bluuebunny/crossref_metadata_2025 huggingface dataset:
MACULAR HAEMORRHAGE IN ADULT ACUTE LEUKAEMIA PATIENTS AT PRESENTATION AND THE RISK OF SUBSEQUENT INTRACRANIAL HAEMORRHAGE
Authors: Jackson Nicholas , Reddy Sagili Chandrasekhara , Harun Mohd Hishamuddin , Quah Soon Hoe , Low Heng Chin
Published: November 2003

Retinal changes are common in adult acute leukaemia patients at presentation, but whether they correlate with the risk of subsequent intracranial haemorrhage is unknown. A 4‐year study has been carried out in 82 newly‐diagnosed acute leukaemia patients, aged 12–77 years, who were studied prospectively for the presence of intra‐retinal haemorrhages (IRH), white‐centred haemorrhages (WCH), cotton‐wool spots (CWS) and macular haemorrhages (MH). Groups with and without these features were compared for their risk of intra‐cranial haemorrhage (ICH) within the first 30 d following diagnosis.

There was no association between the incidence of ICH and the presence of IRH, WCH or CWS. However, 6/13 of those with MH developed ICH, compared to 6/69 of those without MH (relative risk 5.0, CI 95% [2.03–12.33], P=0.003). The only other identifiable risk factor for ICH was the M3 subtype of AML, but if the four cases of M3‐AML were discounted from analysis, MH remained a highly significant risk factor for ICH. Patients with MH should be monitored intensively for the development of ICH, and receive priority in the allocation of platelets where these are in short supply.
Inverse correlation between loss of heterozygosity of the short arm of chromosome 12 and p15ink4B/p16ink4 gene inactivation in childhood acute lymphoblastic leukaemia
Authors: Heyman Mats , Calero Moreno Teresa , Liu Yie , Grandér Dan , Rasool Omid , Borgonova‐Brandter Laura , Merup Mats , Söderhäll Stefan , Einhorn Stefan
Published: November 2003

Seventy‐four patients with acute lymphoblastic leukaemia (ALL) were analysed with limited allelotyping to detect loss of heterozygosity on chromosome segments 6q, 9p, 12p and 13q in order to detect patterns of genetic alteration. In the case of chromosome 9, analyses were also performed to detect inactivation of the p15ink4B and p16ink4 genes by Southern blot and sequencing techniques. The deletion data from these chromosomes were correlated to each other and to clinical features including prognosis. Allelic loss of these chromosomal regions could be detected in 24% (6q), 15% (12p) and 10% (13q) of the patients respectively, whereas aberrations involving 9p were detected in approximately 50% of the cases. There was an inverse correlation between loss of heterozygosity (LOH) for chromosome 12 and inactivation of the p16ink4 gene. This finding may suggest that a leukaemogenic event on chromosome 12p affects the same pathway of cell‐cycle control as p16ink4 inactivation or, alternatively, reflects the fact that these mutations tend to occur in cells of different lineages.
Altered surface expression of effector cell molecules on neutrophils in myelodysplastic syndromes
Authors: OHSAKA AKIMICHI , SAIONJI KATSU , IGARI JUN , WATANABE NORIMICHI , IWABUCHI KAZUHISA , NAGAOKA ISAO
Published: November 2003

The surface expression of effector cell molecules on neutrophils was examined in 18 patients with myelodysplastic syndromes (MDS) and 20 healthy control subjects. The MDS patients were further classified as low clinical risk (L‐MDS, n = 7) and high clinical risk (H‐MDS, n = 11). The expression of Fc receptors for IgG (FcR), complement receptors (CR) and cellular adhesion molecules on neutrophils was determined by flow cytometry and monoclonal antibodies. The effect of granulocyte colony‐stimulating factor (G‐CSF) and tumour necrosis factor‐alpha (TNF) on L‐selectin shedding and CR up‐regulation on neutrophils was also examined. The percentage of FcRI‐positive neutrophils and CD11b/CR3 expression on neutrophils were significantly increased in the H‐MDS patients when compared to the controls. In contrast, the expression of FcRII, FcRIII, L‐selectin, LFA‐1 and CD18 on neutrophils was significantly reduced in the H‐MDS patients compared with the controls. The L‐MDS neutrophils exhibited lower expressions of CR1, L‐selectin, LFA‐1 and CD18 than those of the controls. Neutrophils from some H‐MDS patients showed impaired L‐selectin shedding and CR up‐regulation after stimulation with G‐CSF or TNF, although these were not significantly different when assessed in the whole H‐MDS group. These findings suggest that an altered surface expression of effector cell molecules and an impaired modulation of cellular adhesion molecules on neutrophils may contribute to the increased susceptibility to bacterial infections in MDS patients.
Heterogenous band 3 deficiency in hereditary spherocytosis related to different band 3 gene defects
Authors: Dhermy Didier , Galand Colette , Bournier Odile , Boulanger Laurent , Cynober Thérèse , Schismanoff Pierre Olivier , Bursaux Elisabeth , Tchernia Boivin Gil, Pierre , Garbarz Michel
Published: November 2003

