bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of aspirin and hypothermia on platelet function in vivo
Authors: Michelson Alan , Barnard Marc , Khuri Shukri , Rohrer Michael , MacGregor Hollace , Valeri C.
Published: March 2003

Patients undergoing hypothermic cardiopulmonary bypass are often receiving aspirin therapy. Hypothermia, aspirin and cardiopulmonary bypass can each induce a platelet function defect, but it is not known if the effects of aspirin and hypothermia are additive in this regard. To address this question in humans in vivo, the forearm skin temperature of healthy volunteers was equilibrated and maintained at either normothermia (32°C) or hypothermia (28°C or 22°C) before and 16 h after the ingestion of 650 mg aspirin. A standardized template bleeding time was performed on the forearm and the shed blood emerging from the wound was assayed for platelet surface P‐selectin expression by whole blood flow cytometry (reflecting α granule secretion) and thromboxane B2 (the stable metabolite of thromboxane A2) by radioimmunoassay. Hypothermia resulted in marked prolongation of the bleeding time. Aspirin resulted in prolongation of the bleeding time under normothermic conditions, but only minimally augmented the hypothermia‐induced prolongation of the bleeding time. Platelet surface P‐selectin up‐regulation in shed blood was abolished by hypothermia. Aspirin had no effect on maximal platelet surface P‐selectin expression under normothermic or hypothermic conditions. Both hypothermia and aspirin resulted in markedly reduced shed blood thromboxane B2. Although aspirin slightly augmented the hypothermia‐induced reduction in shed blood thromboxane B2, the concentration of thromboxane generated in shed blood under hypothermic conditions in the absence of aspirin had no effect on platelet surface P‐selectin or platelet aggregation in whole blood. In conclusion, as determined by three independent parameters of the shed blood emerging from a standardized bleeding time wound (bleeding time, platelet surface P‐selectin, and thromboxane B2), aspirin did not significantly augment hypothermia‐induced platelet dysfunction in vivo.
Expression of interleukin‐1β and tumour necrosis factor‐α in plasma cells from patients with multiple myeloma
Authors: Sati Hamdi I. A. , Greaves Mike , Apperley Jane F. , Russell R. Graham G. , Croucher Peter I.
Published: March 2003

Interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) are potent bone resorbing cytokines that may contribute to the development of the osteolytic bone disease observed in patients with multiple myeloma (MM). Although these factors have been identified in cultures of bone marrow mononuclear cells isolated from patients, the identity of the cells responsible for producing IL‐1β and TNFα remains unclear. Using a sensitive dual‐colour fluorescence in situ hybridization (FISH) technique and a two‐colour immunofluorescence method we have investigated the expression of the mRNA and protein, for IL‐1β and TNFα, by individual bone marrow plasma cells from patients with MM and monoclonal gammopathy of undetermined significance (MGUS). The mRNA for IL‐1β and TNFα was identified in all cells expressing the immunoglobulin light chain from all patients with MM and MGUS. However, the IL‐1β protein could not be detected in cytoplasmic light chain positive cells in any of the patients examined. In contrast, the TNFα protein was detected in clonal plasma cells from patients with both MM and MGUS. Interestingly, the IL‐1β and TNFα mRNA and proteins were readily detected within a small proportion of the non‐plasma cells from patients with both MM and MGUS. These data suggest that myeloma cells in vivo are able to produce TNFα but not IL‐1β. In addition, a small proportion of accessory cells are likely to be able to contribute to the production of both IL‐1β and TNFα.
Persistent polyclonal B lymphocytosis with binucleated lymphocytes: a study of 25 cases
Authors: MOSSAFA H. , MALAURE H. , MAYNADIE M. , VALENSI F. , SCHILLINGER F. , GARAND R. , JUNG G. , FLANDRIN G. , TROUSSARD X. ,   et le Groupe Français d'Hématologie Cellulaire (GFHC)
Published: March 2003

Persistent and polyclonal lymphocytosis of B lymphocytes (PPBL) with binucleated lymphocytes is an entity characterized by a polyclonal lymphocytosis. The lymphocytosis is stable for years and binucleated lymphocytes are detected on peripheral blood smears. We previously described +i(3q) as a recurrent chromosomal abnormality in seven PPBL patients. In this study we report a large series of 25 PPBL patients and demonstrated that PPBL was associated with +i(3q) in 77% of cases, premature chromosome condensation (PCC) in 50% and both abnormalities in 41% of cases. Furthermore, we demonstrated that i(3q) was present in a minority of B cells, restricted to B lymphocytes independently of the kappa or lambda light Ig chain expression, and exclusively observed in non‐binucleated cells.

The benign clinical course of PPBL and the lack of biological evolution in the majority of cases suggest that recognition of these disorders is so important that aggressive therapy in PPBL has to be avoided. Whether this syndrome represents a premalignant or benign disease remains unclear. The persistence of cytogenetic abnormalities after stopping tobacco use suggests no association with cigarette smoking.
The incidence of trisomy 3 in splenic lymphoma with villous lymphocytes: a study by FISH
Authors: GRUSZKA‐WESTWOOD ALICJA M. , MATUTES ESTELLA , COIGNET LIONEL J. A. , WOTHERSPOON ANDREW , CATOVSKY DANIEL
Published: March 2003

