bluuebunny/crossref_metadata_2025 huggingface dataset:
HIGH‐DOSE THERAPY WITH PERIPHERAL BLOOD STEM CELL TRANSPLANTATION RESULTS IN A SIGNIFICANT REDUCTION OF THE HaEMOPOIETIC PROGENITOR CELL COMPARTMENT
Authors: Voso M. T. , Murea S. , Goldschmidt H. , Hohaus S. , Haas R.
Published: November 2003

In order to study the effect of high‐dose therapy with peripheral blood stem cell transplantation (PBSCT) on the haemopoietic reserve in man, the number and composition of bone marrow (BM) and peripheral blood (PB)‐derived progenitor cells were examined in 137 cancer patients. In 45 patients, paired samples from BM and PB were obtained before PBSC mobilization and 6–27 months after transplantation. Following PBSCT, the proportion of CD34+ cells was significantly smaller than before mobilization (BM 1.99±0.24 versus 0.8±0.09, P<0.001), and no change was observed at several follow‐up visits thereafter.

The reduction was most pronounced for the primitive BM progenitor subsets such as the CD34+/DR− and CD34+/Thy‐1+ cells. The impairment of hematopoiesis was also reflected by a significant reduction in the plating efficiency of BM and PB samples.

No relationship was found between the decrease in the proportion of CD34+ cells and any particular patient characteristics, kind of high‐dose therapy or the CD34+ cell content in the autograft.

In conclusion, high‐dose therapy with PBSC transplantation is associated with a long‐term impairment of the haemopoietic system. The reduction in the number of haemopoietic progenitor cells is not associated with a functional deficit, as peripheral blood counts post‐transplantation were normal in the majority of patients.
Chronic persistent Epstein‐Barr virus infection of natural killer cells and B cells associated with granular lymphocytes expansion
Authors: Kanegane Hirokazu , Wado Taizo , Nunogami Koji , Seki Hidetoshi , Taniguchi Noboru , Tosato Giovanna
Published: November 2003

B lymphocytes and epithelial cells are the only cell types known to be infected with Epstein‐Barr virus (EBV) in normal individuals. Rarely, EBV also infects other cells, including natural killer (NK) cells, almost always in the context of fatal leukaemias or lymphoproliferative disorders. We report on a 6‐year‐old previously healthy girl who developed fevers and liver function abnormalities for 3 months. The peripheral blood revealed an abnormal expansion of large granular lymphocytes, comprising 24% of the white blood cells. Flow cytometric analysis of the peripheral blood mononuclear cells showed an abnormal increase of CD16‐positive NK cells, 62% of which were EBV‐infected by in situ EBER‐1 hybridization. The circulating B cells were normal in number, but 18% were infected with EBV by in situ EBER‐1 hybridization. Approximately 2 years after resolution of all symptoms and continued good health, 35% of the circulating mononuclear cells were EBV‐infected, indicative of persistent expansion of EBV‐infected cells. We conclude that abnormal expansions of EBV‐infected NK and B cells can be associated with a chronic benign course.
Erythropoietin production by interstitial cells of hypoxic monkey kidneys
Authors: Fisher James W. , Koury Stephen , Ducey Thomas , Mendel Sandra
Published: November 2003

Previous studies which demonstrated that interstitial cells of the peritubular capillary bed of the kidneys are the site of erythropoietin (Epo) production have been performed in non‐primate species. In this study, kidneys from adult rhesus monkeys exposed to 18 h hypoxia (0.42 atm) with high serum (5685 mU/ml) and kidney (814 mU/g, includes serum EPO in the kidney) levels of Epo were compared with a kidney from a nonhypoxic normal rhesus monkey. Localization of Epo mRNA by in situ hybridization was carried out with either anti‐sense or sense RNA probes generated from a 645 base pair KpnI–BglII fragment of a monkey Epo cDNA. Epo mRNA was demonstrated only in interstitial cells in the peritubular capillary bed of the hypoxic and normal monkey kidneys utilizing the antisense probe. The finding that the same type of cell that produces EPO in mice, rats and sheep also produces EPO in a higher primate species strongly supports the contention that renal interstitial cells also produce EPO in the human.
Activated protein C inhibits thrombus formation in a system with flowing blood
Authors: Lozano M. , Escolar G. , Schwarz H. P. , Hernández R. , Bozzo J. , Ordinas A.
Published: November 2003

We studied the effect of increasing concentrations of protein C (PC) and activated protein C (APC) on haemostasis in an in vitro thrombosis model. Blood from healthy donors was anticoagulated with citrate–phosphate–dextrose (final citrate concentration 19 mm) or a low molecular weight heparin (LMWH, 20 IU/ml). Enzymatically denuded rabbit aorta segments were exposed to flowing blood for 10 min in an annular perfusion chamber. PC and APC were added to the perfusate immediately prior to exposure. In citrated blood at a shear rate of 800/s, PC and APC induced a statistically significant decrease in platelet deposit at 16 μg/ml and 32 μg/ml. In perfusions performed with blood anticoagulated with LMWH, there was no effect on platelet deposition at 16 and 32 μg/ml either at shear rates of 300/s or 800/s. Addition of PC showed no effect on fibrin deposition at a shear rate of 300/s; in contrast, a non‐statistically significant 40% reduction was seen at a shear rate of 800/s, compared to controls. Addition of APC caused a 100% reduction in fibrin formation at 16 and 32 μg/ml at both shear rates studied. PC and APC inhibited platelet deposition on the exposed subendothelial surface, in a dose‐dependent manner. Effects of PC and APC on platelet function might be mediated through inhibition of thrombin generation at the platelet microenvironment.
SERIAL QUANTIFICATION OF MINIMAL RESIDUAL DISEASE OF t(8;21) ACUTE MYELOGENOUS LEUKAEMIA WITH RT‐COMPETITIVE PCR ASSAY
Authors: MUTO AKIHIRO , MORI SHIGEHISA , MATSUSHITA HIROMICHI , AWAYA NORIHIRO , UENO HIRONORI , TAKAYAMA NOBUYUKI , OKAMOTO SHIN‐ICHIRO , KIZAKI MASAHIRO , IKEDA YASUO
Published: November 2003