Among 80 hereditary spherocytosis (HS) kindreds studied using denaturing electrophoretic separation of solubilized eythrocyte membrane proteins, we recognized three prominent subsets: HS with isolated spectrin deficiency, HS with combined spectrin and ankyrin deficiency, and HS with band 3 deficiency. These three subsets represent more than 80% of the HS kindreds studied. In this study, eight dominant HS kindreds with band 3 deficiency were investigated for band 3 mutations. In three of these kindreds, linkage analyses confirmed the band 3 gene as the culprit gene. In an attempt to identify the responsible mutations, denaturing gradient gel electrophoresis (DGGE) was used to explore the coding exons (exons 2–20) of band 3 gene. Five different mutations were found in the eight kindreds. In five kindreds we identified substitutions of highly conserved residues, positioned at boundaries of putative transmembrane segments: a C → T substitution at codon 490 changed arginine (CGC) to cysteine (TGC) in three kindreds, a C → T substitution at codon 837 changed threonine (ACG) to methionine (ATG) in two kindreds. In the sixth kindred a G deletion was found in a stretch of five G starting at position 1475, leading to a stop codon either at position 1527 or 1565. In the seventh kindred a T deletion at position 1600 resulted in a stop codon at position 1733 and in the last kindred a T deletion was identified at position 355, leading to a stop codon at position 447. The mutant transcript was present in HS patients bearing missense mutations, whereas only the normal transcript was found in HS patients with frameshift mutations. In the latter group the mean decrease in membrane band 3 content was significantly lower, leading to speculation that missense mutations may have some sort of dominant negative effect.
Cytokines active on granulomonopoiesis: release and consumption by human marrow myeloid stromal cells
Authors: Sensebé Luc , Mortensen Borge T. , Fixe Philippe , Hervé Patrick , Charbord Pierre
Published: November 2003

Haemopoiesis is sustained and preferentially committed to granulomonopoiesis by myeloid stromal cells generated by colony‐derived cell lines (CDCL). Using ELISA and RIA, we studied, in the supernatant of cells from CDCL, the time course of interleukins 3 and 6 (IL‐3, IL‐6), stem cell factor (SCF), granulocyte‐macrophage, granulocyte and macrophage colony stimulating factors (GM‐CSF, G‐CSF and M‐CSF), macrophage‐inflammatory protein‐1α (MIP‐1α) and transforming growth factor β1 (TGF β1). IL‐6, GM‐CSF, M‐CSF and MIP‐1α were released into the supernatant after medium renewal and, except for M‐CSF, addition of IL‐1β. G‐CSF was detected only after addition of IL‐1β. SCF, contained in medium, first declined and then increased 24 h after medium renewal. Release of TGF β1 started 24 h after medium renewal and lasted until day 7. IL‐3, provided by horse serum, declined throughout the 7 d of observation. In conclusion, stromal cells from CDCL synthesized and released into the supernatant. IL‐6, GM‐CSF, G‐CSF, M‐CSF and MIP‐1α after stimulation by seric factor(s) and/or IL‐1β. TGF β1 was synthesized and released without any obvious extraneous stimuli. There is no definite argument for synthesis of soluble SCF and IL‐3. These data support a model where growth factors increase shortly after medium renewal, and negative regulators take over at a later time.
Cancer in relatives of children with myelodysplastic syndrome, acute and chronic myeloid leukaemia
Authors: Hasle Henrik , Olsen JØrgen H.
Published: November 2003

Familial myelodysplastic syndrome (MDS) has been claimed to account for as many as one third of children with MDS, especially among those showing monosomy 7. The present study is the first to provide population‐based estimates of the risk of haematological and other malignancies in relatives of children with MDS. The study was extended to include children with acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML). The index group consisted of 46 children with MDS, 62 with AML, and eight with CML, which is thought to represent all myeloid leukaemias in Danish children, 1980–91. By linkage to the Central Population Register we identified parents (230), siblings (231), grandparents (151), aunts and uncles (132) and cousins (140). Information on the cancer incidence was obtained from the Danish Cancer Registry. 27 cancers were observed versus 26.7 expected (relative risk 1.0). Leukaemia in relatives was observed in only one family. None of 11 children with MDS and monosomy 7 had family members affected by leukaemia. We found no evidence of an increased overall risk of cancer in the relatives. The risk of familial MDS may be considerably lower than previously estimated.
Complementarity determining region‐III is a useful molecular marker for the evaluation of minimal residual disease in mantle cell lymphoma
Authors: Kurokawa Toshiro , Kinoshita Tomohiro , Murate Takashi , Nagasaka Tetsuro , Kagami Yoshitoyo , Ogura Michinori , Nakamura Shigeo , Seto Masao , Hotta Tomomitsu , Saito Hidehiko
Published: November 2003

Bone marrow (BM) and peripheral blood (PB) involvement in 10 patients with mantle cell lymphoma (MCL) was analysed by a polymerase chain reaction (PCR)‐mediated RNase protection assay. The complementarity determining regions (CDR)‐III of all 10 MCLs examined was amplified efficiently with consensus VH and JH primers by PCR, and BM and/or PB involvement was evaluated by RNase protection assay in all 10 patients examined. Our assay showed BM and/or PB of the entire group to have neoplastic cells at presentation, despite the fact that eight patients were found to have BM and/or PB involvement on the basis of morphological examination and/or surface marker analysis. We also examined minimal residual disease (MRD) after conventional chemotherapy, and detected MRD in a patient in complete remission (CR). Although previous studies have shown that t(11;14) breakpoint amplification by PCR was only applicable to about 30–40% of cases, the present study indicates that CDR‐III is a useful molecular marker and the PCR‐mediated RNase protection assay is a good tool for the evaluation of MRD in MCL. It is suggested that BM and PB of MCL patients are quite frequently involved at presentation and even after conventional chemotherapy at the molecular level.
Frequent involvement of chromosomes 1, 3, 7 and 8 in splenic marginal zone B‐cell lymphoma
Authors: Solé Francesc , Woessner Soledad , Florensa Lourdes , Espinet Blanca , Mollejo Manuela , Martín Pedro , Piris Miguel Angel
Published: November 2003

We have studied 19 cases of splenic marginal zone B‐cell lymphoma (SMZBCL) combining cytological features, conventional cytogenetics, and in situ hybridization (ISH) techniques.