Splenic lymphoma with villous lymphocytes (SLVL) is a low‐grade B‐cell lymphoproliferative disorder characterized by splenomegaly and circulating villous lymphocytes. The relationship between SLVL and splenic marginal zone lymphoma (SMZL), a disorder with identical splenic histology to SLVL, is not clear. Previous studies have failed to show a consistent karyotypic abnormality in SLVL whereas trisomy 3 has been reported in patients with SMZL. The presence of trisomy 3 in SMZL and its absence in SLVL has been viewed as evidence that these are different diseases. However, it is possible that the frequency of trisomy 3 in SLVL has been underestimated because previous studies have relied on conventional cytogenetics. We have therefore used interphase fluorescence in situ hybridization (FISH) to re‐assess the frequency of trisomy 3 in SLVL. We studied 70 patients, who were stratified into four groups according to the percentage of circulating villous lymphocytes. Trisomy 3 was found overall in 17% of patients. In particular, trisomy 3 was detected in 13% of cases with >50% of villous lymphocytes and which were considered typical of SLVL. In conclusion, we have demonstrated that some patients with SLVL have circulating cells with trisomy 3, which does not support the view that SLVL and SMZL are different diseases on the basis of the incidence of trisomy 3.
Chlorambucil resistance in B‐cell chronic lymphocytic leukaemia is mediated through failed Bax induction and selection of high Bcl‐2‐expressing subclones
Authors: PEPPER C. , THOMAS A. , HOY T. , BENTLEY P.
Published: March 2003

Our previous data have shown that high Bcl‐2/Bax ratios in chronic lymphocytic leukaemia (B‐CLL) correlate with in vitro apoptosis and clinical resistance. We have now monitored the in vitro viability of B‐CLL cells in relation to Bcl‐2 and Bax expression over a 48 h time course following exposure to chlorambucil. The results showed that Bax up‐regulation was essential for chlorambucil‐induced apoptosis in B‐CLL cells and a 3‐fold increase in expression within 4 h of exposure to drug was typically observed in sensitive cells; resistant cells failed to up‐regulate Bax at all. In contrast, the constitutively high levels of Bcl‐2 found in B‐CLL cells were found to be down‐regulated in apoptotic cells but the mean Bcl‐2 expression in viable cells was increased, probably as a result of the loss of lower Bcl‐2‐expressing cells into the apoptotic compartment. Taken together, these data add further weight to the suggestion that Bcl‐2/Bax ratios may be pivotal in determining the fate of B‐CLL cells. Furthermore, the Bcl‐2/Bax ratios found in apoptotic B lymphocytes were remarkably similar in the treated, untreated and normal control cells, which suggests that there is a universal Bcl‐2/Bax ratio threshold for cell survival and cell death.
Syndecan‐1 (CD138) expression in human non‐Hodgkin lymphomas
Authors: Sebestyén Anna , Berczi Lajos , Mihalik Rudolf , Paku Sándor , Matolcsy András , Kopper László
Published: March 2003

Syndecan‐1, an important transmembrane heparan sulphate proteoglycan, is expressed in distinct stages of differentiation of normal lymphoid cells :  in pre‐B cells and Ig‐producing plasma cells; however, its normal function, or presence in lymphoid malignancies, is still largely unknown. The expression of syndecan‐1 (CD138) was studied in 57 human non‐Hodgkin lymphomas using immunocytochemistry and immunohistochemistry. Positive expression of syndecan‐1 was found in the plasma cells in chronic lymphoblastic leukaemia (B‐CLL) cases, in different plasmocytoid lymphomas as well as in myeloma. All normal and malignant T cells, or CD5+ cells other than B‐CLL proved to be negative. These results strongly suggest that syndecan‐1 expression is a characteristic phenotypic marker for B‐CLL and lymphoplasmocytoid lymphomas and could be used for diagnostic purposes.
CD59‐deficient blood cells and PIG‐A gene abnormalities in Japanese patients with aplastic anaemia
Authors: AZENISHI YASUHIKO , UEDA ETSUKO , MACHII TAKASHI , NISHIMURA JUN‐ICHI , HIROTA TOSHIYUKI , SHIBANO MASARU , NAKAO SHINJI , KINOSHITA TAROH , MIZOGUCHI HIDEAKI , KITANI TERUO
Published: March 2003

Patients with aplastic anaemia (AA) frequently develop paroxysmal nocturnal haemoglobinuria (PNH) as a late complication. We investigated the frequency of the development of PNH features including a glycosyl phosphatidylinositol (GPI) anchoring defect in 73 Japanese patients with AA. A deficient expression of CD59 was found on erythrocytes and/or granulocytes in 21/73 (28.8%) of the patients. A Ham/sugar water test was positive in 13/21 patients. We also examined mutations of the PIG‐A gene in 11 patients with CD59 deficiency. A heteroduplex analysis detected PIG‐A gene abnormality in 10/11 patients tested. Nucleotide sequencing was performed in six patients and identified eight mutations including three mutations in one patient. The mutations of the PIG‐A gene were all different and included two single‐base insertions, one single‐base deletion, two two‐base deletions, and one each of eight‐base insertion and nine‐ and ten‐base deletions. All mutations but one caused frameshifts. Our findings indicate that a high proportion of Japanese patients with severe AA have a GPI‐anchoring defect and that the PIG‐A gene is mutated in the AA patients who had a GPI deficiency. We found no significant difference in the pattern of the PIG‐A gene mutation between the AA patients with a GPI deficiency and those with de novo PNH.
Metabolic indicators of oxidative stress correlate with haemichrome attachment to membrane, band 3 aggregation and erythrophagocytosis in β‐thalassaemia intermedia
Authors: CAPPELLINI MARIA DOMENICA , TAVAZZI DARIO , DUCA LORENA , GRAZIADEI GIOVANNA , MANNU FRANCA , TURRINI FRANCO , ARESE PAOLO , FIORELLI GEMINO
Published: March 2003