The chromosomal translocation (8;21)(q22;q22) in the AML M2 subtype according to the FAB classification, results in the production of a novel fusion gene AML1/ETO. The chimaeric AML1/ETO transcript is useful for the detection of minimal residual disease (MRD). Recently, several studies on the detection of AML1/ETO transcripts in t(8;21) AML have been reported. However, the clinical significance of a small number of AML1/ETO transcripts by a reverse transcription–polymerase chain reaction (RT‐PCR) remains to be elucidated. We have developed a novel quantitative RT‐competitive PCR assay and evaluated the clinical usefulness of this method by the monitoring of MRD in eight patients with t(8;21) AML. In four patients in first continuous complete remission (CR) the value of MRD was always <0.1 fg of the competitor dose throughout their courses, whereas in four relapsed patients there was an increase in the value of MRD to <0.1 fg of the competitor dose before cytogenetic relapse. We conclude that the detection of the presence of cells with AML1/ETO fusion transcripts by our RT‐competitive PCR assay may be useful to monitor disease progression and to predict subsequent relapse.
Quantification of PML‐RARα transcripts in acute promyelocytic leukaemia: explanation for the lack of sensitivity of RT‐PCR for the detection of minimal residual disease and induction of the leukaemia‐specific mRNA by alpha interferon
Authors: Seale James R. C. , Varma Subhash , Swirsky David M. , Pandolfi Pier‐Paolo , Goldman John M. , Cross Nicholas C. P.
Published: November 2003

The RT‐PCR technique for the identification of the PML‐RARα fusion mRNA is widely used for the detection of minimal residual in acute promyelocytic leukaemia (APL). A positive result after remission induction is highly predictive of early relapse, but the vast majority of patients have no detectable disease by this technique after chemotherapy consolidation, despite the fact that many later relapse. We report a quantitative PCR technique for the PML‐RARα cDNA which was used to show that less than 1000 PML‐RARα molecules are obtained from 1 μg of diagnostic bone marrow RNA derived from approximately 1 million APL blasts. The lack of sensitivity of currently employed RT‐PCR methods may therefore be explained by their poor yield of PML‐RARα cDNA. Minor modifications to the reverse transcription procedure improved this yield 3 fold. Furthermore, expression of the leukaemia‐specific transcript increased by approximately one order of magnitude after incubation of the patients’ cells for 24 h in vitro with 100 iu/ml alpha interferon.
AUTOANTIBODIES AND ANTI‐MOUSE ANTIBODIES IN THROMBOCYTOPENIC PATIENTS AS ASSESSed by DIFFERENT MAIPA assays
Authors: Clofent‐Sanchez Gisèle , Lucas Sophie , Laroche‐Traineau Jeanny , Rispal Patrick , Pellegrin Jean‐Luc , Nurden Paquita , Nurden Alan
Published: November 2003

Two MAIPA (monoclonal antibody [MAb] immobilization of platelet antigen) assays were performed to determine (a) autoantibodies to platelet glycoproteins (GP) and (b) serum antibodies recognizing mouse MAbs used in the assay. In MAIPA I, control platelets were incubated simultaneously with human serum and a mouse MAb to a platelet glycoprotein (GP IIb–IIIa, Ib–IX, Ia–IIa, IV and p24). In MAIPA II, the control platelets were incubated first with the human serum and then, after washing, with the selected mouse MAb. A series of 25 patients with autoimmune thrombocytopenic purpura (ATP) associated or not with other autoimmune states were examined. Autoantibodies (both MAIPA I and MAIPA II positive) or anti‐mouse Abs (MAIPA I positive and MAIPA II negative) were frequent in both groups of patients. Statistically significant differences existed in the incidence of anti‐mouse Abs between patients (56.5%) and healthy donors (10%). This suggests that their production may be related to thrombocytopenias associated with autoimmune disease. We speculate that the presence of anti‐mouse antibodies could reflect an abnormality in the immunological modulation of the idiotypic network.
Role of factor VIII on activated protein C resistance ratio in inflammatory diseases
Authors: Mathonnet Florence , De Mazancourt Philippe , Denninger Marie‐Hélène , Morot Mireille , Benattar Norbert , Giudicelli Yves
Published: November 2003

Activated protein C resistance ratio (APC‐Rr), factor VIIIC (FVIIIC) and plasma fibrinogen levels were studied in patients with inflammatory disease. The patient mean APC‐Rr was significantly lower than in the control group. This decreased ratio in inflammatory diseases appeared to be connected with increased FVIIIC. Moreover, supplementation of plasmas with purified factor VIII decreased the APC‐Rr in plasma from both groups, and suppressed the difference between groups. These data suggest that factor VIIIa and factor Va compete for protein C‐catalysed cleavage. Ratios were identical in both groups when FVIIIC level was lowered by dilution in factor V deficient plasma.
DELETIONS OF CHROMOSOME 20q AND THE PATHOGENESIS OF MYELOPROLIFERATIVE DISORDERS
Authors: Aimakopoulos F. A. , Green A. R.
Published: November 2003

Myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS) represent an overlapping spectrum of clonal preleukaemic conditions in which mutations have resulted in dysregulation of multipotent haemopoietic progenitors. An interstitial deletion of the long arm of chromosome 20 is a recurring abnormality associated with both of these classes of disorders. The association of 20q deletions with ‘stem cell’ disorders suggests that the deletion may mark the site of one or more genes, loss or inactivation of which perturbs the regulation of haemopoietic progenitors. The identification and study of the target gene(s) on chromosome 20q is therefore likely to illuminate the regulation of normal haemopoietic differentiation as well as the genesis of clonal myeloid disorders. This article provides a review of progress in the search for critical genes harboured within 20q deletions, together with a summary of several insights that these studies have provided into the biology of MPD and MDS.
STEM CELL MOBILIzATION IN NORMAL DONORS FOR ALLOGENEIC TRANSPLANTATION: ANALYSIS OF SAFETY AND FACTORS AFFECTING EFFICACY
Authors: Miflin Gail , Charley Carole , Stainer Claire , Anderson Sally , Hunter Ann , Russell Nigel
Published: November 2003

The use of peripheral blood stem cells instead of bone marrow as the source of haemopoietic cells for allogeneic transplantation is being increasingly explored. We have analysed data from 17 normal donors who underwent stem cell mobilization for allogeneic transplantation with an identical protocol using G‐CSF at a dose of 10 μg/kg/d, with the first leukapheresis (LP) on the day following the fourth dose of G‐CSF. Both G‐CSF administration and leukapheresis were well tolerated. Donors underwent a median of two leukaphereses (range one to three) and a median of 6.80 × 106 CD34+ cells/kg recipient weight (range 2.4–15.6 × 106) were collected. The median number of CD34+ cells per kg donor weight was 6.05 × 106, when corrected for a 12 litre leukapheresis, this gave a median total of 3.89 × 106 CD34+ cells/kg donor weight. When analysed with respect to factors which might influence the efficacy of mobilization, male donors were associated with a superior yield. The median number of CD34+ cells/kg/LP harvested was 4.96 × 106 in males and 2.79 × 106 in females (P<0.05). The results suggested that, given a recipient of 75 kg, in a male donor a single 12 litre leukapheresis should yield sufficient CD34+ cells (4 × 106/kg), whereas a female donor would be likely to need two leukaphereses. Age was not found to affect donor yield. In summary, these data confirm that leukapheresis is a safe procedure in normal donors and suggest that males may be more efficient mobilizers of stem cells than females.
p190bcr/abl rearrangement in myelodysplastic syndromes: two reports and review of the literature
Authors: Lesesve J. F. , Troussard X. , Bastard C. , Hurst J. P. , Nouet D. , Callat M. P. , Lenormand B. , Piguet H. , Flandrin G. , Macintyre E.
Published: November 2003

We describe two patients with myelodysplastic syndrome (MDS) and the Philadelphia chromosome (Ph). The patients were 64‐ and 69‐year‐old men who were diagnosed as having refractory anaemia with excess of blasts. During the terminal phase, the MDS evolved to myeloblastic leukaemia. Chromosome analysis showed normal karyotypes mixed with metaphases containing a classic Ph chromosome t(9;22)(q34;q11). Surprisingly, molecular studies showed breakpoint cluster region rearrangement between exons e1 and a2, compatible with a p190bcr/abl breakpoint, as observed in acute lymphoblastic leukaemia. We discuss the correlation between MDS and acquisition of the Ph chromosome, and the occurrence of p190bcr/abl in MDS.
Therapy with CD7 monoclonal antibody TH‐69 is highly effective for xenografted human T‐cell ALL
Authors: Baum Wolfgang , Steininger Helmuth , Bair Hans‐Jürgen , Becker Wolfgang , Hansen‐Hagge Thomas E. , Kressel Michael , Kremmer Elisabeth , Kalden Joachim R. , Gramatzki Martin
Published: November 2003

Human T‐cell acute lymphocytic leukaemia (ALL) was established in athymic nude or severe combined immunodeficient (SCID) mice by injecting CEM or MOLT‐16 cells. When nude mice bearing approx. 2 g of tumour were treated with a single injection of CD7 antibody TH‐69, 82.6% reached complete remission within 10 d whereas 13.0% showed partial remission. Similarly, in SCID mice with advanced disease a significant prolongation of survival was seen. The therapeutic effects were dependent upon dose and affinity of the antibody. TH‐69 is a high‐affinity antibody (7.6 × 109 M−1) that rapidly induced modulation during treatment. The Fc‐portion of the antibody was required for effective tumour cell killing. Complement deposition was found on tumour sections after TH‐69 treatment and in part may account for tumour destruction. There was no evidence for antibody‐dependent cellular cytotoxicity (ADCC). The kinetics of tumour disappearance suggested the initiation of a programmed cell death (PCD), despite the lack of significant DNA fragmentation. Unmodified high‐affinity antibodies to the T‐cell antigen CD7 have potential for T‐cell ALL therapy.
Red blood cell phenotypes in the α+ thalassaemias from early childhood to maturity
Authors: Williams T. N. , Maitland K. , Ganczakowski M. , Peto T. E. A. , Clegg J. B. , Weatherall D. J. , Bowden D. K.
Published: November 2003

