santelises Miguel
Published: November 2003

Platelets are physiologically anucleated cells, derived from megakaryocytes, that undergo vesiculation and transformation into small particles when they are stimulated in vitro by ionomycin and other agents. Electron microscopy images suggest a similarity to apoptosis in cells with nuclei, which ends with cell disintegration and formation of apoptotic bodies. By PCR, we have demonstrated mRNA expression of bcl‐2, baxα and p53 in highly purified non‐stimulated platelets. A side‐scatter shift and a decrease in the Bcl‐2/Bax protein ratio were observed by flow cytometry analysis after stimulation with ionomycin. The ionomycin‐induced modifications were inhibited by the calpain I inhibitor calpeptin and, less effectively, by VAD‐cmk, a broad‐spectrum caspase inhibitor. However, caspase 3‐like activity was very low, with only a twofold increase after ionomycin stimulation, as measured by the cleavage of the fluorogenic peptide substrate DEVD‐AMC. Our data indicate that platelets may constitute a natural model for the analysis of cytoplasmic events in apoptosis.
Decreased half‐life time of plasma von Willebrand factor collagen binding activity in essential thrombocythaemia: normalization after cytoreduction of the increased platelet count
Authors: Van Genderen Perry J. J , Prins Fransisco J , Lucas Irene S. , Van De Moesdijk Desiree , Van Vliet Huub H. D. M. , Van Strik Roel , Michiels Jan J.
Published: November 2003

Patients with essential thrombocythaemia (ET) exhibit a decrease of large von Willebrand factor (VWF) multimers in plasma, which is inversely related to the platelet count. In the present study we investigated whether the decrease of large VWF multimers in plasma with increasing platelet counts is the consequence of increased turnover of large VWF multimers in vivo. To that end we measured the half‐life times of endogenously released VWF:Ag and VWF:CBA (collagen binding activity) after intravenous administration of desmopressin (DDAVP) to nine ET patients and nine control subjects (N). In addition, the half‐life times of VWF:Ag and VWF:CBA were also measured in four ET patients after cytoreduction of the increased platelet count to normal or nearly normal values. Estimated half‐life times of VWF:Ag did not differ between ET patients and normals (11.0 ± 4.0 h v 12.4 ± 2.5 h, P  > 0.05). Estimated half‐life times of VWF:CBA were significantly lower in ET patients as compared with normal individuals (6.1 ± 2.0 h v 8.4 ± 2.5 h, P  < 0.05). After cytoreduction of the increased platelet count to (nearly) normal values in all four ET patients the half‐life time of VWF:CBA significantly (P = 0.014) increased from 5.2 ± 1.2 h to 8.7 ± 2.0 h. Our data suggest that platelets may play a role in the homeostasis of circulating von Willebrand factor, which may compromise normal haemostasis at fairly increased platelet counts.
t(14;18) chromosomal translocation in follicular lymphoma: an event occurring with almost equal frequency both at the D to JH and at later stages in the rearrangement process of the immunoglobulin heavy chain gene locus
Authors: Stamatopoulos Kostas , Kosmas Christos , Belessi Chrysoula , Papadaki Theodora , Afendaki Stavroula , Anagnostou Dimitra , Loukopoulos Dimitris
Published: November 2003

bcl‐2/IgH fusion is considered a genetic error which occurs at the diversity (D) to joining (JH) stage of the gene rearrangement process in the immunoglobulin heavy chain (IgH) gene locus. Translocations of the bcl‐2 protooncogene to the IgH locus at ontogenetically later IgH gene rearrangements are thought to represent exceptions. In the present study we analysed the junctional nucleotide sequence of 18 bcl‐2/IgH fusion genes identifiable by polymerase chain reaction performed on DNA extracted from diagnostic lymph node tissue of 14 follicular lymphoma patients. In all clones studied, segments of variable length were found interposed between bcl‐2 and JH gene sequences. Nucleotide sequence data analysis and comparisons performed with the corresponding germline sequences using the GenBank/EMBL database revealed the presence of D segments in most of the bcl‐2/IgH fusion genes under study (13/18). By the same kind of computer‐aided analysis, previously unrecognized D segments were identified in many published junctional sequences. These results suggest that bcl‐2/IgH fusion events are very prevalent in rather more differentiated stages in B‐cell ontogeny than previously recognized.
Comparative detection and quantitation of human CDK inhibitor mRNA expression of p15INK4B, p16INK4A, p16β, p18INK4C, p19INK4D, p21WAF1, p27KIP1 and p57KIP2 by RT‐PCR using a polycompetitive internal standard
Authors: Schwaller J. , Pabst Th. , Bickel M. , Borisch B. , Fey M. F. , Tobler A.
Published: November 2003