Haematological data, genotype, transfusion requirements, metabolic indicators of oxidative stress (flux via hexose‐monophosphate shunt (HMPS); steady state level of GSH and GSSG, NADPH and NADP; activity of anti‐oxidant enzymes), parameters of membrane damage (aggregated band 3; membrane‐bound haemichromes, autologous immunoglobulins (Igs) and C3 complement fragments) and erythrophagocytosis were measured in erythrocytes (RBC) of 15 β‐thalassaemia intermedia patients (nine splenectomized) with low, if any, transfusion requirements. Patients presented increased aggregated band 3, bound haemichromes, Igs and C3 complement fragments, and increased erythrophagocytosis. Bound haemichromes strongly correlated with aggregated band 3. Anti‐band 3 Igs were predominantly associated with aggregated band 3. Erythrophagocytosis positively correlated with aggregated band 3, haemichromes and Igs, suggesting the involvement of haemichrome‐induced band 3 aggregation in phagocytic removal of β‐thalassaemic RBC. Splenectomized patients showed higher degrees of membrane damage and phagocytosis, significantly higher numbers of circulating RBC precursors, and tendentially higher numbers of reticulocytes. Basal flux via HMPS was increased twofold, but HMPS stimulation by methylene blue was decreased, as was the glucose flux via HMPS. GSH was remarkably decreased, whereas NADPH was increased. Except for unchanged catalase and glutathione reductase, anti‐oxidant enzymes had increased activity. Negative correlation between HMPS stimulation by methylene blue and bound haemichromes indicated that the ability to enhance HMPS may counteract haemichrome precipitation and limit consequent membrane damage leading to erythrophagocytosis.
Increased adhesion of the promyelocytic leukaemia cell line, NB4, to fibronectin and thrombospondin upon all‐trans‐retinoic acid treatment
Authors: Touhami Myriem , Bourge Jean‐François , Legrand Chantal
Published: March 2003

We have evaluated the effects of all‐trans‐retinoic acid (RA) on the adhesion of the human promyelocytic cell line NB4 to various components of the extracellular matrix. NB4 cells, radiolabelled with 111Indium, showed a 2–3‐fold increase (P < 0.001) in adhesion to fibronectin and thrombospondi‘n upon RA (3 × 10−7 m) treatment, whereas adhesion to collagen I, laminin and vitronectin was not modified. The increase in cell adhesion, observed as early as day 1, preceded cell differentiation and was concomitant with tyrosine phosphorylation events. Using flow cytometry, we analysed the expression of major receptors for fibronectin (α4β1 and α5β1) and for thrombospondin (αvβ3, αIIbβ3, CD36 and CD47) on NB4 cells before and after RA treatment. Except for αIIbβ3, which was induced on RA‐treated cells, we found no significant increase in the expression of the other receptors, and a decrease in the expression of CD36, upon RA treatment. Preincubation of RA‐treated cells with blocking antibodies demonstrated a role for α4β1 and α5β1 in cell adhesion to fibronectin and α5β1, αIIbβ3, CD36 and CD47 in cell adhesion to thrombospondin. Experiments with the synthetic peptides GRGDS (0.2 mm) and CSVTCG (0.2 mm) confirmed the participation of integrins, and integrins and CD36, in adhesion of RA‐treated cells to fibronectin and thrombospondin, respectively. Further inhibition by heparin (10 μg/ml) and/or recombinant heparin‐binding domain of thrombospondin (TSP18) indicated the additional participation of heparin‐like receptors in cell adhesion to thrombospondin. Our results indicate that increase in NB4 cell adhesion to fibronectin and thrombospondin upon RA treatment is likely to occur through a modulation of the functional state of several receptors for these proteins.
Alterations of the retinoic acid receptor α(RARα) gene in myeloid and lymphoid malignancies
Authors: Parrado Antonio , West Robert , Jordan Denise , Bastard Christian , McKenna Sharon , Whittaker Jack A. , Bentley Paul , White Denise , Chomienne Christine , Padua Rose Ann
Published: March 2003

The retinoic acid receptor α (RARα) protein plays a central role in myeloid differentiation, and chromosomal translocations disrupting the RARα gene are implicated in the development of acute myeloid leukaemia (AML). To identify haemopoietic malignant disorders which may also be linked to RARα abnormalities, Southern blot analysis was performed in DNA from 153 patients with haematological malignancies other than AML FAB type M3 using RARα cDNA probes. Alterations of RARα were detected in 1/42 myelodysplastic syndromes (MDS), 2/24 AML, 3/47 B‐chronic lymphocytic leukaemias (B‐CLL), 0/40 lymphomas and 0/60 normal individuals. These data strongly suggest that alterations of RARα might predispose for myeloid and lymphoid disorders.
Prognostic value of vertebral lesions detected by magnetic resonance imaging in patients with stage I multiple myeloma
Authors: Mariette Xavier , Zagdanski Anne‐Marie , Guermazi Ali , Bergot Catherine , Arnould Anne , Frija Jacques , Brouet Jean‐Claude , Fermand Jean‐Paul
Published: March 2003

We assessed the role of spinal magnetic resonance imaging (MRI) and bone densitometry as prognostic factors in patients with asymptomatic stage I multiple myeloma (MM) and negative skeletal survey. 55 consecutive patients underwent spinal MRI and 41 of them underwent bone densitometry by dual‐energy X‐ray absorptiometry (DEXA).

Spinal MRI studies showed evidence of bone marrow involvement in 17/55 patients (31%). A diffuse pattern was present in three patients and a focal pattern in 14 patients, nine of them with only one nodular lesion. During a median follow‐up of 25 months, 10 patients had disease progression, 8/17 patients with abnormal MRI and 2/38 patients with normal MRI. Median time to disease progression was not reached in both groups but was significantly different for patients with normal and those with abnormal patterns on MRI (P < 0.0001). Lumbar BMD was only slightly decreased compared with normal people (median lumbar Z score − 0.43) and was not of prognostic value. Using a multivariate analysis the only two independent significant prognostic parameters were abnormal MRI (P < 0.001, HR 30.4, 95% CI 4.3–213) and bone marrow plasmacytosis > 20% (P = 0.004, HR 16.4, 95% CI 2.6–104).