The α+ thalassaemias are the most common single gene disorders of humans, yet little is known about their haematological characteristics in childhood. Blood samples have been collected randomly from more than 2000 individuals in village communities in Vanuatu in the South West Pacific and analysed for α thalassaemia and associated haematological changes. Here we describe the haematological effects of the α+ thalassaemias from early childhood through to maturity in this population. Mean cell volume (MCV) and mean cell haemoglobin (MCH) levels in individuals of normal, heterozygous and homozygous genotype differed significantly from one another throughout the entire age range (2P < 0.05). In contrast, haemoglobin levels in heterozygous and homozygous individuals were well maintained throughout development. Adults of normal genotype attain Hb levels which are indistinguishable from Caucasian reference values, a finding made all the more remarkable given the high frequency of clinical malaria in this population. It is clear from these findings that haematological data are valuable in screening for carriers of α+ thalassaemia in this population. MCH is clearly the most sensitive discriminator. None of the homozygous adults tested had an MCH of >27 pg, whereas <10% of normals had a value of <27 pg. These data provide reference values for areas in which the α+ thalassaemias are common and often confused with iron‐deficiency anaemia.
A novel human leukaemic cell line, CTS, has a t(6;11) chromosomal translocation and characteristics of pluripotent stem cells
Authors: KAKUDA HARUMI , SATO TAKEYUKI , HAYASHI YASUHIDE , ENOMOTO YASUHIRO , TAKAYAMA JUN , OHIRA MUTSURO , SETO MASAO , UEDA RYUZO , FUSE AKIRA , NIIMI HIROO
Published: November 2003

A novel human leukaemic cell line, designated CTS, was established from the peripheral blood of a 13‐year‐old girl suffering from acute myeloblastic leukaemia (AML) in relapse. CTS cells expressed CD7, CD13, CD33, CD34 and HLA‐DR antigens, and showed ultrastructural myeloperoxidase activity. In addition, CTS cells showed DNA rearrangements of the immunoglobulin heavy chain gene and the light κ chain gene, and deletions of the T‐cell receptor δ1 gene. Cytogenetic analysis revealed a human female diploid karyotype with a t(6;11)(q27;q23) chromosomal translocation. Molecular studies demonstrated a DNA rearrangement of the MLL gene, the expression of a truncated 11.0 kb MLL mRNA and the detection of the MLL/AF‐6 fusion transcript in CTS cells. To our knowledge, this cell line is the first report of a human leukaemic cell line with a t(6;11) chromosomal translocation. CTS cells showed no significant proliferative response to the cytokines, IL‐2, IL‐3, IL‐6, IL‐11, GM‐CSF, G‐CSF, EPO, SCF, but were induced to differentiate to the T‐cell, B‐cell, erythroid or megakaryocytic lineage in the presence of particular cytokines. This CTS cell line may provide a useful tool in the study of the oncogenesis of mixed lineage leukaemia with 11q23 abnormalities and for the analysis of growth and differentiation of pluripotent stem cells.
Simplified strategies for minimal residual disease detection in B‐cell precursor acute lymphoblastic leukaemia
Authors: Landman‐Parker J. , Aubin J. , Delabesse E. , Tabone M‐D. , Adam M. , Millien C. , Leboeuf D. , Buzyn‐Veil A. , Dollfus C. , Leverger G. , Macintyre E. A.
Published: November 2003

We have developed a simplified fluorescent run‐off (FluRO) based IgH PCR strategy in order to facilitate follow‐up of large numbers of B‐cell precursor (BCP) acute lymphoblastic leukaemias (ALL) in a routine molecular diagnostic laboratory. DNA samples from 26 BCP‐ALL and one B‐cell line were amplified using IgH FR1 and FR2 consensus primers and analysed in parallel either by ethidium bromide non‐denaturing PAGE or, after rendering the PCR products fluorescent with an internal JH consensus primer, by high‐resolution analysis on an automated fragment analyser. The latter led to a minimum of one log increase in sensitivity of detection in 62% of alleles from 19 samples (16/28 in FR1; 11/15 in FR2) tested in parallel on log DNA dilutions, and to at least a 10−2 level of sensitivity of detection in 15/19. The improved resolution allowed an approximate 20% increase in the number of clonal alleles detected, and consequently doubled the incidence of oligoclonality (6/26; 23%). Using these strategies, 6/17 (35%) of children analysed prospectively showed residual IgH positivity in the post induction complete remission bone marrow sample. Both early deaths occurred within this subgroup of patients and of the three of four surviving patients tested, two remained positive 2–3 months later. Although this simplified strategy is, as expected, less sensitive than anti‐V‐D‐J junction specific strategies, it enables detection of a category of ‘slow‐remitters’ which may have prognostic significance at a stage where therapeutic decisions are taken.
The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis
Authors: Choi Esther S. , Hokom Martha M. , Chen Jen‐Li , Skrine James , Faust Judy , Nichol Janet , Hunt Pamela
Published: November 2003

Thrombopoietin (TPO), the ligand for the c‐Mpl cytokine receptor, is a recently identified cytokine with potent effects on platelet production. The receptor‐binding portion of c‐Mpl ligand is encompassed in another molecule known as megakaryocyte growth and development factor, or MGDF. Although it is clear that the administration of TPO or MGDF to animals dramatically increases the platelet count, the specific stage(s) of thrombopoiesis during which these molecules are principally active have not been unambiguously determined. Pharmacology studies administering MGDF at doses ranging from 0.1 to 630 μg/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 μg/kg/d resulted in platelet counts 5‐fold above normal. However, doses >60 μg/kg/d resulted in less‐than‐optimal platelet production. This phenomenon was investigated in vitro. Using an established culture system for the generation of human megakaryocytes and platelets, MGDF was shown to be optimally and equivalently active in the generation of mature megakaryocytes at concentrations from 10 to 1000 ng/ml. However, the cytokine was not required for proplatelet formation and in fact was inhibitory to that process in a dose‐dependent manner. When MGDF was added to human megakaryocytes at concentrations of 200 ng/ml or greater, proplatelet formation was inhibited to 30% of control values. MGDF‐mediated inhibition was specific, since the addition of the truncated form of the c‐Mpl receptor reversed the inhibition in a dose‐dependent manner. Other recombinant factors, interleukin‐6, interleukin‐11 and erythropoietin had no significant positive or negative effects in this human proplatelet assay. Together, these data suggest that although TPO and MGDF promote the full spectrum of megakaryocyte growth and development, they are not necessary for proplatelet formation, and may in part regulate platelet shedding by their absence.
Interferon‐γ secretion defects in haemophilia A patients receiving highly purified plasma‐derived or recombinant factor VIII
Authors: K. Newton‐Nash Debra , Tollerud David , Guevarra Lucille , C. Gill Joan
Published: November 2003