A clonal chromosome abnormality was found in 11/19 patients (58%). The more frequent recurrent abnormalities were: del(3), del (7q), and involvement of chromosomes 1, 3, 7 and 8. No patient showed the translocation t(11;14)(q13;q32). An outstanding finding was the low incidence of trisomy 3 (36%) compared to patients with MALT lymphoma. These findings support the interpretation that SMZBCL is a distinct lymphoproliferative disorder.
Specificity and sensitivity of RHD genotyping methods by PCR‐based DNA amplification
Authors: Aubin Jean‐Thierry , Le Van Kim Caroline , Mouro Isabelle , Colin Yves , Bignozzi Christiane , Brossard Yvon , Cartron Jean‐Pierre
Published: November 2003

We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I–III were the most sensitive and gave a detectable signal with D‐pos/D‐neg mixtures containing only 0.001% D‐positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10–50 times less. Investigation of D variants indicated that false‐negative results were obtained with method II (DIVb variant), method III (DVI and DFR variants) and method IV (DVI variants), but not method I. Weak D (Du) was correctly detected as D‐positive by all methods, but most cases of Rhnull appeared as false‐positives, as they carry normal RH genes that are not phenotypically expressed. Some false‐positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD‐neg but carrying a C‐ or E‐allele, whereas a high incidence of false‐positives was found among non‐Caucasian Rh‐negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.
Detection of BCR/ABL RNA transcripts using the polymerase chain reaction is highly predictive for relapse in patients transplanted with unrelated marrow grafts for chronic myelogenous leukaemia
Authors: Drobyski William R. , Endean Debra J. , Klein John P. , Hessner Martin J.
Published: November 2003

Relapse after allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukaemia (CML) is thought to result from residual leukaemia cells which survive the intensive conditioning regimen and are not eradicated by donor‐derived immune effector cells capable of mediating a graft‐versus‐leukaemia (GVL) effect. Early relapse can be detected using highly sensitive assays such as the polymerase chain reaction (PCR) which have been shown to have predictive value for subsequent relapse in selected patient populations. The validity of PCR for predicting CML relapse in unrelated marrow transplant recipients where the GVL effect appears to be augmented due to increased HLA disparity between donor and recipient, however, has not been well defined. In this study we assessed the prognostic value of PCR in a cohort of 52 patients transplanted with T‐cell‐depleted unrelated marrow grafts for CML. The actual probability of relapse at 3 years was 71% in patients with at least one positive assay versus 6% in patients with no positive assays post‐transplant. Patients with one or more positive assays at any time post‐transplant had a 56‐fold increased risk of relapse which was significantly higher (P = 0.0002) than that observed in patients who remained persistently PCR negative. Moreover, PCR detected relapse a median of 5 months earlier than cytogenetic analysis in a subgroup of patients in whom concurrent sampling had been performed. These data validate the use of PCR as a prognostic test in this patient population and may help to identify a cohort of patients to be considered as candidates for pre‐emptive adoptive immunotherapy.
Increased frequency of somatic mutations at glycophorin A loci in patients with aplastic anaemia, myelodysplastic syndrome and paroxysmal nocturnal haemoglobinuria
Authors: Hattori Hideki , Machii Takashi , Ueda Etsuko , Shibano Masaru , Kageyama Takashi , Kitani Teruo
Published: November 2003

Paroxysmal nocturnal haemoglobinuria (PNH), aplastic anaemia (AA) and myelodysplastic syndrome (MDS) are haemopoietic stem cell disorders. These disorders have some features in common, and a percentage of cases progress to acute leukaemia. We speculated that changes in gene stability are involved in the pathogenesis of these haemopoietic stem cell disorders. Therefore we investigated in vivo mutation frequencies in these disorders by erythrocyte glycophorin A (GPA) mutation assay. The assay enumerates NO or NN variant cells in 106 erythrocytes of the MN type using a flowcytometric technique. Patients undergoing chemotherapy known to be at risk of hypermutageneity were also studied. Events exceeding the 95th percentile of healthy donors (≧ 32 and 34 events, respectively for NO and NN variants) were defined as abnormal. Abnormal events in the NO variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, two out of seven patients with MDS, and four out of nine patients with PNH. Abnormal events in the NN variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, one out of seven patients with MDS, and two out of nine patients with PNH. These results suggest that not only PIG‐A, but also other genes including the GPA gene, are hypermutable in haemopoietic stem cell disorders, and that mutagenic pressure and/or gene instability can contribute to the pathogenesis of these disorders.
The significance of minimal residual disease in hairy cell leukaemia treated with deoxycoformycin: a long‐term follow‐up study
Authors: Matutes E. , Meeus P. , McLennan K. , Catovsky D.
Published: November 2003

We investigated the clinical significance and long‐term follow‐up of detecting minimal residual disease (MRD) in hairy cell leukaemia (HCL) in complete remission (CR) after treatment with deoxycoformycin (DCF). MRD was assessed in 23 patients by immunophenotyping peripheral blood and bone marrow frozen sections using a panel of antibodies, CD11c, CD25, CD103 and HC2, which detect hairy cells. 31 cases with active HCL were used as controls. 10/23 patients (43%) had MRD in bone marrow (seven), blood (one) or both sites (two) which was not detected on haematoxylin–eosin bone marrow sections nor by immunohistochemistry on paraffin sections in six cases tested. Sequential studies in four cases did not show changes in the amount of MRD. At a median follow‐up of 72 months (range 15–108), 5/23 (22%) patients have relapsed with a median time of 59 months (range 15–105). MRD was detected in three of the five patients who relapsed. In the two patients with negative MRD, one relapsed with an abdominal mass and the other was a late relapse at 84 months. MRD was also documented in 7/18 patients who continued in clinical CR for a median of 80 months (range 63–98). There were no statistical differences in disease‐free survival between MRD+ and MRD− patients (P  =  0.8). Our findings indicate that relapse after long‐term remission achieved with DCF cannot be predicted when MRD is detected by sensitive methods. A search for other parameters such as proliferative rate of the residual cells or chest and abdominal CT scan might identify patients with a higher probability of disease recurrence.
Detection of donor‐derived Langerhans cells in MHC class II immunodeficient patients after allogeneic bone marrow transplantation
Authors: Emile J. F. , Haddad E. , Fraitag S. , Canioni D. , Fischer A. , Brousse N.
Published: November 2003