Thus, spinal MRI but not bone densitometry, appeared to be justified in patients with stage I asymptomatic MM and negative skeletal survey.
Induction‐consolidation with an idarubicin‐containing regimen, unpurged marrow autograft, and post‐graft chemotherapy in adult acute lymphoblastic leukaemia
Authors: Bassan Renato , Lerede Teresa , Di Bona Eros , Rambaldi Alessandro , Rossi Giuseppe , Pogliani Enrico , Oriani Alessandro , D'Emilio Anna , Izzi Teodosio , Lambertenghi‐Deliliers Giorgio , Corneo Gianmarco , Barbui Tiziano
Published: March 2003

Between 1991 and 1993 we conducted a collaborative trial in adult acute lymphoblastic leukaemia, introducing an idarubicin (IDA)‐containing regimen for induction and early consolidation, and increasing consolidation intensity with an autologous bone marrow transplantation phase (ABMT, patients aged <51 years) followed by further chemotherapy for 12 weeks and low‐dose maintenance for 6 months (ABMT patients) or 18 months. 96 patients were evaluable for antileukaemic response after induction with vincristine–prednisone–l‐asparaginase plus cumulative IDA 36 or 20 mg/m2 (IVAP‐1 and IVAP‐2), and for disease‐free survival (DFS) after a minimum follow‐up >3.5 years with an off‐therapy interval >1.5 years. The response rate was 44% (7/16) with IVAP‐1 and 90% (72/80) with IVAP‐2 (P = 0.0001), due to regimen‐related toxicities. Post‐remission therapy was administered as planned to most cases but protocol violation was registered in some patients eligible to ABMT and post‐graft chemotherapy. The 5‐year disease‐free survival (DFS) rate was 31%. Multivariate analysis indicated that DFS was improved in patients receiving a transplant (11 allogeneic, DFS 70%; 32 ABMT, 36%; 37 neither, 17%; P < 0.001) and was negatively affected by high‐risk features such as blast cell count >25 × 109/l, T‐cell or mature B‐cell immunophenotype, and t(9;22)/t(4;11) (all P values <0.05). The 5‐year DFS rate was 54% for 26 patients with no high‐risk factor, 26% for 35 patients with any one, and 6% for 18 patients with any two (P < 0.005). IVAP‐2 brought about a high complete response rate and post‐remission treatment including ABMT was feasible and modestly toxic. In spite of the short post‐graft chemotherapy phase, the long‐term DFS rate was good in cases with no high‐risk feature. However, because autografting may be redundant in the standard‐risk category, its role requires further investigation for high‐risk cases.
Definition of a standard‐risk group in children with AML
Authors: CREUTZIG U. , ZIMMERMANN M. , RITTER J. , HENZE G. , GRAF N. , LÖFFLER H. , SCHELLONG G.
Published: March 2003

To define paediatric AML patients with a favourable outcome in order to design a risk‐adapted therapy, we analysed 489 children under 17 years of age treated similarly in studies AML‐BFM 83 and 87. 369 patients (75.4%) achieved remission. Estimated probabilities of survival, event‐free survival (EFS) and disease‐free survival (DFS) at 5 years were 50% (SE 2%), 43% (SE 2%) and 58% (SE 3%), respectively. Multivariate analysis revealed bone marrow blasts on day 15, morphologically defined risk groups and hyperleucocytosis to be of prognostic value. EFS at 5 years estimated for patients with 5% and >5% blasts on day 15 were 56% (SE 3%) v 27% (SE 4%); for the favourable morphological subgroups (M1/M2 with Auer rods, M3 and M4eo) it was 60% (SE 4%) compared with other patients (33%, SE 3%), P (Kaplan‐Meier)  =  0.0001 each. Hyperleucocytosis proved to be an independent prognostic factor, indicating a high risk, especially for early failure. The specific karyotypes t(8;21), t(15;17) and inv16 were closely related to the favourable morphology and outcome was in the same range. We conclude that for the definition of a standard‐risk group a combination of morphological and response criteria may be sufficient. The standard‐risk group defined by favourable morphology and a blast cell reduction on day 15 (not required for M3) comprises 31% of all patients, P survival, pEFS and pDFS at 5 years were 73% (SE 4%), 68% (SE 5%) and 76% (SE 4%), respectively.
Severe inclusion body β‐thalassaemia with haemolysis in a patient double heterozygous for β°‐thalassaemia and quadruplicated α‐globin gene arrangement of the anti‐4.2 type
Authors: Beris Ph. , Solenthaler M. , Deutsch S. , Darbellay R. , Tobler A. , Bochaton‐Pialat M.‐L. , Gabbiani G.
Published: March 2003

We describe a new case of an association of α‐globin gene quadruplication of the anti‐4.2 type with β °‐thalassaemia. The patient, a young woman of mixed Brazilian–Portuguese origin, suffered from chronic haemolytic anaemia with splenomegaly. Bone marrow supravital staining with brilliant cresyl blue and electron microscopy studies showed large inclusion bodies in about 3% of erythroblasts. Upon immunofluorescent staining these inclusions reacted with a monoclonal antibody to α‐ but not to β‐globin. Analysis of α‐globin cluster by Southern blotting showed the presence of pathologic fragments specific for the anti‐4.2 α‐globin gene quadruplication. α/β mRNA ratio was higher than in cases combining α‐globin triplication and β°‐thalassaemia or in cases of β°‐thalassaemia heterozygous state alone (18, 14.7 and 10.1 respectively). Our data confirmed the hypothesis that the clinically detectable haemolysis in this β°‐thalassaemic patient was due to an unusually high amount of precipitated α‐globin in erythroid precursors. This considerable excess of α‐globin chains was due partly to the β‐globin deficit caused by the presence of the β°‐thalassaemic gene, but also to the presence of 6 active α‐globin genes resulting from α‐globin gene quadruplication in one chromosome.
The cytoskeletal linker protein moesin: decreased levels in Wiskott‐Aldrich syndrome platelets and identification of a cleavage pathway in normal platelets
Authors: Shcherbina Anna , Bretscher Anthony , Rosen Fred S. , Kenney Dianne M. , Remold‐O'Donnell Eileen
Published: March 2003