For comparative and quantitative analysis of human cyclin‐dependent kinase inhibitor gene expression (CKI; p15INK4B, p16INK4A, p16β, p18INK4C, p19INK4D, p21WAF1, p27KIP1 and p57KIP2) we set up an RT‐PCR assay with a construct termed pCKIquant producing polycompetitive RNA as an internal standard. We demonstrated the reproducibility, accuracy and high sensitivity of the assay in the in vitro model of myeloid leukaemic HL‐60 cells. We also showed that the pCKIquant CKI assay is an excellent tool for the assessment of CKI mRNA expression in clinical samples, e.g. single cryostat sections of lymphoma biopsies.
Thrombotic thrombocytopenic purpura in a patient with genetic haemochromatosis, liver cirrhosis and an iron‐free focus
Authors: Killick S. , Jeffery S. , Otter M. , Rist C. , Bevan D.
Published: November 2003

We report a patient with thrombotic thrombocytopenic purpura and genetic haemochromatosis. The patient was shown by the polymerase chain reaction to be homozygous for the Cys282Tyr mutation of the HFE gene. Liver biopsy showed micronodular cirrhosis and the presence of an iron‐free focus which was thought to be pre‐neoplastic.
Transforming growth factor‐beta 1 delays formation of granulocyte‐macrophage colony‐forming cells, but spares more primitive progenitors during ex vivo expansion of CD34+ haemopoietic progenitor cells
Authors: Garbe Annette , Spyridonidis Alexandros , Möbest Dieter , Schmoor Claudia , Mertelsmann Roland , Henschler Reinhard
Published: November 2003

Ex vivo culture and expansion of autologous haemopoietic transplants has been developed to improve tumour cell purging and accelerate haemopoietic reconstitution by transplantation of increased progenitor cell numbers. We studied the effect of the negative haemopoietic regulator, transforming growth factor beta‐1 (TGF‐beta 1) on primitive precursors during ex vivo expansion of CD34+ cells. When added directly to methylcellulose colony‐forming assays, TGF‐beta 1 potently suppressed the development of granulocyte‐macrophage colonies from CD34+ enriched peripheral blood progenitor cells (80–90% inhibition). In contrast, expansion of total nucleated cells and granulocyte‐macrophage colony‐forming cells (GM‐CFC) from CD34+ progenitors in liquid culture in the presence of stem cell factor (SCF), interleukin (IL)‐1 beta, IL‐3, IL‐6 and erythropoietin (EPO) was inhibited to 32–65% of control culture levels within 14 d when TGF‐beta 1 was added, and still produced an average 3.3‐fold absolute amplification of GM‐CFC. The inhibitory effect of TGF‐beta 1 on GM‐CFC generation was reversed when it was washed out on day 6 of ex vivo expansion cultures, and total numbers of GM‐CFC generated from expansion cultures then reached levels of untreated controls by day 16. Long‐term bone marrow culture‐initiating cell (LTCIC) numbers were preserved, at least at input levels, over a culture period of 14 d both in control and TGF‐beta‐1‐treated expansion cultures. These findings suggest that TGF‐beta 1, a cytokine which induces apoptosis or terminal differentiation in a number of malignant cell types, may be added to ex vivo expansion cultures without loss of primitive cells from autologous haemopoietic transplants.
Severe erythrocyte adenylate kinase deficiency due to homozygous A → G substitution at codon 164 of human AK1 gene associated with chronic haemolytic anaemia
Authors: Qualtieri A. , Pedace V. , Bisconte M. G. , Bria M. , Gulino B. , Andreoli V. , Brancati C.
Published: November 2003