T‐cell dependent immune response is initiated by dendritic cells, which are the only leucocytes able to prime naive CD4‐positive T cells. Langerhans cells (LC) are dendritic cells characterized by their localization within the epidermis, their dendritic shape, and their expression of specific markers such as major histocompatibility complex (MHC) class II molecules, CD1a and S100 protein. We retrospectively studied the phenotype of LC in the skin of eight children with MHC class II deficiency (bare lymphocyte syndrome) after allogeneic bone marrow transplantation (BMT). The presence of donor‐derived MHC class II positive LC within the epidermis was studied by immunohistochemistry on skin biopsies performed for the determination of graft‐versus‐host disease. MHC class II positive LC were undetectable in the epidermis of a child who did not engraft and of three children 13–18 d after HLA‐mismatched BMT, despite engraftment. However, donor‐derived MHC class II positive LC were detected in four children 9–43 d after HLA‐identical BMT. Our results demonstrate that LC can differentiate or expand very quickly, as early as within 9 d after BMT.
Granulocytopenia in Cohen syndrome
Authors: Kivitie‐Kallio Satu , Rajantie Jukka , Juvonen Eeva , Norio Reijo
Published: November 2003

Cohen syndrome is an autosomal recessive disorder characterized by mental retardation, microcephalia and typical craniofacial features, myopia and chorioretinal dystrophy. As some patients were reported to have leucopenia, we collected the haematological data of 26 Finnish Cohen patients. They all had experienced periods of isolated granulocytopenia from an early age. Granulocytopenia was mild to moderate, non‐cyclic and never fatal. Most patients suffered from prolonged or repeated gingival or skin infections. We restudied 16 patients. Bone marrow examination revealed in all patients a normo‐ or hypercellular marrow, with a left‐shifted granulopoiesis in 8/16 patients. The response to adrenaline stimulation was subnormal in 12/14 and to hydrocortisone in 8/16 patients, but administration of rhG‐CSF caused granulocytosis in the three patients studied. No bone marrow malignancies were seen.
Paroxysmal nocturnal haemoglobinuria due to an 88 bp direct tandem repeat insertion in the PIG‐A gene
Authors: Pavlu J. , Mortazavi Y. , Tooze J. , Marsh J. C. W. , Gordon‐Smith E. C. , Rutherford T. R.
Published: November 2003

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired stem cell abnormality which frequently develops in patients with aplastic anaemia. The disease is due to somatic mutations in the PIG‐A gene, and a variety of mutations have been reported. The majority are point mutations, or small insertions and deletions resulting in a frameshift. Previous insertions reported have all been within the range of 1–10 bp. We describe here a patient with PNH due to a large insertion of 88 bp; DNA sequencing showed this to be a tandem repeat of PIG‐A sequences. The same mutation could be found in granulocytes and lymphocytes, indicating a pluripotent stem cell origin.
High serum levels of CA125 and interleukin‐6 in a patient with Ki‐1 lymphoma
Authors: Kubonishi Ichiro , Bandobashi Kentaro , Murata Naoaki , Daibata Masanori , Ido Eiji , Sonobe Hiroshi , Ohtsuki Yuji , Miyoshi Isao
Published: November 2003

We report a 53‐year‐old‐man with an aggressive Ki‐1 lymphoma who had high serum CA125, a marker protein of the epithelial ovarian cancer, and interleukin‐6 (IL‐6) concentrations. Both CA125 and IL‐6 levels decreased after chemotherapy and elevated with disease progression. The patient's lymphoma cells obtained before chemotherapy grew continuously in vitro, were IL‐6 dependent and were found to secrete CA125 in culture medium. These results indicate that CA125 can be secreted by Ki‐1 lymphoma cells and IL‐6 may promote the growth of Ki‐1 lymphoma cells.
Crossover study of the haematological effects and pharmacokinetics of glycosylated and non‐glycosylated G‐CSF in healthy volunteers
Authors: Watts M. J. , Addison I. , Long S. G. , Hartley S. , Warrington S. , Boyce M. , Linch D. C.
Published: November 2003

A cross‐over study of glycosylated and non‐glycosylated G‐CSF was performed in 20 healthy male volunteers to compare the effects of the different forms of G‐CSF, the extent of inter‐individual progenitor cell mobilization and to determine whether any differences observed were related to the serum concentrations of G‐CSF attained. The peak WBC achieved during 6 d of G‐CSF administration at a dose of 5 μg/kg/d was significantly higher with the glycosylated than the non‐glycosylated product (P = 0.02) as was the peak level of granulocyte‐monocyte colony forming cells (GM‐CFC) (P = 0.03). The average GM‐CFC count on days 5, 6 and 7 was 28% higher with the glycosylated product (P = 0.003). Serum concentrations of G‐CSF achieved were significantly higher with the non‐glycosylated G‐CSF, however, suggesting that the difference in bio‐efficacy was not due to a difference in G‐CSF stability. Marked inter‐individual variation in progenitor mobilization was observed, but this was not related to serum G‐CSF levels. The G‐CSF concentrations on day 6 were approximately one third of those on day 1, with both forms of G‐CSF.
Alterations of p53 and Rb genes in a novel human GM‐CSF‐dependent myeloid cell line (OHN‐GM) established from therapy‐related leukaemia
Authors: Nagai Masami , Fujita Mitsuhiro , Ikeda TakashiI , Ohmori Minoru , Kuwabara Hiroko , Yamaoka Genji , Tanaka Kimio , Kamada Nanao , Taniwaki Masafumi , Inoue Tokiko , Irino Shozo , Takahara Jiro
Published: November 2003