The Wiskott‐Aldrich syndrome (WAS) is a severe disease of platelets (small size, thrombocytopenia) and lymphocytes (immunodeficiency) arising from mutations of the X‐chromosome gene WASP. Because of the prominent role of cytoskeletal abnormalities, particularly the paucity of surface microvilli, in the cellular pathology of this disease, blood cells from WAS patients were examined for moesin, a cytoskeletal linker protein that stabilizes cell surface microvilli, filopodia and lamellipodia. Comparison of patient and normal lymphocytes by immunofluorescence microscopy and immunoblotting showed normal levels and distribution of moesin in lymphocytes of WAS patients. In contrast, platelets from WAS patients stained only dimly for moesin relative to normal platelets. Quantitation by immunoblot revealed significantly decreased moesin levels in WAS patient platelets relative to normal platelets (63.5 ± 4.9% of normal levels, n = 8, P < 0.0001). A novel reaction of normal platelets was discovered that may play a role in the depletion of moesin in patient platelets, namely the cleavage of moesin as a late event in platelet activation in response to certain platelet agonists.
Repeated peripheral stem cell mobilization in healthy donors: time‐dependent changes in mobilization efficiency
Authors: Tichelli André , Passweg Jakob , Hoffmann Till , Gregor Michael , Kühne Thomas , Favre Geneviève , Wodnar‐Filipowicz Alexandra , Gratwohl Alois
Published: March 2003

Mobilization of peripheral blood stem cells was analysed in 10 consecutive healthy donors undergoing repeated stem cell mobilization for allogeneic transplantation. Donors received recombinant G‐CSF at a dose of 10 μg/kg/d for both mobilizations. Collection of stem cells was started on day 5 of G‐CSF administration. To compare the efficiency of first and second mobilization, we determined the leucocyte and CD34+ cell counts in peripheral blood, and the yield of nucleated cells and CD34+ cell counts in the apheresis product. CD34+ cell numbers in peripheral blood were (median) 81.2 × 106/l during the first and 50.4 × 106/l during the second mobilization (P = 0.007). Likewise, CD34+ cells in the apheresis product decreased from 319.8 × 106 to 275.7 × 106 (P = 0.02). Decrease in CD34+ cell counts in peripheral blood and in the apheresis product was associated with the time interval between first and second mobilization. In a regression analysis there was a correlation between the ratios of CD34+ cell counts of first and second mobilization and the inverse of time interval between procedures (r2 = 0.51 peripheral blood; r2 = 0.74 apheresis product). Thus, stem cell yield is reduced when healthy donors receive repeated mobilization within a short time. Nevertheless, an adequate number of stem cells may repeatedly be mobilized within 2 months.
Preliminary assessment of whole‐blood, red‐cell and platelet‐ leucodepleting filters for possible induction of prion release by leucocyte fragmentation during room temperature processing
Authors: Prowse  , Hornsey  , Drummond  , MacGregor  , Pepper  , Barclay  , Bethel  , Walker  , Barnard  , Kirby  , Hope
Published: March 2003

Universal leucodepletion is being introduced in the U.K. to reduce a theoretical risk of Creutzfeldt‐Jakob disease (CJD) transmission. If CJD infectivity is associated with leucocytes, any cell fragmentation associated with filtration could reduce the potential benefit. Four types each of whole blood, red cell and platelet leucodepletion filters were assessed after holding of blood units for at least 4 h at 22°C. In all cases the mean residual leucocyte content was <1 000 000 per unit, with only two individual filtered whole blood units having a leucocyte content exceeding this. Evidence of leucocyte fragmentation during filtration was sought but not found by assay of soluble elastase, beta‐thromboglobulin and normal prion protein, as well as by isotopic labelling of leucocyte external membrane. These preliminary studies indicate that it was possible to prepare leucodepleted blood components by filtration at room temperature, and that this appeared not to be associated with overt cell fragmentation. Definitive demonstration that fragmentation does not occur requires the development of improved general (non‐specific) assays for cell membrane fragments.
Induction of apoptosis and caspase activation in cells obtained from familial haemophagocytic lymphohistiocytosis patients
Authors: Fadeel Bengt , Orrenius Sten , Henter Jan‐Inge
Published: March 2003

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare and uniformly fatal disorder of early childhood characterized by fever, hepatosplenomegaly, cytopenia and widespread infiltration of vital organs by activated lymphocytes and macrophages. In order to test whether the massive accumulation of immune cells in these patients is associated with a perturbation of apoptosis, lymphocytes were isolated from eight patients and subjected to the chemotherapeutic agent etoposide or agonistic anti‐Fas monoclonal antibodies in vitro. These stimuli elicited a normal apoptotic response in FHL patient cells when compared to healthy controls, as determined by phosphatidylserine exposure, DNA fragmentation, in vitro cleavage of the caspase‐3‐like substrate aspartate‐glutamate‐valine‐aspartate‐7‐amino‐4‐methyl‐coumarin (DEVD‐AMC) and proteolysis of the anti‐apoptotic protein Bcl‐2. In addition, the degree of constitutive and Fas‐triggered apoptosis in freshly isolated neutrophils was monitored in three children, with similar results. These studies indicate that immune cells derived from FHL patients are not inherently resistant to apoptosis induction. Specifically, etoposide‐induced and Fas‐triggered activation of intracellular caspases appears to remain intact in these individuals. However, the degree of spontaneous activation of caspase‐3‐like enzymes in activated lymphocytes was attenuated in three of the four patients tested prior to initiation of therapy, suggesting a possible biological deficiency in these individuals.
GBV‐C/HGV and HCV infection in mixed cryoglobulinaemia
Authors: Crovatto Marina , Mazzaro Cesare , Mishiro Shunij , Santini Gianfranco , Baracetti Stefano , Zorat Francesca , Pozzato Gabriele
Published: March 2003

Recently, a new, suspected hepatotropic virus has been identified. Named GBV‐C/HGV, this virus shares with the hepatitis C virus (HCV) routes of transmission and molecular organization. Indeed, a proportion of HCV‐infected patients (10–25%) are also carriers of GBV‐C/HGV. Since mixed cryoglobulinaemia (MC) is closely associated with HCV infection, the aim of this study was to determine the prevalence of GBV‐C/HGV infection in MC patients, and to investigate whether the double infection influenced the clinical and/or laboratory aspects of the disease.