The outcome of developing immune responses is influenced by interactions among a large and complex network of secreted cytokines. T‐cell secretion of interferon‐γ (IFN‐γ), tumour necrosis factor α (TNF‐α) and TNF‐β, or lymphotoxin contributes to the development of cell‐mediated immunity, whereas secretion of interleukin (IL)‐4, IL‐5 and IL‐6 contributes to development of humoral immunity. Humoral immunity to factor VIII (FVIII) develops in approximately 25% of severe haemophilia A patients. The aim of our research was to understand the underlying immune response to FVIII in patients with FVIII inhibitors. We report a defect in IFN‐γ secretion by peripheral blood mononuclear cells (PBMC) derived from haemophilia A patients, which was accompanied by a low level of mitogen‐induced proliferation and a significant decrease in the percentage of natural killer (NK) cells. All of the observed defects were found in haemophilia A patients, both with and without FVIII inhibitors, who were free of viral infection and had been treated predominantly or exclusively with monoclonal antibody‐purified or recombinant FVIII.
REGULATION OF CLINICAL CHEMORESISTANCE BY bcl‐2 AND BAX ONCOPROTEINS IN B‐CELL CHRONIC LYMPHOCYTIC LEUKAEMIA
Authors: Pepper Chris , Bentley Paul , Hoy Terry
Published: November 2003

The bcl‐2 gene was first shown to be dysregulated in the majority of follicular lymphomas in which a t(14;18) chromosomal translocation is present, but is also over‐expressed in the absence of gene rearrangements in most cases of B‐cell chronic lymphocytic leukaemia (B‐CLL). The bcl‐2 oncoprotein is a regulator of apoptosis and the activity of this protein is opposed by bax, a homologous protein that accelerates the rate of cell death. B‐lymphocyte bcl‐2 and bax protein levels were found to be significantly altered in B‐CLL patients when compared to those of a normal control group. Increased bcl‐2/bax ratios were observed in both the treated and untreated patients when compared to those of normal controls. These alterations were particularly pronounced in those treated patients found to be clinically unresponsive to chemotherapy.
Differential expression of cell proliferation regulatory proteins in B‐ and T‐lineage acute lymphoblastic leukaemias
Authors: WoŁowiec Dariusz , Mekki Yahia , Ffrench Patrick , Manel Anne‐Marie , Bertrand Yves , Rimokh Ruth , Philippe Noel , Bryon Paul‐André , Ffrench Martine
Published: November 2003

In order to better understand the molecular background of differences between the clinical picture of T‐ and B‐lineage ALLs, we studied the expression of several proteins involved in the regulation of cell proliferation in bone marrow blast cells from 30 cases of previously untreated acute lymphoblastic leukaemia (ALL); 14 cases were T‐ and 16 B‐cell lineage ALLs. We studied several cyclin‐dependent kinases (cdk1, cdk2, cdk4, cdk6) and cyclins (cyclin A, cyclin B1, cyclin D3 and cyclin E). We also studied proliferating cell nuclear antigen (PCNA) and Bcl‐2 expression, the latter protein known to be involved in the prolonged survival of B‐lineage ALL blasts. Proteins obtained from cell lysates were resolved on polyacrylamide gel followed by immunodetection and densitometry of specific bands. Expression of cdk1 and PCNA, markers of proliferative activity, was significantly higher in T‐ than in B‐lineage ALL. Cdk6, which was highly correlated to PCNA, was also higher in T‐cell ALL. In contrast, B‐lineage ALL displayed a higher expression of anti‐apoptotic protein Bcl‐2. We hypothesize that those particularities may reflect differential roles of cell multiplication and apoptosis in the neoplastic proliferation of B‐ and T‐lineage ALL.
Primary proliferating immature myeloid cells from CML patients are not resistant to induction of apoptosis by DNA damage and growth factor withdrawal
Authors: Albrecht Tarja , Schwab Renate , Henkes Martin , Peschel Christian , Huber Christoph , Aulitzky Walter E.
Published: November 2003

Induction of apoptosis by growth factor deprivation or gamma‐irradiation‐induced DNA damage was directly studied in proliferating primary haemopoietic cells derived from CD34‐positive cells of 13 CML patients and 12 normal controls. CD34‐positive cells were cultured in the presence of appropriate concentrations of SCF and G‐CSF for 5–7 d. After gamma irradiation with 500 rad or growth factor deprivation, the fraction of apoptotic cells was assessed by two independent methods applying either measurement of cells incorporating FITC‐labelled dUTP by terminal transferase or assessment of the fraction of cells with a less than 2N DNA content in flow cytometry.