A child of Italian origin with a congenital haemolytic anaemia had spectrophotometrically undetectable erythrocyte adenylate kinase (AK) activity. Her parents and brother had approximately 50% normal AK activity, and AK electrophoresis of red blood cell (RBC) crude extract on cellulose acetate strips showed the presence of the normal allele AK1‐1. No AK band was detected in the AK electrophoresis of the proband, in whom the erythrocyte 2,3‐diphosphoglycerate (2,3DPG) and glutathione (GSH) concentrations were normal whereas adenosine triphosphate (ATP) concentration, pyruvate kinase (PK) and glucose‐6P‐dehydrogenase (G6PD) activities were increased, reflecting the high reticulocyte count (6.9%). No other evident enzymatic defect was detected by standard procedures. Analysis of AK gene exons, based on polymerase chain reaction–single‐strand conformational polymorphism (PCR‐SSCP), clearly showed an abnormality in the fragment containing exon 6. The subsequent sequence analysis of this abnormal fragment revealed homozygous and heterozygous A → G substitutions in the proband and in the parents and brother respectively at codon 164, corresponding to a tyrosine → cysteine substitution in the AK protein.
Quantitative characterization and potential function of membrane Fas/APO‐1 (CD95) receptors on leukaemic cells from chronic B and T lymphoid leukaemias
Authors: Kamihira Shimeru , Yamada Yasuaki , Hirakata Yoichi , Tsuruda Kazuto , Sugahara Kazuyuki , Tomonaga Masao , Maeda Takahiro , Tsukasaki Kunihiro , Atogami Sunao , Kobayashi Nobuyuki
Published: November 2003

The expression and function of the Fas‐receptor (Fas‐R) were examined in chronic lymphocytic leukaemia (CLL), hairy cell leukaemia‐variant (HCL‐v) and adult T‐cell leukaemia (ATL). The expression of Fas‐R in freshly isolated leukaemic cells was qualitatively and quantitatively different between each disease; faint in B‐CLL, moderate in HCL‐v and strong in ATL. Both full‐length and alternatively spliced truncated forms of Fas mRNA were detected even in CLL B cells with faint to negative Fas‐R, and Fas mRNA was also shown to be capable of increasing in vitro expression, i.e. the message was functional. In contrast, Fas‐R expression on ATL cells was heterogenous and usually intense with a mean density approximately 3‐fold higher than that of normal T cells. Fas‐R was confirmed to have the potential function for anti‐Fas monoclonal antibody‐mediated cell death in vitro in Fas‐R+ ATL cells. The expression level of Fas‐R on the cells was higher in chronic than acute ATL (10360 v 6260 antibody‐binding capacity per cell, mFasABC; P < 0.05) and was inversely correlated with serum LDH activity, suggesting that the strong Fas‐R accounts for the slow progression of chronic ATL and the negative Fas‐R protects from Fas‐mediated cell death. These results show that Fas‐R expression on leukaemic cells is valuable in their characterization and perhaps their function, and may contribute to the progression and immune evasion of malignant clones.
One‐tube method for complete HPA‐1 genotyping by PCR using sequence‐specific primers
Authors: Boldt B. , Skogen B. , Agostini T. , Roscetti D. , McFarland J.
Published: November 2003

Human platelet antigens (HPA) can be targets for antibody responses that cause life‐threatening thrombocytopenia following platelet transfusions or pregnancy. As an aid to diagnosis and prevention, serologic and DNA‐based methods have been developed to type HPA. Of the DNA‐based strategies, those using the polymerase chain reaction (PCR) are very sensitive, but often require processing of amplification products. Sequence‐specific primers (SSP) in the PCR eliminate the need for extensive handling of reaction products beyond gel electrophoresis. However, current methods require a separate reaction for each allele being typed. In this report we describe a method to simultaneously and completely genotype both alleles of HPA‐1 in a single PCR. In addition, because the absence of an amplification product might also show the failure of a SSP, we introduced a recombinant template that can only be amplified by the SSP, thus ensuring primer performance and the identified genotype.
Intermittent exposure to doxorubicin in vitro selects for multifactorial non‐P‐glycoprotein‐associated multidrug resistance in RPMI 8226 human myeloma cells
Authors: Wyler Beat , Shao Ying , Schneider Erasmus , Cianfriglia Maurizio , Scheper Rik J. , Frey Beat M. , Gieseler Frank , Schmid Luzius , Twentyman Peter R. , Lehnert Manfred
Published: November 2003