52 patients affected by MC were studied. 100 patients affected by HCV‐positive chronic liver disease (CLD) without MC were used as control group. To determine the prevalence of GBV‐C/HGV infection in general population, 150 blood donors were studied, as well as 80 patients affected by non‐A–E CLD.

Among the MC patients, only five (9.6%) were positive for both HCV and GBV‐C/HGV infection. No difference was found between patients with and without double infection as regards main clinical and laboratory aspects. Among HCV‐positive CLD cases, 27 were positive for double infection. Among blood donors, the prevalence of GBV‐C/HGV infection was 8.0%, whereas in cases with cryptogenetic CLD the prevalence was 5.0%.

In conclusion, these data show that GBV‐C/HGV infection does not play any role in the pathogenesis of MC.
Aberrant expression and reverse signalling of CD70 on malignant B cells
Authors: Lens Susanne M. A. , Drillenburg Paul , Den Drijver Bianca F. A. , Van Schijndel Gijs , Pals Steven T. , Van Lier René A. W. , Van Oers Marinus H. J.
Published: March 2003

In normal lymphoid tissues the tumour necrosis factor‐receptor family member CD27 and its ligand CD70 have a restricted expression pattern. Previously, we reported that expression of CD27 is deregulated in B‐cell leukaemias and lymphomas. Here we show that, although infrequently expressed by normal human B cells in vivo, CD70 is found on 50% of B‐CLLs, 33% of follicle centre lymphomas, 71% of large B‐cell lymphomas, and 25% of mantle cell lymphomas. Interestingly, in the majority of leukaemias and lymphomas examined, CD70 was found to have a capped appearance, a feature that coincided with co‐expression of CD27. Functional analysis showed that a subset of B‐CLLs could proliferate vigorously in response to CD70 mAb but not to CD27 mAb. This response was synergistically enhanced by ligation of CD40 but inhibited by the presence of IL‐4. Additional experiments indicated that the proliferative response was due to an agonistic signal delivered via CD70, rather than blocking of negative signalling by CD27. Thus, next to its role as ligand, in a subset of malignant B cells CD70 can operate as receptor and as such might contribute to progression of these B‐cell malignancies.
Use of the haemopoietic progenitor cell count of the Sysmex SE‐9500 to refine apheresis timing of peripheral blood stem cells
Authors: Pollard Y. , Watts M. J. , Grant D. , Chavda N. , Linch D. C. , Machin S. J.
Published: March 2003

The Sysmex SE‐9500 automated cell counter provides an estimate of immature cells referred to as ‘haemopoietic progenitor cells’ (HPC). The aim of this study was to relate the HPC count to CD34+ cell levels in mobilized peripheral blood and to determine whether the HPC count was valuable in predicting apheresis yields of CD34+ cells. Studies were performed on 114 samples from 67 patients undergoing progenitor cell mobilization.

HPC cells were undetectable in the steady state. On the day of apheresis the HPC and CD34 counts were weakly correlated, with the median HPC count being 2.3‐fold greater than the CD34+ cell count. The HPC count did not include the CD34+ cells as immunomagnetic depletion of CD34+ cells did not significantly reduce the HPC count. CD34+ cell counts predicted for apheresis yield (r = 0.773) on that day as did the HPC count (r = 0.623). The optimal strategy to prevent unnecessary harvesting while minimizing the risk of missing an adequate harvest, and minimizing laboratory investigations, was to screen all samples for HPC and limit CD34+ cell measurements to those with an HPC count <10 × 106/l (19/114 samples). If the CD34+ cell count was also <10 × 106/l then harvesting should not be carried out.
FADD expression and caspase activation in B‐cell lymphomas resistant to Fas‐mediated apoptosis
Authors: Xerri Luc , Devilard Elisabeth , Bouabdallah Reda , Stoppa Anne‐Marie , Hassoun Jacques , Birg Françoise
Published: March 2003

We have previously shown that malignant B cells from non‐Hodgkin's lymphomas (NHL) are resistant to Fas‐mediated apoptosis. To determine the mechanisms underlying this resistance, we analysed by Western blotting the expression of several apoptotic regulators, caspase 3, caspase 8, FADD and poly(ADP‐ribose) polymerase (PARP) in fresh lymphoma cells, isolated from 16 B‐NHL biopsy samples of different histological subtypes, and displaying variable levels of Fas expression. The profiles of expression of these apoptotic regulators were monitored in cell lysates at different times following Fas with or without CD40 stimulation. Expression of FADD and of the uncleaved forms of PARP, caspase 3 and caspase 8 were detected in all untreated NHL samples. Low levels of PARP cleavage were noted in three untreated samples. Fas stimulation alone induced neither significant apoptosis nor significant changes in the expression profiles of FADD, caspases 3 and 8 and PARP in the 16 samples, except for variations in FADD and caspase 8 expression levels in a minority of samples. Fas/CD40 co‐stimulation induced apoptosis and cleavage of caspase 3, caspase 8 and PARP in the five NHLs tested; expression of FADD was not modified. Our results showed (1) that induction of apoptosis in B‐NHLs by Fas/CD40 co‐stimulation used the same caspase executioner machinery as the normal Fas pathway, and (2) that NHL cells which resisted Fas‐mediated apoptosis displayed no defect in either expression or functionality of caspases 3 and 8, nor in FADD expression. The dysfunction underlying NHL resistance to apoptosis must therefore lie upstream of caspase 8, or could alternatively be influenced by anti‐apoptotic regulators of the Bcl‐2 family.
P53 deletion is not a frequent event in multiple myeloma
Authors: Avet‐Loiseau Hervé , Li Jian‐Yong , Godon Catherine , Morineau Nadine , Daviet Axelle , Harousseau Jean‐Luc , Facon Thierry , Bataille Régis
Published: March 2003