Proliferating CML cells were not resistant to the induction of apoptosis either after gamma irradiation or subsequent to growth factor deprivation. A similar fraction of normal and CML cells underwent apoptosis 48 h after withdrawal of growth factors. CML cells displayed an increased susceptibility to induction of apoptosis after DNA damage. A significantly higher proportion of apoptotic cells were detected in samples derived from CML patients after irradiation with 0.5 Gy. These results, in conjunction with conflicting observations by other investigators, on the induction of apoptosis by gamma irradiation in various bcr‐abl positive cells, suggest that bcr‐abl‐dependent effects on apoptosis strongly depend on the cells used. Our observations in CML cells derived exclusively from newly diagnosed CML patients demonstrate that bcr‐abl expression per se is not sufficient to induce resistance to apoptosis.
Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance
Authors: Liebman Howard A. , Sutherland Douglas , Bacon Rachael , McGehee William
Published: November 2003

Resistance to the anticoagulant effects of activated protein C (APC) is now considered the most prevalent cause of inherited thrombophilia. The great majority of patients with activated protein C resistance (APCR) have a missense mutation in the factor V molecule (factor V Leiden, FVR506Q) resulting in defective inactivation of factor Va due to a loss of an APC cleavage site. The diagnosis of APCR has been based upon the inability of APC to prolong the activated partial thromboplastin (aPTT) clotting time in subjects with APCR. However, this assay has a number of deficiencies which limit its general use. We have evaluated a newly described one‐stage tissue factor dependent factor V coagulation assay for APCR in 117 patients and controls and compared the results of this assay in a blinded manner to a polymerase chain reaction (PCR) based assay for the molecular defect of factor V Leiden. 43% (50 /117) of the patients studied were receiving coumadin or heparin, or had a lupus anticoagulant. The tissue factor dependent factor V assay had 100% specificity and sensitivity for factor V Leiden and successfully predicted a homozygous state in the three documented homozygotes. The PCR‐based assay for factor V Leiden resulted in a single false positive assay due to a silent A to C transition at nucleotide 1692 resulting in the loss of the Mnl restriction endonuclease cleavage site. The single‐stage tissue factor dependent factor V assay is a highly sensitive and generally applicable assay for APCR.
Superantigen‐mediated cellular cytotoxicity is dependent on antigen expression, but independent of the P‐glycoprotein multidrug resistance phenotype
Authors: Zehrer Cornelia , Beck James , Gekeler Volker , Ihle Johannes , Holzer Ursula , Dohlsten Mikael , Kalland Terje , Niethammer Dietrich , Dannecker GU¨nther E.
Published: November 2003

Superantigen‐activated T cells can be targeted by monoclonal antibodies (mAb) to lyse MHC class II negative tumour cells. In this study we determined the susceptibility of the T‐lymphoblastoid leukaemic cell line CCRF‐CEM and its multidrug resistant sublines CCRF VCR100, CCRF VCR1000 and CCRF ADR5000 to lysis by monoclonal antibody‐targeted and superantigen‐activated T cells (superantigen‐dependent cellular cytotoxicity, SDCC). A recombinant fusion protein of protein A and the superantigen Staphylococcus enterotoxin A (SEA) was used together with the mAbs anti‐CD7, anti‐CD38, anti‐CD45RA and 4E3 (anti‐P‐glycoprotein) to correlate susceptibility to SDCC with expression of the MDR1‐gene product. Our results demonstrated SDCC to be independent of MDR1‐gene expression. This was further confirmed by blocking the function of Pgp in the leukaemic cell lines with a cyclosporine A derivative, which had no influence on SDCC. As expected, expression of the respective cell surface antigens on target cells had a strong impact on SDCC, although other factors seem to influence efficiency of SDCC as well.
The triplicated α‐globin gene locus in β‐thalassaemia heterozygotes: clinical, haematological, biosynthetic and molecular studies
Authors: Traeger‐Synodinos J. , Kanavakis E. , Vrettou C. , Maragoudaki E. , Michael Th. , Metaxotou‐Mavromati A. , Kattamis C.
Published: November 2003

Excess α‐globin chains play a major role in the pathophysiology of homozygous β‐thalassaemia. In β‐thalassaemia carriers a minor effect of α‐globin chain excess is reflected in a minimal or mild anaemia without clinical symptoms. Factors that increase α‐chain excess in heterozygotes are expected to accentuate the severity of the clinical and haematological phenotype.

We report the clinical, haematological, biosynthetic and molecular data in three β‐thalassaemia heterozygotes with the rare interaction of homozygosity for α‐globin gene triplication, and in 17 heterozygotes with a single additional α‐globin gene. The three patients homozygous for the α‐globin gene locus (anti 3.7 kb arrangement) had β °‐thalassaemia mutations and a diagnosis of thalassaemia intermedia, preserving haemoglobin levels around 7–8 g/dl. Of the 17 β‐thalassaemia heterozygotes (six children and 11 adults), 16 had severe β‐thalassaemia mutations interacting with an additional α‐globin gene (13 with αααanti‐37 and four with αααanti‐42). Compared to simple β‐thalassaemia heterozygotes, they had lower haemoglobin levels and red cell indices, but higher α/β biosynthesis, HbF levels and reticulocytes.