The purpose of the present study was to evaluate whether intermittent exposure to a constant dose of doxorubicin selects for multidrug resistance (MDR) in RPMI 8226 human myeloma cells and, if so, to determine the molecular mechanism. In an attempt to approximate clinical doxorubicin treatment in vitro, cells were exposed to a fixed dose of doxorubicin for 4 d alternating with growth in drug‐free medium for 17 d. An MDR subline emerged, termed 8226/DOXint5, which was 3–4‐fold resistant to doxorubicin, etoposide and m‐AMSA, and 1.6‐fold resistant to vincristine. Sensitivity to docetaxel, melphalan and cisplatin was normal. Verapamil normalized vincristine sensitivity but had little effect on resistance to the other agents. Cellular uptake and retention of daunorubicin and vincristine were reduced by approximately 10%. The 8226/DOXint5 cells showed diminished DNA topoisomerase IIα expression and increased expression of the multidrug resistance protein MRP. Expression of MDR1/P‐glycoprotein was not detected. Immunostaining showed 70% of the cells to over‐express the lung‐resistance protein LRP. This new MDR myeloma cell line may prove to be a useful model for the development of strategies to overcome low‐level, multifactorial MDR, which might be a common phenomenon in clinical myeloma treated with doxorubicin.
Spectrin Cosenza: a novel β chain variant associated with Sp αI/74 hereditary elliptocytosis
Authors: Qualtieri A. , Pasqua A. , Bisconte M. G. , Le Pera M. , Brancati C.
Published: November 2003

A Calabrian family (Southern Italy) with Sp αI/74 hereditary elliptocytosis (HE) in the heterozygous state was studied. Sp αI/74 HE is associated with asymptomatic elliptocytosis, a defect in spectrin dimer self association and an increase of the αI/74 kD fragment from the α chain after partial tryptic digestion of spectrin. To identify the underlying molecular defect, we analysed exons V, W, X, Y, Z of the β gene and exon 2 of the α gene by single‐strand conformational polymorphism (SSCP) of the amplification products. Direct DNA sequencing of the mutant exon showed a C →G substitution at position 6284 of the β gene. The corresponding substitution at the protein level was Arg → Pro in the 2064 position of the β‐spectrin chain.
A phenylalanine‐55 to serine amino‐acid substitution in the human glycoprotein IX leucine‐rich repeat is associated with Bernard‐Soulier syndrome
Authors: NORIS Patrizia , SIMSEK Suat , STIBBE Jean , Kr. VON DEM BORNE Albert E. G.
Published: November 2003

The platelet membrane glycoprotein (GP) Ib–IX–V complex, the major von Willebrand factor receptor on platelets, is absent or dysfunctional in patients with the Bernard‐Soulier syndrome (BSS). The four single subunits of the GPIb–IX–V complex (GPIbα, Ibβ, IX and V) are molecular products of different genes. Several point mutations and deletions affecting the GPIbα gene have been identified as the cause of BSS, whilst in four BSS families a GPIX gene defect has been reported. Moreover, a single case of BSS has been associated with a genetic defect of GPIbβ. We investigated the molecular basis of another case of BSS with a deficient expression of GPIX, as detected by immunofluorescence studies. After amplification of the entire GPIX coding region, nucleotide sequence analysis showed a homozygous single point mutation predicting a phenylalanine to serine substitution at position 55 of the mature GPIX within its unique leucine‐rich repeat. By allele‐specific oligonucleotide hybridization we confirmed the homozygosity of the patient as well as the carrier state of two out of three of his children studied. Although the parents of the patient, who were first cousins, were no longer alive and thus not available for study, we speculate that the molecular defect observed in the proband was inherited from both parents, who probably were heterozygous for this GPIX gene defect.

This study confirms that BSS may be caused by many different subtle molecular defects that often prevent the assembly and expression of a functional GPIb–IX–V complex.
Multiple myeloma cells are protected against dexamethasone‐induced apoptosis by insulin‐like growth factors
Authors: Xu Fenghao , Gardner Agnes , Tu Yiping , Michl Patrick , Prager Diane , Lichtenstein Alan
Published: November 2003

Multiple myeloma cell lines express functional receptors for insulin‐like growth factors (IGFs) and several cell types that make up the bone marrow microenvironment produce these cytokines. This suggests that IGFs may play a role in survival and/or expansion of the malignant clone within the marrow in patients with multiple myeloma. We tested the effects of these growth factors on myeloma cells challenged with dexamethasone. Dye exclusion and MTT assays demonstrated that both IGF‐I and IGF‐II protected the 8226 and dox‐40 myeloma cell lines and three primary myeloma cultures from dexamethasone‐induced cytotoxicity in a dose‐dependent fashion. Morphologic studies of target cells and their nuclei as well as DNA electrophoresis confirmed the IGFs afforded protection against dexamethasone‐induced apoptosis. Insulin also protected but was less impressive and required much higher concentrations. IGFs also protected against cycloheximide‐induced apoptosis but were ineffective against serum starvation, topoisomerase II inhibitors, or anti‐fas antibodies. IGF‐induced protection against dexamethasone was not associated with any alteration in quantitative or qualitative expression of BCL‐2, BAX or BCL‐X proteins. These data indicate that insulin‐like growth factors may play a role in maintenance of the malignant clone in patients with myeloma by protecting tumour cells from apoptotic death.
Treatment of dyskeratosis congenita with Granulocyte Colony‐Stimulating Factor and Erythropoietin
Authors: Alter Blanche P. , Gardner Frank H. , Hall Robert E.
Published: November 2003