Recently a high incidence of interstitial deletion of the P53 locus has been reported in multiple myeloma (MM) patients. Considering the importance of such an event, we analysed 79 patients with advanced‐stage disease using fluorescence in situ hybridization (FISH). Strikingly, we found only 7/79 patients with a P53 deletion. In order to rule out any differences in probe selection, we reanalysed all the patients with the same probe as that used in a previous study, and confirmed the low incidence of P53 deletion (7/79, 9%). The only explanation is a difference in hybridization efficiency. Since hybridization is far less efficient on malignant plasma cells than on other bone marrow cells we suggest that this poor hybridization efficiency may lead to a false P53 deletion.
Acute lymphoblastic leukaemia occurring as second malignancy: report of the GIMEMA Archive of Adult Acute Leukaemia
Authors: Pagano Livio , Pulsoni Alessandro , Tosti Maria Elena , Annino Luciana , Mele Alfonso , Camera Andrea , Martino Bruno , Guglielmi Cesare , Cerri Raffaella , Di Bona Eros , Invernizzi Rosangela , Castagnola Carlo , Bassan Roberto , Mele Luca , Todeschini Giuseppe , Leone Giuseppe , Mandelli Franco
Published: March 2003

Between July 1992 and June 1996, 901 new cases of adult acute lymphoblastic leukaemia were recorded in the GIMEMA Archive of Adult Acute Leukaemia; 21 of them (2.3%) had a previous primary malignancy (PM). We found that secondary acute lymphoblastic leukaemia cases (sALL) presented with older age, a high incidence of pre‐pre‐B immunophenotype and a significantly higher prevalence of cancer among relatives compared to de novo ALL.

The leukaemogenic activity of the cytotoxic drugs employed for the treatment of PM may have played a potential role in only a proportion of patients, opening the possibility that some sALL patients may have developed two or more malignancies due to individual predisposing factors.
Serum interleukin‐6 has no discriminatory role in paraproteinaemia nor a prognostic role in multiple myeloma
Authors: Schaar  , Kaiser  , Snijder  , Ong  , Hermans  , Franck  , Kluin‐Nelemans
Published: March 2003

We determined interleukin‐6 (IL‐6) levels in the serum of 212 well‐defined patients with newly diagnosed paraproteinaemia and evaluated its discriminatory value and prognostic role in multiple myeloma (MM). Results were compared with serum neural cell adhesion molecule and β‐2‐microglobulin, both established prognostic MM markers. Paraproteinaemia‐related diagnoses were: MM (60), other haematological diseases (46), solid tumours (35), autoimmune diseases (17) and monoclonal gammopathy of unknown significance (MGUS) (54). The range of IL‐6 levels in all diagnostic groups overlapped widely and did not serve as a discriminatory marker in newly diagnosed paraproteinaemia even when patients with infection or fever (42) were excluded. In MM high IL‐6 levels (50 pg/ml) were not associated with a shorter survival (P = 0.24). We compared our results with 20 published studies on serum IL‐6 in paraproteinaemia and/or MM. IL‐6 data have to be related to the assay used (bio‐ or immunoassay) and to the status of MM (newly diagnosed, during therapy, progressive disease). We conclude that serum IL‐6 is not specific for paraproteinaemia‐related diseases and will not serve as a reliable discriminatory or prognostic marker in paraproteinaemia and MM.
The PNH phenotype cells that emerge in most patients after CAMPATH‐1H therapy are present prior to treatment
Authors: Rawstron  , Rollinson  , Richards  , Short  , English  , Morgan  , Hale  , Hillmen
Published: March 2003

Paroxysmal nocturnal haemoglobinuria (PNH) cells are deficient in glycosylphosphatidylinositol (GPI) linked antigens due to a somatic mutation of the PIG‐A gene in a haemopoietic stem cell. It appears that a PNH clone reaches detectable proportions only when there is selection in its favour. GPI‐deficient T lymphocytes have been identified in patients treated with CAMPATH‐1H, a monoclonal antibody against the GPI‐linked CD52 molecule. CAMPATH‐1H selects for cells that are deficient in CD52 (such as PNH‐like cells) promoting the development of a PNH‐like clone (analogous to PNH). We report that 10/15 patients with chronic lymphocytic leukaemia developed PNH‐like lymphocytes after therapy with CAMPATH‐1H. The remaining five patients developed no PNH‐like cells at any stage, including one patient who received 12 weeks of therapy. The inactivating PIG‐A mutation has been identified in one patient. This mutation was detectable by an extremely sensitive mutation‐specific PCR‐based analysis in the patient's mononuclear cells prior to CAMPATH‐1H therapy. The frequency and phenotype of GPI‐deficient lymphocytes after CAMPATH‐1H and the detection of a PIG‐A mutation in the lymphocytes prior to CAMPATH‐1H therapy indicated that such mutations were present in a very small proportion of cells prior to selection in their favour by CAMPATH‐1H. This suggests that a large proportion of individuals have cells with PIG‐A mutations that are not detectable by flow cytometry and thus may have the potential to develop PNH.
Hepatitis B and C virus infection in Romanian non‐Hodgkin's lymphoma patients
Authors: Cucuianu Andrei , Patiu Mariana , Duma Milena , Basarab Carmen , Soritau Olga , Bojan Anca , Vasilache Anca , Mates Mihaela , Petrov Ljubomir
Published: March 2003