Our results suggest that homozygous α‐gene triplication interacts with a severe β‐thalassaemia mutation to cause an α‐chain excess equivalent to that observed in homozygous β‐thalassaemia intermedia. In heterozygotes for severe β‐thalassaemia mutations with one additional α‐globin gene, the α‐chain excess causes a more pronounced degree of anaemia than is usually seen in simple β‐thalassaemia heterozygotes.
TEL‐AML1 fusion in acute lymphoblastic leukaemia of adults
Authors: AGUIAR RICARDO C. T. , SOHAL JASTINDER , Van Rhee FRITS , CARAPETI MELINA , Franklin Ian M. , Goldstone Anthony H. , Goldman John M. , Cross Nicholas C. P.
Published: November 2003

A number of fusion genes have been identified by study of acquired chromosomal translocations. Their detailed characterization has provided insights into mechanisms of leukaemogenesis and has enabled the development of molecular methods to assist in the diagnosis and monitoring of residual disease after treatment. The TEL‐AML1 fusion gene is associated with a cryptic t(12;21)(p12;q22) translocation, and is the commonest known genetic abnormality in childhood B‐cell precursor acute lymphoblastic leukaemia (ALL), occurring in about 25% of cases. We have used RT‐PCR, followed by Southern blotting and direct sequencing, to establish the incidence of TEL‐AML1 rearrangement in 131 adults with acute leukaemia (101 with ALL and 30 with chronic myeloid leukaemia in blastic crisis). Three patients were positive for TEL‐AML1 transcripts. All three had common‐ALL. All other patients were negative for TEL‐AML1. We conclude that the TEL‐AML1 fusion gene is found in adult ALL, though less commonly than in children.
A (15;17) translocation not associated with acute promyelocytic leukaemia
Authors: Di Bona Eros , Montaldi Anna , Guercini Nicola , Rossi Vincenzo , Luciano Anna , Biondi Andrea , Rodeghiero Francesco
Published: November 2003

We report a woman with acute myeloid leukaemia (AML) type M2 according to FAB classification, showing a t(15;17) apparently identical to that of acute promyelocytic leukaemia (APL) on conventional cytogenetic analysis. Fluorescence in situ hybridization (FISH) using cosmidic probes specific for RARα and PML regions did not show a fusion signal as in APL. The breakpoints were assigned to 15q24.3 and 17q21.1. Detailed molecular analyses did not reveal any involvement of RARα and PML genes. The patient was resistant to several front‐line AML treatments and to all‐trans retinoic acid (ATRA). These findings reinforce FISH and RT‐PCR as useful tools for the characterization of a t(15;17) as the translocation specifically associated with APL.
An improved animal model for studying desferrioxamine
Authors: Steward A. , Williamson I. , Madigan T. , BRETNALL A. , Hassan F.
Published: November 2003

A hamster model has been developed for studying desferrioxamine. The hamster shows many similarities to man in terms of plasma stability and metabolites formed from desferrioxamine. The [59Fe]ferritin derived from rats has been shown to be sequestered into liver parenchymal cells when injected intravenously into hamsters. The technique has proved sufficiently sensitive to enable detection of differences of < 1% of the radioactivity administered in the elimination of iron. Alterations in iron excretion were seen when dosing desferrioxamine via different routes. The principal route of iron excretion was into the intestines. The effectiveness of the dosing routes for desferrioxamine in removing iron were subcutaneous (10.5%) > intravenous (6.25%) > oral (3.66%) > control (2.19%). A dose–response relationship was demonstrated using the intravenous dose route. The model offers a simple method for comparing the efficacy of administration routes for determining the optimal use of desferrioxamine.
Neutrophil apoptosis is delayed by the diadenosine polyphosphates, Ap5A and Ap6A: synergism with granulocyte‐macrophage colony‐stimulating factor
Authors: Gasmi Lakhdar , McLennan Alexander G. , Edwards Steven W.
Published: November 2003

In addition to ATP, platelets and other cell types can secrete high quantities of diadenosine polyphosphates Ap3A, Ap4A, Ap5A and Ap6A. There is increasing evidence to show that these molecules can function as novel modulators of cell function. For this report we have measured the effects of the diadenosine polyphosphates Ap5A and Ap6A on neutrophil apoptosis. These molecules can themselves delay neutrophil apoptosis (as assessed by morphology, function, CD16 expression and chromatin integrity), and are as effective on a molar basis as ATP, Ap3A and Ap4A. Moreover, these dinucleotides act synergistically with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) to delay neutrophil apoptosis. Thus, diadenosine polyphosphates may act, in concert with cytokines, as novel modulators of neutrophil function and survival in certain types of inflammatory conditions.
Combination chemotherapy with CHOP for recurrent thrombotic thrombocytopenic purpura
Authors: Spiekermann K. , Wörmann B. , Rumpf K.‐W. , Hiddemann W.
Published: November 2003

We report the case of a 42‐year‐old woman with chronic recurrent thrombotic thrombocytopenic purpura. Therapy with corticosteroids, high‐dose immunoglobulins, plasma exchange and cyclophosphamide only induced short‐lasting remissions during a course of almost 3 months. Following a severe relapse on day 90 after the start of symptoms, polychemotherapy with cyclophosphamide, adriamycin, vincristine and prednisolone (CHOP) was begun. After two cycles of CHOP the patient has stayed in complete remission, with normal platelet counts for more than 9 months to date.
Prognosis in monoclonal gammopathy of undetermined significance
Authors: Vuckovic Josko , Ilic Anita , Knezevic Nebojsa , Marinkovic Marija , Zemunik Tatijana , Dubravcic Mirko
Published: November 2003

Eighty‐seven patients with monoclonal gammopathy of undetermined significance (MGUS) were followed for a period of 1–20 years, median 91 months. Transformation to multiple myeloma occurred in 14 patients of whom seven died as a consequence of the disease. There were 13 unrelated deaths. The actuarial probability of survival was 80% at 10 years and 44% at 15 years and the probabilities of malignant conversion for the same periods were 17% and 30% respectively. The most significant factor influencing the probability of malignant conversion was the increase of monoclonal protein above the level of 30 g/l during the observation period (P<0.001), followed by an increase of M‐protein to more than 50% above the baseline level (P=0.02) and a decreased level of uninvolved immunoglobulins (P=0.054).
Photopheresis in paediatric patients with drug‐resistant chronic graft‐versus‐host disease
Authors: Dall'Amico R. , Rossetti F. , Zulian F. , Montini G. , Murer L. , Andreetta B. , Messina C. , Baraldi E. , Montesco M. C. , Dini G. , Locatelli F. , Argiolu F. , Zacchello G.
Published: November 2003