Aplastic anaemia is both frequent and difficult to manage in patients with dyskeratosis congenita (DC). We recently treated a 23‐year‐old male for a year with granulocyte colony‐stimulating factor (G‐CSF) and erythropoietin (Ep), with an excellent neutrophil response, and a transient effect on haemoglobin levels. G‐CSF alone or combined with other cytokines may provide at least a partial effect in pancytopenic patients with DC.
Influence of thrombopoietin on platelet activation in myeloproliferative disorders
Authors: USUKI KENSUKE , IKI SEIKO , ENDO MITSUE , IZUTSU KOJI , INOUE KAORU , NISHIMURA TOSHIHARU , URABE AKIO
Published: November 2003

Although thrombopoietin itself does not influence platelet aggregation, it enhances platelet activation in response to certain agonists. We evaluated the effects of thrombopoietin on platelet activation using platelet‐rich plasma from 16 patients with myeloproliferative disorders (MPD group) and 16 healthy volunteers (control group). Preincubation with thrombopoietin significantly enhanced platelet aggregation stimulated by ADP, collagen, or epinephrine in the MPD group as well as the control group. However, aggregation induced by 3 μM ADP or 16 μM epinephrine showed significantly less augmentation by thrombopoietin in the MPD group than in the control group. Thrombopoietin significantly shortened the lag time between the addition of 3 μM ADP or 16 μM epinephrine and initiation of secondary aggregation and the lag time between addition of 2 μg/ml collagen and initiation of aggregation in both groups. When platelet‐rich plasma was used without adjustment of the platelet count, thrombopoietin itself induced aggregation in two patients. Hypoaggregation after addition of 0.5 μg/ml collagen was observed in seven out of nine patients with normal thrombopoietin levels and only one of six patients with high levels (P=0.04). Enhancement of 0.5 μg/ml collagen‐induced aggregation by thrombopoietin was seen in five out of nine patients with normal thrombopoietin levels and none of the six patients with elevated levels (P=0.04). These results indicate that platelet activation by certain agonists is enhanced by thrombopoietin in patients with these diseases as well as in normal controls and that the serum thrombopoietin level may regulate the function of circulating platelets in vivo.
Prevalence of GBV‐C/HGV, a novel ‘hepatitis’ virus, in patients with aplastic anaemia
Authors: Brown Kevin E. , Wong Susan , Young Neal S.
Published: November 2003

GBV‐C or hepatitis G virus (GBV‐C/HGV) is a novel RNA virus with similarities to members of the Flaviviridae family, especially hepatitis C. Viral RNA is detected in about 1.5% of American blood donors, with higher prevalence in multiply transfused patients and in individuals with hepatitis or liver disease. Some cases of aplastic anaemia follow apparent non‐A, non‐B, non‐C viral hepatitis, and GBV‐C viraemia has been described in three case reports of hepatitis‐associated aplastic anaemia. We tested clinical samples from patients with aplastic anaemia with or without recent hepatitis for the presence of GBV‐C/HGV. Virus was detected in a total of 15/57 (26.3%) of patients with aplastic anaemia and 12/52 (23.1%) of multiply transfused control patients. Sequencing of the 188 base pair NS3 helicase PCR product in the serum of five individuals indicated the same high degree of sequence variation as has been seen among other isolates of the virus. GBV‐C/HGV does not appear to be implicated in the aetiology of aplastic anaemia.
Follicular lymphoma immunoglobulin κ light chains are affected by the antigen selection process, but to a lesser degree than their partner heavy chains
Authors: Stamatopoulos Kostas , Kosmas Christos , Papadaki Theodora , Pouliou Evi , Belessi Chrysoula , Afendaki Stavroula , Anagnostou Dimitra , Loukopoulos Dimitris
Published: November 2003