A novel GM‐CSF‐dependent myeloid cell line, OHN‐GM, was established from a patient who developed acute myelogenous leukaemia (AML) as a consequence of myelodysplastic syndrome (MDS). As the patient had previously received cytotoxic chemotherapy for Hodgkin's disease, the MDS and AML were probably related to such therapy. Sequential karyotypic analysis established a del(5q) as the initial cytogenetic abnormality. Additional alterations, including t(10;13)(q24;q14), had developed subsequently during disease progression. Southern blot analysis of OHN‐GM cells suggested deletion of one allele of the IRF‐1 gene, although no aberrant transcripts were detected. Fluorescence in situ hybridization analysis revealed the deletion of the Rb gene due to the t(10;13)(q24;q14) translocation, and Western blot analysis demonstrated the absence of Rb protein in OHN‐GM cells. Finally, the OHN‐GM cells exhibited two missense point mutations in highly conserved regions of the p53 gene. These observations suggest that a multistep process, involving alterations of Rb and p53 genes, may have contributed to the patient's disease development and progression. To our knowledge, OHN‐GM is the first cell line derived from a therapy‐related AML. These cells may aid the investigation of leukaemogenesis as well as the biology of secondary leukaemia.
A randomized comparison of liposomal versus conventional amphotericin B for the treatment of pyrexia of unknown origin in neutropenic patients
Authors: Prentice H. G. , Hann I. M. , Herbrecht R , Aoun M , Kvaloy S. , Catovsky D. , Pinkerton C. R. , Schey S. A. , Jacobs F , Oakhill A. , Stevens R. F. , Darbyshire P. J. , Gibson B. E. S.
Published: November 2003

One hundred and thirty‐four adults and 204 children were randomized in two prospective, parallel comparative multicentre trials to receive either conventional amphotericin B 1 mg/kg/d (c‐AMB), liposomal amphotericin B 1 mg/kg/d (L‐AMB1) or liposomal amphotericin B 3 mg/kg/d (L‐AMB3). Patients were entered if they had a pyrexia of unknown origin (PUO) defined as temperature of 38°C or more, not responding to 96 h of systemic broad‐spectrum antibiotic treatment, and neutropenia (< 0.5 × 109/l). The safety and toxicity of liposomal amphotericin B was compared with that of conventional amphotericin B. Efficacy of treatment was assessed, with success defined as resolution of fever for 3 consecutive days (< 38°C) without the development of any new fungal infection. Clinical and laboratory parameters were collected for safety analysis. In both the paediatric and adult populations, L‐AMB treated patients had a 2–6‐fold decrease in the incidence (P  0.01) of test‐drug‐related side‐effects, compared to c‐AMB. Severe trial‐drug‐related side‐effects were seen in 1% of L‐AMB treated patients, in contrast to 12% of patients on c‐AMB (P < 0.01). Nephrotoxicity, in the patient subset not receiving concomitant nephrotoxic agents, defined as a doubling from the patients baseline serum creatinine level, was not observed in the L‐AMB1 arm whereas the incidence was 3% in patients on L‐AMB3 and 23% in those on c‐AMB (P < 0.01). Moreover, time to develop nephrotoxicity was longer in both L‐AMB arms than c‐AMB (P < 0.01). Severe hypokalaemia was observed less frequently in both L‐AMB arms (P < 0.01).

Analysis was by intention‐to‐treat and included all patients randomized. Success was defined by a minimum of 3 consecutive days with fever (< 38°C) continuing to study end indicated by recovery of neutrophils to 0.5 × 109/l. Addition of systemic antifungal therapy or development of systemic fungal infection were failures as was persistent fever to study end. Efficacy assessments indicated success in 49% of the total group treated with c‐AMB. 58% of patients responded to L‐AMB1 and 64% to L‐AMB3. A statistically significant difference was found between c‐AMB and L‐AMB3 (P = 0.03) but a Kaplan‐Meier analysis of time to deffervescence of fever showed there was no significant difference between the arms.

It was concluded that liposomal amphotericin at either 1 or 3 mg/kg/d was significantly safer than conventional amphotericin B in children and adults. The main aim of this open‐label study was to compare safety between the three trial arms. However, we provide evidence for an equivalent or possibly superior efficacy of liposomal amphotericin with regard to resolution of fever of unknown origin. Subsequent trials should compare amphotericin preparations in defined fungal infections.
A potential determinant of enhanced crystallization of HbC: spectroscopic and functional evidence of an alteration in the central cavity of oxyHbC
Authors: Hirsch Rhoda Elison , Rybicki Anne C. , Fataliev Nazim A. , Lin Margaret J. , Friedman Joel M. , Nagel Ronald L.
Published: November 2003

The structural basis of the crystallizing tendencies of oxyHbC (β6Glu → Lys), that produces haemolytic anaemia in homozygotes, is unknown. Using a fluorescent organic phosphate analogue (8‐hydroxy‐1,3,6‐pyrenetrisulphonate), and conventional oxygen equilibrium studies, data suggest that the binding of inositolhexaphosphate (IHP) to oxyHbC differs from HbA, indicating perturbations of the oxyHbC central cavity, which was predicted from our earlier spectroscopic findings. To define the relationship between this conformational change in oxyHbC and its tendency to crystallize, the effect of four central cavity ligands on the crystallization rate was studied: a peptide containing 11 residues from the N‐terminal portion of band 3, the full cytoplasmic domain of band 3, 2,3‐diphosphoglycerate and IHP. OxyHbC crystallization was accelerated by all these central cavity ligands and not by the appropriate controls. These central cavity changes become an excellent candidate for the dramatic increase in the crystallization rate of oxyHbC.
An alternative to continuous subcutaneous infusion of desferrioxamine in thalassaemic patients
Authors: Di Gregorio Felicia , Romeo Maria A. , Pizzarelli Giuseppe , Aiello Giuseppa , Russo Giuseppe
Published: November 2003