Photopheresis (ECP) is a new type of photochemotherapy, used for the treatment of oncological and autoimmune diseases. Lymphocytes are drawn from the patients by leukapheresis, treated with 8‐methoxypsoralen (8‐MOP) and ultraviolet light A (UVA) in an extracorporeal system and then reinfused. Skin exposure to 8‐MOP and UVA (PUVA) has been shown to relieve cutaneous symptoms of graft‐versus‐host disease (GVHD) in bone marrow transplant (BMT) recipients. ECP, which is similar in some ways to PUVA, has been used in this study to treat four paediatric patients who developed chronic GVHD following BMT and in whom GVHD had failed to respond to conventional immunosuppressive therapy. Following ECP, skin lesions cleared almost completely and pulmonary function tests improved in two of three patients with cutaneous and lung involvement. Serum bilirubin and transaminases gradually normalized, and γGT decreased considerably in the remaining patient who had a severe cholestatic hepatopathy. The Karnofsky performance score increased to 90% in the three patients with positive responses to ECP and remained unchanged (40%) in the patient who did not respond. Immunosuppressive therapy was reduced in three patients and eventually discontinued in two. No significant side‐effects were observed during the treatment. Our results suggest that ECP is a non‐aggressive treatment that may benefit patients with chronic GVHD who do not respond to standard immunosuppressive therapy.
Avascular necrosis of bone after allogeneic bone marrow transplantation: analysis of risk factors for 4388 patients by the Société Française de Greffe de Moëlle (SFGM)
Authors: Socié G. , Cahn J. Y. , Carmelo J. , Vernant J. P. , Jouet J. P. , Ifrah N. , Milpied N. , Michallet M. , Lioure B. , Pico J. L. , Witz F. , Molina L. , Fischer A. , Bardou V. J. , Gluckman E. , Reiffers J.
Published: November 2003

Increasing numbers of patients are surviving after allogeneic bone marrow transplantation and are therefore at risk for developing late complications. Among these complications, avascular necrosis of bone has been reported, but only two single‐centre studies included sufficient patients to enable analysis of the risk factors for developing avascular necrosis. In this multicentre retrospective study the aim was to assess risk factors for this complication in a large number of patients.

The population consisted of 4388 patients, recorded in the Société Française de Greffe de Moelle (SFGM) data base, who had undergone a single allogeneic bone marrow transplant between January 1974 and December 1993. 77 patients developed avascular necrosis leading to a 4.3% projected incidence at 5 years. Symptoms developed 2–132 months after transplantation. In these 77 patients a mean of 1.87 joints per patient were affected (range 1–7). The hip joint was the most often affected (88% of patients) and 48% of the patients required joint replacement. All but two patients received steroids for acute and/or chronic graft‐versus‐host disease (GvHD) over a mean period of 15 months. In univariate analysis, among eight factors tested as risk factors for developing avascular necrosis, six were shown to be linked to an increased risk: older age, diagnosis of aplastic anaemia or acute leukaemia, an irradiation‐based conditioning regimen, type of GvHD prophylaxis regimens, acute and chronic GvHD. In multivariate logistic regression analysis, five factors remained significantly associated with an increased risk for developing avascular necrosis: chronic GvHD (odds ratio (OR) 3.52), acute GvHD (OR 3.73), age>16 years (OR 5.81), aplastic anaemia (OR 3.90), and acute leukaemia (OR 1.72).

Avascular necrosis is not a rare late complication of allogeneic bone marrow transplantation causing significant morbidity. In this study, older age, initial diagnosis, and GvHD and/or its treatment with steroids emerged as significant risk factors.
The role of immunoglobulin G and fibrinogen in platelet aggregation by Streptococcus sanguis
Authors: Ford I. , Douglas C. W. I. , Cox D. , Rees D. G. C. , Heath J. , Preston F. E.
Published: November 2003

Previous work has shown that the type strain of Streptococcus sanguis, NCTC 7863, induces aggregation of normal platelets by a complement‐dependent mechanism. We investigated the roles of IgG and fibrinogen in the aggregation process. Plasma depleted of IgG by passage through protein A–sepharose failed to support platelet aggregation, as did plasma absorbed at 0°C with whole bacteria. However, absorption of plasma with a non‐aggregating strain of S. sanguis, SK96, did not remove aggregating activity for NCTC 7863. Supplementing 0°C‐absorbed plasma with purified IgG restored the aggregation supporting activity. A monoclonal antibody to the FcγRII receptor inhibited platelet aggregation by the bacteria, indicating a requirement for bacteria–IgG complexes interacting with the Fc receptor in platelet aggregation. There was a lag time to the onset of platelet aggregation of 7–19 min depending upon the platelet donor, but the length of this lag did not correlate with either total IgG concentration recognizing NCTC 7863 in subjects’ plasma, or the concentration any of the four IgG subclasses or with IgG avidity levels.

Fibrinogen was shown to bind rapidly to the bacterial cell surface. Monclonal antibody to GPIIb/IIIa, RGDS peptide, and a specific antagonist for the platelet fibrinogen receptor, GPIIb/IIIa, FK633, inhibited platelet aggregation by NCTC 7863, indicating that platelet aggregation is fibrinogen dependent. These data suggest that platelet aggregation by some strains of S. sanguis requires multiple stimuli/agonists, including IgG–Fc receptor interaction, complement and fibrinogen.