Follicular lymphoma cells carry surface immunoglobulin whose heavy chain variable (VH) regions exhibit considerable divergence from the aminoacid sequence predicted by the germline nucleotide sequence as a result of the somatic hypermutation process. The present study examined the extent of somatic hypermutation in follicular lymphoma κ light chain variable region (Vκ) genes about which the available data is limited. DNA extracted from fresh frozen lymph node tissue of 14 patients with follicular lymphoma at diagnosis was subjected to polymerase chain reaction (PCR) amplification aimed at detecting clonal VH and VL (L; light chain) gene rearrangements. Clonal Vκ gene rearrangements were detected in 10/14 cases. Amplified VH and Vκ genes of these 10 cases were directly sequenced by the dideoxy‐chain termination method. In all cases, rearranged VH genes demonstrated numerous mutations clustering in the complementarity determining regions (CDRs), in keeping with previous reports. The degree of divergence of the rearranged Vκ genes from the closest homologous germline Vκ genes varied significantly. Furthermore, two patterns of mutations were observed: (i) in six cases (60%), mutations were most often of the replacement (R) type (changing the aminoacid sequence of the encoded polypeptide) in the CDRs and of the silent (S) type (leaving the aminoacid sequence of the encoded polypeptide unchanged) in the framework regions (FWRs) resulting in R:S ratios significantly greater than would have occurred by chance; (ii) in four cases (40%), very few or no mutations were observed and the distribution of mutations as well as the R:S mutation ratios did not differ significantly from what would have occurred by chance alone. These findings imply that, compared to their partner heavy chains, the κ light chains of follicular lymphoma neoplastic B‐cells' surface immunoglobulin (sIg): (i) are less affected by somatic hypermutation; (ii) play a less significant role in the antigen selection process.
Serum oncostatin M in multiple myeloma: association with prognostic factors
Authors: KOSKELA KARI , PELLINIEMI TARJA‐TERTTU , REMES KARI , RAJAMÄKI ALLAN , PULKKI KARI
Published: November 2003

The serum concentration of oncostatin M (OSM) was measured in 40 multiple myeloma patients at diagnosis. Serum OSM level exceeded the sensitivity limit of the ELISA assay in eight (20%) of these patients (OSM+ patients). The serum levels of IL‐6, another member of the gp130 cytokine family, and C‐reactive protein (CRP) as a surrogate of IL‐6 were significantly higher in OSM+ patients. There was a trend towards higher serum β2M concentration in OSM+ patients, whereas there was no difference in the serum sIL‐6R level or clinical data (age, gender, myeloma protein or stage) between the two groups. Two human myeloma cell lines secreted OSM and IL‐6, but not IL‐11 or leukaemia inhibitory factor (LIF), which suggests an important role for OSM and IL‐6 in supporting growth of myeloma cells.
Analysis of the p16INK4A, p15INK4B and p18INK4C genes in multiple myeloma
Authors: Tasaka Taizo , Berenson James , Vescio Robert , Hirama Toshiyasu , Miller Carl W. , Nagai Masami , Takahara Jiro , Koeffler H. Phillip
Published: November 2003

To study the structural integrity of the cyclin‐dependent kinase inhibitors known as INK4A (p16), INK4B (p15) and INK4C (p18) in multiple myeloma, we examined 20 primary myeloma samples (including one case of plasma cell leukaemia) using polymerase chain reaction–single strand conformation polymorphism, and 17 samples were examined by Southern blot analysis. The plasma cell leukaemia sample had homozygous deletions of the p15 and p16 genes (6%). One myeloma case had a p15 gene homozygous deletion (6%) with an intact p16 gene. This sample also had a p18 homozygous deletion, suggesting that the deletion of both genes may be important in either the development or progression of myeloma. No point mutations of these INK4 genes were found in the 20 samples. This is the first report that indicates that deletions of p15, p16 and p18 genes occur in some individuals with multiple myeloma (2/17 cases).
Generation of multinuclear tartrate‐resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors
Authors: Pierelli Luca , Scambia Giovanni , D'Onofrio Giuseppe , Ciarli Marina , Fattorossi Andrea , Bonanno Giuseppina , Menichella Giacomo , Battaglia Alessandra , Benedetti Panici Pierluigi , Tommasi Maria , Mancuso Salvatore , Leone Giuseppe
Published: November 2003

Circulating CD34+ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34− contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3+ T‐lymphocytes (range 2–12%), CD3−/CD16+/CD56+ natural killer cells (range 2–11%) and rare mature CD15+/CD11b+ granulocytes (range 1–2%). CD34+ cells were cultured in liquid medium in the presence of interleukin‐3, granulocyte‐macrophage colony stimulating factor, stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate‐resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34+ cells until day 35.