Sixteen patients with thalassaemia major were treated with subcutaneous desferrioxamine (DF) 50 mg/kg/d, 5 consecutive days a week, for 8 weeks. Every other week the total dose was administered by 12 h infusion pump or by rapid injection of the same dose (25 × 2 mg/kg) twice a day. The two methods of DF administration produced no significant differences in urinary iron excretion. No significant changes in serum ferritin levels were observed at the end of the study. Compared with continuous infusion, rapid injection is equally efficacious, does not induce serious side‐effects, is better accepted by the patients, and can improve their compliance to the iron‐chelating therapy.
Relapse of Philadelphia chromosome positive acute lymphoblastic leukaemia after marrow transplantation: sustained molecular remission after early and dose‐escalating infusion of donor leucocytes
Authors: Keil Felix , Kalhs Peter , Haas Oskar A. , Fritsch Gerhard , Reiter Elisabeth , Mannhalter Christine , Lechner Klaus , Leitner Gerda , Greinix Hildegard T.
Published: November 2003

We present a patient who underwent sibling allogeneic BMT because of refractory Ph+ve ALL and remained BCR‐ABL‐positive after marrow grafting. Haemopoietic precursor cells were predominantly BCR‐ABL‐negative and of donor origin. In T cells an exclusively donor genotype was demonstrated. Despite donor leucocyte infusion (DLI), 20 weeks after BMT BCR‐ABL fusion mRNA increased in semiquantitative polymerase chain reaction and leukaemic infiltration of the patient's bone marrow was seen. After a second course of DLI the patient achieved sustained molecular remission but he developed severe graft‐versus‐host disease (GvHD) and died from bacterial sepsis 9 months after DLI.
Granulocyte‐macrophage colony‐stimulating factor abrogates transforming growth factor‐β1‐mediated cell cycle arrest by up‐regulating cyclin D2/Cdk6
Authors: Ohtsuki Fumiko , Yamamoto Masuji , Nakagawa Toshitaro , Tanizawa Takakuni , Wada Hiroyoshi
Published: November 2003

The role of positive and negative cytokine interactions in G1 cell cycle regulation of haemopoietic cells was analysed by determination of the expression patterns of D‐type cyclins and cyclin‐dependent kinases (cdks) in SKM‐1 myelodysplastic syndrome (MDS) cells incubated with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and/or transforming growth factor‐β1 (TGF‐β1). TGF‐β1 inhibited SKM‐1 cell proliferation due to the cell cycle arrest in G1 phase. GM‐C18SF abrogated the TGF‐β1‐mediated G1 arrest in these cells. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis indicated that TGF‐β1‐mediated G1 arrest correlated with the down‐regulation of cdk4, cdk6 and cyclin D2, and that abrogation of TGF‐β1‐mediated G1 arrest by GM‐CSF correlated with the constitutive over‐expression of cyclin D2 and cdk6 but not cdk4. These results suggest the importance of cyclin D2/cdk6 levels in abrogating G1 arrest in cells exposed to TGF‐β1, and raise the possibility that the GM‐CSF‐mediated up‐regulatory pathway of signal transduction through cyclin D2/cdk6 differs from the TGF‐β1‐cdk4‐mediated pathway in SKM‐1 cells. This signal transduction pathway through cyclin D2/cdk6 might play an important role in haemopoietic regulation by the cytokine network.
Interferon‐gamma induced proliferation of human myeloid leukaemia cell lines
Authors: Drexler Hans G. , Zaborski Margarete , Quentmeier Hilmar
Published: November 2003

Interferon‐gamma (IFN‐γ) is a pleiotropic cytokine involved in the regulation of various phases of immune and inflammatory responses; it also has anti‐viral and anti‐proliferative activity. Using continuous human leukaemia cell lines as model systems, we found that IFN‐γ stimulated the proliferation of leukaemic myeloid cells; this effect was specifically neutralized by an anti‐IFN‐γ monoclonal antibody (McAb). No proliferative response was seen in autonomously growing cell lines; however, 11/19 constitutively growth factor‐dependent cell lines showed a significant response in short‐term proliferation assays upon incubation with IFN‐γ. The stimulation indices ranged from 2 to 37 compared with the untreated control cells; the EC50 values for these cell lines were in the range of 0.1–0.6 ng/ml IFN‐γ. Flow cytometric analysis demonstrated heterogeneity in the expression of the IFN‐γ receptor, as it was found on 37–97% of the cells per cell line. The effects of IFN‐γ on proliferation triggered by a spectrum of 10 other cytokines were variable, and both stimulation and attenuation of the proliferative responses were seen in different cell lines. Under serum‐free culture conditions, IFN‐γ acted as a survival factor suppressing apoptosis. As has been described for other functional processes triggered by IFN‐γ, the proliferation‐inducing activity of IFN‐γ also led to activation of the signal transducing element STAT 1. Thus, IFN‐γ can induce myeloid leukaemia cells to proliferate and can modulate their proliferative response to other cytokines. Therefore IFN‐γ may be a pathologically relevant ligand for leukaemic cell proliferation in vivo. In physiological settings, IFN‐γ might be a bifunctional regulator of haemopoietic cell proliferation, depending on other differential co‐signals from the micro‐environment.
Fibrinogen Otago: a major α chain truncation associated with severe hypofibrinogenaemia and recurrent miscarriage
Authors: Ridgway Hayley J. , Brennan Stephen O. , Faed James M. , George Peter M.
Published: November 2003