We determined the hepatitis C virus (HCV) antibodies (anti‐HCV) and the hepatitis B virus (HBV) surface antigen (HBsAg) in a cohort of 68 consecutive non‐Hodgkin's lymphoma (NHL) patients diagnosed and treated in our institution between December 1997 and March 1999. 27 cases were diagnosed as low‐grade, 33 as intermediate‐grade, and eight as high‐grade NHL. In 35 cases (51.4%) we found evidence of either HCV or HBV infection. Anti‐HCV antibodies were found in 20 patients (29.5%) and HBsAg was found in 21 patients (30.8%). In six patients both anti‐HCV and HBsAg were present. Anti‐HCV were present in 12/27 low‐grade NHL cases (44.4%) and in 8/41 intermediate/high‐grade (aggressive) NHL cases (19.5%, P < 0.03). HBsAg was found in 10/27 low‐grade NHL cases (37%) and in 11/41 aggressive NHL cases (26.8%). Evidence of liver disease, as reflected by elevated aminotransferases or typical alterations at liver biopsy, was present in eight patients. Cryoglobulins were present in six patients, all anti‐HCV positive and with low‐grade NHL. The prevalence of both HCV antibodies and HBsAg was significantly higher (P < 0.0001) in our NHL cases than in a sample of the general Romanian population, where the prevalence of anti‐HCV was 4.9% and that of HBsAg was 6.3%. It is difficult to say whether either HCV or HBV had actually been involved in lymphomagenesis or if α‐interferon treatment would be effective in this subset of patients.
Clonal selection of CD56+ t(8;21) AML blasts: further suggestion of the adverse clinical significance of this biological marker?
Authors: Daniels Jasmine T. , Davis Brad J. , Houde‐McGrail Lisa , Byrd John C.
Published: March 2003

Although patients with acute myelogenous leukaemia (AML) and t(8;21)(q22;q22) have a favourable prognosis, a subset die despite receiving appropriate treatment. Recent reports suggest that expression of the CD56 antigen might predict for both extramedullary disease and poor outcome in these patients. We describe a patient who presented with CD56‐negative t(8;21) AML who achieved a complete remission and was subsequently treated with three consolidative courses of high‐dose cytarabine therapy. She relapsed 9 months later with extramedullary and bone marrow involvement of CD56‐positive t(8;21) AML. This case demonstrates clonal evolution and provides further support that blast expression of CD56 might be an unfavourable prognostic factor in t(8;21) AML.
Characterization of 12p molecular events outside ETV6 in complex karyotypes of acute myeloid malignancies
Authors: La Starza Roberta , Stella Mario , Testoni Nicoletta , Di Bona Eros , Ciolli Stefania , Marynen Peter , Martelli Massimo Fabrizio , Mandelli Franco , Mecucci Cristina
Published: March 2003

Acute myeloid disorders with rearrangements of 12p outside the ETV6 gene were characterized by fluorescence in situ hybridization (FISH) with a panel of DNA probes. Seven patients with de novo acute myeloid leukaemia (AML), one with secondary acute myeloid leukaemia (sAML), and one in the blast phase of chronic myeloid leukaemia (CML‐BP) were enrolled in the study. All AML cases showed multiple karyotypic changes. Chromosome 5 and/or 7 deletions were the most frequent accompanying changes. FISH revealed amplification, cryptic translocation, and fragmentation of chromosome 12, not discernible at karyotypic level. Different karyotypic rearrangements of 12p showed a common molecular event. Among the seven cases in which breakpoints could be determined, six were telomeric and one centromeric to ETV6. In three AML cases a new recurrent breakpoint in the telomeric region was identified distally to locus D12S158 and to pac 922B22 which is the most telomeric probe available for 12p. Accompanying cryptic deletions were also detected in five patients and the commonly deleted region, of around 700 kb, included the ETV6 gene and the D12S391 locus.
Chronic myelogenous leukaemia with p185BCR/ABL expression: characteristics and clinical significance
Authors: Ravandi Farhad , Cortes Jorge , Albitar Maher , Arlinghaus Ralph , Qiang Guo Jie , Talpaz Moshe , Kantarjian Hagop M.
Published: March 2003

We investigated the significance of p185BCR/ABL expression in patients with chronic myelogenous leukaemia (CML) in relation to disease features, therapy and outcome. Results of Western blot analysis for 1384 patients referred with a diagnosis of CML to our institution from 1989 to 1997 were reviewed. Clinical characteristics, results of cytogenetic analysis and RT‐PCR for BCR rearrangement were analysed. Five patients with Ph‐positive CML expressing the p185BCR/ABL hybrid protein were identified. By RT‐PCR, bone marrow specimens of these patients were confirmed to have an e1a2 junction. The median age at diagnosis of these patients was 55 years (range 43–76). All had elevated white cell counts at diagnosis (median 50 × 109/l, range 11.7–163 × 109/l). Four patients had monocytosis (range 10–16%) with a low neutrophil/monocyte ratio in the peripheral blood (range 3.4–5.7). Patients presented with various stages of the disease (two in chronic–phase CP, two in accelerated–phase AP, and one in blastic‐phase BP). The clinical course and therapy of the patients varied, with one patient receiving hydroxyurea only, three patients receiving hydroxyurea followed by interferon‐α based regimens and bone marrow transplantation. The patient presenting in BP was treated with combination chemotherapy. The clinical outcome of the patients was also varied with one patient alive and in complete remission (with complete cytogenetic remission after transplant) and four patients dead after progression to more advanced stages.

We conclude that patients with Ph‐positive p185BCR/ABL CML frequently present with monocytosis and a low neutrophil/monocyte ratio in the peripheral blood, aiding the speculation that the presence of the p185BCR/ABL hybrid protein may contribute to a phenotype intermediate between CML and CMML. Of interest, the only other specific clinical feature identified was the absence of splenomegaly in four of five patients. There was no definite association with transformation to lymphoid blast phase.