These observations suggest that circulating progenitors have a multilineage potential in vitro and contribute to the clarification of osteoclast development in humans; additionally, they provide the basis for the future development of optimized osteoclast culture techniques in liquid medium and the basic culture system, to test the distinct activity of 1,25(OH)2D3, parathyroid hormone, interleukin‐11 and of other cytokines on osteoclast development in humans.
Purified unfractionated G‐CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor
Authors: PIERELLI Luca , Scambia Giovanni , Menichella Giacomo , Fattorossi Andrea , Ciarli Marina , Bonanno Giuseppina , Battaglia Alessandra , D'Onofrio Giuseppe , Benedetti Panici Pierluigi , Iacone Antonio , Mancuso Salvatore , Leone Giuseppe
Published: November 2003

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G‐CSF)/chemotherapy mobilized peripheral blood CD34+ cells to selective erythroid differentiation in liquid culture with an average 28‐fold increase in the total cell number after 21 d. From day 6 of culture, cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34+ cells were cultured with EPO plus SCF in serum‐free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34+ cells underwent granulocytic/monocytic differentiation in liquid culture in response to granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin‐3 (IL‐3) and SCF, demonstrating that these CD34+ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34+ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34+ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G‐CSF/chemotherapy mobilized CD34+ progenitor cells in liquid culture. The absence of cytokines such as GM‐CSF and IL‐3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.
Serum markers of bone metabolism in multiple myeloma: prognostic value of the carboxy‐terminal telopeptide of type I collagen (ICTP)
Authors: Abildgaard N. , Bentzen S. M. , Nielsen J. L. , Heickendorff L.
Published: November 2003

This study was performed to evaluate the prognostic significance of serum markers of bone and collagen metabolism in multiple myeloma. Serum C‐terminal telopeptide of type I collagen (ICTP) reflects degradation of bone, whereas serum osteocalcin, together with serum C‐terminal propeptide of procollagen type I (PICP) and serum bone‐specific alkaline phosphatase (bAP) reflect synthesis of bone matrix. The N‐terminal propeptide of procollagen type III (PIIINP) in serum reflects synthesis of type III collagen. We analysed frozen sera from 109 patients with newly diagnosed multiple myeloma. Serum ICTP was elevated (>5.0μg/l) in most patients (median 6.6 μg/l, range 1.4–29.4 μg/l). Serum PIIINP was elevated (>4.2μg/l) in 46% (median 4.0 μg/l, range 1.4–20.1 μg/l). Serum PICP was generally within the reference limits, whereas serum osteocalcin and serum bAP were elevated in 19% and 37%, respectively. Serum ICTP correlated with serum PIIINP, serum β2‐microglobulin (β2m), serum calcium, performance status, and stage. In univariate analysis, the test variables serum ICTP (P=0.026) and serum osteocalcin (P=0.036) were found to be of prognostic value, but PIIINP, PICP, or bAP in serum were not. Serum ICTP and serum β2m had a similar prognostic value. In multivariate analysis, serum calcium showed the highest prognostic significance, and serum β2m was the only other variable of independent prognostic value. However, in normocalcaemic patients, serum ICTP showed the highest prognostic significance, followed by serum osteocalcin. Thus, the serum levels of ICTP and osteocalcin seem related to bone turnover and calcium metabolism, and provide further information about myeloma activity, particularly in normocalcaemic patients.
Myeloid differentiation is impaired in transgenic mice with targeted expression of a dominant negative form of retinoid X receptor β
Authors: SUNAGA SHINJI , MAKI KAZUSHIGE , LAGASSE ERIC , BLANCO JORGE C. G. , OZATO KEIKO , MIYAZAKI JUN‐ICHI , IKUTA KOICHI
Published: November 2003