A woman with a preliminary diagnosis of afibrinogenaemia was later found to have a functional fibrinogen of 0.06 mg/ml and markedly prolonged thrombin and reptilase times. The stoichiometry of fibrinopeptide release was normal but there was a gross delay in the polymerization of purified fibrin. Plasma protein electrophoresis showed an absence of normal fibrinogen and a novel anodal component which was confirmed as fibrinogen by immunofixation. Western blots of non‐reducing SDS‐PAGE gels indicated a molecular weight of 270 kD, compared to 340 kD for normal fibrinogen and similar analysis of reducing gels showed that the expected 67 kD Aα chain was missing and replaced by a 30 kD band. This aberrant chain was not detected by the monoclonal antibody F‐103, which recognizes the epitope formed by residues 259–276 of the Aα chain. Cycle sequencing of the DNA encoding the F‐103 epitope revealed the homozygous insertion of cytosine at position 4133 of the gene sequence. Predictably this translates as three new amino acids (268Gln‐Glu‐Pro) before termination at a new (TAG) stop codon. No abnormal Aα chains could be detected in plasma from the woman's heterozygous son. The hypofibrinogenaemia observed is likely to be the result of diminished assembly and/or secretion of the truncated Aα chains rather than enhanced extracellular degradation.
Genetic heterogeneity of AIDS‐related small non‐cleaved cell lymphoma
Authors: Gaidano Gianluca , Pastore Cristina , Gloghini Annunziata , Canzonieri Vincenzo , Capello Daniela , Franceschi Silvia , Saglio Giuseppe , Carbone Antonino
Published: November 2003

AIDS‐related small noncleaved cell lymphoma (AIDS‐SNCCL) includes Burkitt's lymphoma (BL) and high‐grade B‐cell Burkitt‐like lymphoma (BLL). Due to the marked polymorphism of AIDS‐related non‐Hodgkin's lymphomas (AIDS‐NHL), the morphologic distinction between these two types of lymphomas is frequently controversial, although it may bear clinical relevance. Although the molecular features of AIDS‐BL have been clarified to a certain extent, the genetic peculiarities of AIDS‐BLL have not been investigated in detail. In this study we have compared morphologic and genetic features of AIDS‐BL and AIDS‐BLL in a blind coded fashion. Molecular studies were focused on the genetic lesions known to be implicated in AIDS‐NHL, including alterations of c‐MYC, BCL‐6, p53, deletions of 6q, as well as infection by EBV and HHV‐8. Alterations of c‐MYC occurred in 10/10 AIDS‐BL, whereas they were restricted to 2/10 AIDS‐BLL (P < 0.01). Mutations of p53 were present in 5/10 AIDS‐BL, whereas they were consistently absent among AIDS‐BLL (n = 10; P < 0.05). Infection by EBV occurred in 30% of both AIDS‐BL and AIDS‐BLL. Rearrangements of BCL‐6, deletions of 6q and infection by HHV‐8 scored consistently negative in both AIDS‐BL and AIDS‐BLL. Based on the genetic lesions tested, the molecular profile of AIDS‐BLL appears to be closer to that of AIDS‐related diffuse large cell lymphoma (AIDS‐DLCL) than to that of AIDS‐BL. In contrast to AIDS‐BLL, however, AIDS‐DLCL carried rearrangements of BCL‐6 in a fraction of cases (2/9). This study, the largest of its kind reported so far, suggests that AIDS‐BL and AIDS‐BLL have a different molecular pathogenesis and that characterization of genetic lesions may help to distinguish between these two lymphomas.
Factors influencing collection of peripheral blood progenitor cells following high‐dose cyclophosphamide and granulocyte colony‐stimulating factor in patients with multiple myeloma
Authors: Goldschmidt H. , Hegenbart U. , Wallmeier M. , Hohaus S. , Haas R.
Published: November 2003

We treated 103 multiple myeloma (MM) patients with 7 g/m2 cyclophosphamide (Cy) followed by 300 μg G‐CSF/d to harvest peripheral blood progenitor cells (PBPC). PBPC autografts containing > 2.0 × 106 CD34+ cells per kg body weight were obtained at the first attempt from 90/100 evaluable patients. The most significant factor predicting impairment of PBPC collection was the duration of previous melphalan treatment (P < 0.0001). In multivariate discriminate analysis, treatment with melphalan during the most recent chemotherapy cycles prior to mobilization (P = 0.0727) and previous radiotherapy (P = 0.0628) had a marginally significant negative influence on the efficacy of PBPC collection. We found no reduced functional capacity of CD34+ cells to restore haemopoiesis after myeloablative treatment related to the duration of melphalan exposure. At the time of best response to conventional treatment, a median paraprotein reduction of 21% was achieved following high‐dose cyclophosphamide (HD‐Cy). Two heavily pretreated patients died and one patient developed pulmonary toxicity W.H.O. grade IV following HD‐Cy. Potential transplant candidates should undergo mobilization and harvesting of PBPC before melphalan‐containing treatment. Combinations of haemopoietic growth factors and their dose modifications should be investigated to improve PBPC collection, to allow a dosage reduction of the mobilization chemotherapy.
A murine model of human cold agglutinin disease
Authors: Dumas Gérard , Pritsch Otto , Pereira Arturo , Gallart Teresa , Dighiero Guillaume
Published: November 2003