To investigate the in vivo function of retinoid X receptor (RXR) on myelopoiesis, we generated transgenic (Tg) mice with targeted expression of a dominant negative form of RXR β in myeloid cells. In these Tg mice the transgene is expected to suppress the function of heterodimeric receptors composed of RXR and its counterparts, such as retinoic acid receptor. Out of 12 mice analysed, one Tg mouse exhibited a severe maturation arrest at the promyelocytic stage. Three other Tg mice showed a mild inhibition of myeloid differentiation, which was further augmented when mice were treated with 5‐fluorouracil (5‐FU). Furthermore, four Tg mice showed impaired myeloid differentiation in response to the treatment by 5‐FU or granulocyte‐colony stimulating factor (G‐CSF), although they exhibited apparently normal myelopoiesis in the untreated state. The phenotype of Tg mice observed after G‐CSF treatment correlated with the expression level of the transgene, although the correlation was not found in untreated mice. These results indicated that myeloid differentiation is perturbed in the Tg mice by the dominant negative effect of the transgenic RXR, indicating that RXR plays a role in myelopoiesis.
Ongoing immunoglobulin gene mutations in mantle cell lymphomas
Authors: Du Ming‐Qing , Diss Tim C. , Xu Chun‐Fang , Wotherspoon Andrew C. , Isaacson Peter G. , Pan Lang‐Xing
Published: November 2003

Mantle cell lymphomas (MCL) frequently show a vaguely follicular growth pattern. This phenomenon is thought to result from the colonization of reactive B‐cell follicles by tumour cells. In view of the unique property of the germinal centre environment, antigen stimulation may play a role in the expansion of the tumour. To assess this, we have examined ongoing Ig mutations, which are genetic markers of B cells in persistent response to antigen stimulation, in five MCLs including two cases derived from the gastrointestinal tract known as lymphomatous polyposis (LP). We have specifically analysed Ig ongoing mutations in tumour cells from multiple lesions in one case and in tumour cells microdissected from colonized follicles in two cases. The consensus Ig VH sequences in four MCLs were identical, or almost identical (three cases 100%, one case 99% homology) to the published germlines, which in each case were those frequently employed by autoantibodies. The consensus Ig VH sequence in the remaining case displayed 95.5% homology to the closest published germline. This may represent derivation from an unknown VH germline or a rare instance of somatic mutations. Extensive sequencing of the rearranged Ig genes revealed ongoing mutations within the tumour clone in two cases: one was a LP with multiple lesions of the gastrointestinal tract and the other was a nodal MCL in which tumour cells from colonized follicles were analysed. Our results indicate that MCLs are derived from pre‐germinal centre B cells, possibly autoreactive B‐cell clones. The ongoing mutations identified suggest a possible involvement of antigen stimulation in the clonal expansion of a proportion of MCLs.
Evolution of blood coagulation and fibrinolysis parameters after abrupt versus gradual withdrawal of acenocoumarol in patients with venous thromboembolism: a double‐blind randomized study
Authors: Tardy Bernard , Tardy‐Poncet Brigitte , Laporte‐Simitsidis Silvy , Mismetti Patrick , Decousus Hervé , Guyotat Denis , Bertrand Jean C.
Published: November 2003

A double‐blind randomized trial was conducted to research a hypercoagulable state rebound after abrupt versus gradual withdrawal of acenocoumarol. 20 patients were included: 10 in the abrupt withdrawal group (AW) and 10 in the gradual withdrawal group (GW). Between days 1 and 15, F1+2 was higher in group AW (P<0.002). A sig‐nificant increase of D‐dimer with time was found (P<0.001) without difference between the two groups. tPA and PAI‐1 levels remained stable throughout without difference between the two groups. No rebound phenomenon was observed. Four thrombotic recurrences were observed: group AW: 1, group GW: 3 (P=0.29). There is neither clinical nor biological support for a gradual anticoagulation withdrawal.
Feasibility of prenatal diagnosis and carrier detection in South African haemophilia A patients
Authors: Dangerfield Bruce T. F. , Manga Prashiela , Field Stephen P. , Hartman Ella , Jenkins Trefor , Krause Amanda
Published: November 2003

The feasibility of DNA diagnosis for haemophilia A was tested in South African patients and families by screening for the common inversion mutation in the factor VIII gene and for the intragenic microsatellite markers in introns 13 and 22. The allele frequencies at the two microsatellite loci were significantly different, with informativity being higher in the Negroid (100%) than the Caucasoid group (67%). In severely affected haemophiliacs the inversion was found in 43% (6/14) of Negroids but in only 32% (13/41) of Caucasoids. Presence of a second common unidentified mutation may account for the low frequency in the latter. Haplotype analysis shows a disproportionately high frequency of an (AC)20 intron 13–(AC)26 intron 22 inversion negative Caucasoid haemophilia chromosome, supporting a founder effect.