Progress has been limited in the treatment of cold agglutinin (CA) disease by the absence of an animal model. We have recently studied at the molecular level one CA displaying the rare anti‐Sia‐1b specificity (CAGAS). CAGAS displays strong CA activity and is able to haemolyse mouse RBC in the presence of complement, thus constituting a suitable Ab for creating a murine model of CA disease. In the present work we introduced CAGAS VH and VL domains into eukaryotic expression vectors and transfected them into the non‐secreting mouse myeloma X63 cell line. Clones expressing complete engineered pentameric IgMκ CAGAS (eCAGAS) recapitulating the characteristics of serum CA (sCAGAS), could be obtained. The i.p. injection of eCAGAS to normal BALB/c mice induced a typical haemolytic anaemia, as demonstrated by the presence of spontaneous cold agglutination of RBC, induction of anaemia and significant reticulocytosis. Of interest, conspicuous bilateral ear loss was observed in one of these animals. In addition, i.p. injection of X63 transfected line into BALB/c nude mice induced ascites, typical haemolytic anaemia, and shortening of the mean RBC survival. These findings validate the practical interest of constructing a transgenic mouse model expressing eCAGAS.
Effects of all‐trans‐retinoic acid on superoxide generation in intact neutrophils and a cell‐free system
Authors: Koga Hiroyuki , Fujita Ichiro , Miyazaki Sumio
Published: November 2003

To determine the pathophysiology of the retinoic acid syndrome which occurs during all‐trans‐retinoic acid (ATRA) treatment of acute promyelocytic leukaemia patients, we investigated the direct effects of ATRA on the function of human neutrophils. We found that ATRA (10–200 μM) dose‐dependently stimulated superoxide (O−2) generation in intact neutrophils. The maximal activity of ATRA‐stimulated O−2 generation was 3.0 nmol/min/106 cells. Adding EGTA to the assay mixture did not affect the activity nor was the intracellular free calcium concentration changed upon stimulation. The treatment of neutrophils with 0.1 μM staurosporine, an antagonist of protein kinase C, for 10 min, enhanced the activity of ATRA‐stimulated O−2 generation up to 186% of that for control samples. Wortmannin (1 μM), an inhibitor of phosphatidylinositol 3‐kinase (PI 3‐kinase), reduced this stimulatory activity by 67%. These results suggest that ATRA activates the signalling pathway related to PI 3‐kinase rather than that utilizing calcium and protein kinase C. ATRA enhanced the O−2 generated in a sodium‐dodecyl‐sulphate (SDS) cell‐free system, resulting in rates up to 288% higher than that seen with SDS alone. This enhancement was not affected by pretreatment with staurosporine or wortmannin. ATRA may thus directly activate and/or enhance the function of neutrophils.
Low‐dose aspirin in polycythaemia vera: a pilot study
Authors: ITALIANO STUDIO POLICITEMIA (GISP) GRUPPO
Published: November 2003

In this pilot study, aimed at exploring the feasibility of a large‐scale trial of low‐dose aspirin in polycythaemia vera (PV), 112 PV patients (42 females, 70 males, aged 17–80 years) were selected for not having a clear indication for, or contraindication to, aspirin treatment and randomized to receive oral aspirin (40 mg/d) or placebo. Follow‐up duration was 16 ± 6 months. Measurements of thromboxane A2 production during whole blood clotting demonstrated complete inhibition of platelet cyclooxygenase activity in patients receiving aspirin. Aspirin administration was not associated with any bleeding complication. Within the limitations of the small sample size, this study indicates that a biochemically effective regimen of antiplatelet therapy is well tolerated in patients with polycythaemia vera and that a large‐scale placebo‐controlled trial is feasible.
Aplastic anaemia in a case of hereditary neutrophil Fcγ receptor IIIb deficiency
Authors: Tournilhac Olivier , Kiladjian Jean‐Jacques , Cayuela Jean‐Michel , Noguera Maria‐Eléna , Zini Jean‐Marc , Daniel Marie‐Thérèse , Maarek Odile , Gluckman ÉLiane , Socié Gérard , Sigaux François
Published: November 2003

CD16 antibodies recognize Fcγ receptors III of a and b types. In a patient with severe idiopathic aplastic anaemia (AA), polymorphonuclear cells, which in normal subjects express FcγRIIIb, were found to be CD16 negative. The FcγRIIIb gene configuration was analysed by PCR on peripheral blood mononuclear cells. Bi‐allelic deletion encompassing at least part of the coding exon 5 was found in the patient and his brother, suggesting a hereditary defect. The patient underwent successful bone marrow transplantation from his HLA‐matched brother despite a similar phenotype and genotype. This observation suggests that FcγRIIIb hereditary deficiency in donor and/or recipient does not impair engraftment and justifies the use of other monoclonal antibodies in addition to CD16 in the study of GPI‐anchored antigen expression.
Expression of Apo‐1/Fas (CD95), Bcl‐2, Bax and Bcl‐x in myeloma cell lines: relationship between responsiveness to anti‐Fas mab and p53 functional status
Authors: Egle Alexander , Villunger Andreas , Marschitz Ingrid , Kos Marion , Hittmair Anton , Lukas Peter , Grünewald Kurt , Greil Richard
Published: November 2003

The down‐regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl‐2, which inhibits apoptosis, of Bax, which counteracts Bcl‐2, of Bcl‐xL and Bcl‐xS, which act in an anti‐ and pro‐apoptotic fashion, respectively, and of Apo‐1/Fas, which induces programmed cell death, when activated by the Apo‐1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl‐2, but displayed different amounts of Bax and Bcl‐x proteins. The Apo‐1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo‐1/Fas correlated with that of Bax, but not with that of Bcl‐2 or Bcl‐x subtypes. Furthermore, the effectiveness of the Apo‐1/Fas mab was associated with the relative expression levels of the Apo‐1/Fas and with that of the Bax antigen, but not with that of the Bcl‐2 and Bcl‐x antigens. We further showed that wild‐type p53 function is not required for Apo‐1/Fas‐induced apoptosis, nor is it necessary for the expression of Bax or Apo‐1/Fas antigens in myeloma.  In conclusion, our results suggest a p53‐independent co‐regulation of Apo‐1/Fas and Bax, as well as a role for Bax in Apo‐1/Fas‐induced apoptosis in myeloma.