bluuebunny/crossref_metadata_2025 huggingface dataset:
Pegylated asparaginase (OncasparTM) in children with ALL: drug monitoring in reinduction according to the ALL/NHL‐BFM 95 protocols
Authors: Müller Hans‐Joachim , Löning Lutz , Horn Alexander , Schwabe Dirk , Gunkel Martin , Schrappe Martin , Von Schütz Volker , Henze Günter , Casimiro da Palma João , Ritter Jörg , Pinheiro João Paulo Vieira , Winkelhorst Monika , Boos Joachim
Published: March 2003

Hypersensitivity reactions are relevant adverse effects of asparaginase therapy. Therefore, children treated with native Escherichia coli asparaginase in induction therapy of acute lymphoblastic leukaemia (ALL) or non‐Hodgkin's lymphoma (NHL) were switched to the pegylated enzyme for reinduction under drug monitoring. Seventy children, including four patients with allergic reactions during induction, were given one dose of OncasparTM 1000 U/m2 intravenously. Activity was determined every third or fourth day until it dropped below the limit of quantification. In current reinduction protocols [ALL/NHL‐Berlin–Frankfurt–Münster (BFM) 95 trials], four doses of 10 000 U/m2E. coli asparaginase deplete asparagine for about 2–3 weeks, therefore activities of ≥ 100 U/l up to day 14 and ≥ 50 U/l up to day 21 were targeted. In 66 patients without an allergic reaction during induction, the mean activity was 606 ± 313 U/l, 232 ± 211 U/l and 44 ± 50 U/l after 1, 2 and 3 weeks respectively. In 44/66 patients, activity was ≥ 100 U/l after 14 d. A rapid decline in activity was seen in the remaining 22 patients, including 8/22 patients who showed no activity after 1 week. Toxicity was low and comparable to the native enzymes but, in contrast to about 30% of hypersensitivity reactions with conventional reinduction therapy, no allergic reaction was seen. Substituting 4 × 10 000 U/m2 asparaginase medac for one dose of 1000 U/m2 OncasparTM was safe and well tolerated. Comparable pharmacokinetic treatment intensity was achieved in about two‐thirds of patients.
Evidence for two alternatively spliced forms of phospholipase C‐β2 in haematopoietic cells
Authors: Mao Guang Fen , Kunapuli Satya P. , Rao A. Koneti
Published: March 2003

Alternatively spliced forms have been reported for several phospholipase C (PLC) isozymes, but not for PLC‐β2, the most abundant PLC‐β in platelets. PLC‐β2 cDNA cloned from the HL‐60‐cell cDNA library is 3543 bases long, coding for 1181 amino acids. Compared with the published sequence, a deletion of 45 nucleotides (2755–2799 nt, amino acids 864–878) was detected in platelet and leucocyte mRNA amplified by reverse transcription (RT) polymerase chain reaction (PCR) using primers corresponding to 1814–1838 nt (forward) and 3328–3352 nt (reverse). Amplification of genomic DNA using primers corresponding to 2575–2596 nt and 2864–2885 nt yielded a ∼ 750 bp product; restriction analysis and sequencing revealed the 45‐bp exon flanked by introns of 198 bp and 118 bp. Amplification of leucocyte and platelet cDNA using the same primers yielded products of ∼ 310 nt and ∼ 265 nt, with (PLC‐β2a) and without (PLC‐β2b) the 45‐nt sequence. Thus, two alternatively spliced forms (1181 and 1166 amino acids) of PLC‐β2 are generated in haematopoietic cells. They differ in the carboxyl terminal sequence implicated in interaction of PLC‐β enzymes with Gαq, particulate association and nuclear localization. We propose that the PLC‐β2 splice variants may be regulated differentially with distinct roles in signal transduction.
Quantitative deficiency of monocyte‐specific esterase (MSE) mRNA in monocyte esterase deficiency (MED)
Authors: Boyle C. , Cosby S. L. , Markey G. M. , Alexander H. D. , Morris T. C. M.
Published: March 2003

Cytochemical staining of monocyte‐specific esterase (MSE) is widely used for identification of the monocytic lineage in leukaemias. Deficiency of this enzymatic activity occurs as a familial trait and the deficiency has been shown to occur with greater frequency in patients with lymphoproliferative or gastrointestinal malignant neoplastic diseases than in normal blood donors. Reverse transcriptase polymerase chain reaction (RT‐PCR), sequencing and quantification by Northern blot analysis was conducted on the MSE mRNA of 12 subjects with monocyte esterase deficiency (MED) and seven MSE‐positive subjects to examine whether mutations were present or whether the defect was quantitative. Mutations were not found in the mRNA sequences. However, MED subjects had significantly less MSE mRNA than MSE‐positive subjects (P = 0·001). These findings show that deficiency of monocyte esterase activity in MED is not as a result of the presence of inactive isoenzymes and may be owing to an abnormality in the regulation of mRNA production.
Familial thrombocytosis as a recessive, possibly X‐linked trait in an Arab family
Authors: Stuhrmann M , Bashawri L. , Ahmed M. A. , Al‐Awamy B. H. , Kühnau W. , Schmidtke J. , El‐Harith E. A.
Published: March 2003

Familial thrombocytosis (FT) has previously been described as an autosomal‐dominant disorder with manifestations similar to those of sporadic essential thrombocythaemia. We studied an Arab family consisting of four brothers, aged 4–8 years, who had either sustained markedly elevated (> 1000 × 109/l) or moderately elevated (> 500 × 109/l) platelet counts, two healthy sisters and their parents who had normal platelet counts. The four brothers with FT had normal plasma thrombopoietin levels and are currently not presenting with any thrombotic or haemorrhagic complications. Mutation analysis at the thrombopoietin gene (THPO) of the affected family members failed to detect the intron 3 G→C splice mutation that had been described as causing FT. In addition, segregation analysis using a polymorphic CA marker revealed completely discordant THPO alleles among the affected brothers. We postulate the existence of a new locus for FT whereby the disease is transmitted as a recessive, possibly X‐linked trait.
Granulocyte colony‐stimulating factor‐mobilized peripheral blood CD34+ cells from children contain the same levels of long‐term culture‐initiating cells producing the same numbers of colony‐forming cells as those from adults, but display greater in vitro monocyte/macrophage potential
Authors: Boiret Nathalie , Kanold Justyna , Bons Jean‐Michel , Rapatel Chantal , Halle Pascale , Tournilhac Olivier , Guilhouard Laurent , Guérin Jean‐Jacques , Travade Philippe , Demeocq François , Bonhomme Jean , Berger Marc G.
Published: March 2003

Autologous peripheral blood progenitor cell (PBPC) transplantation is now commonly used in children. The ontogenic differences in haematopoiesis published in recent years suggest differences in the categories of mobilized PBPCs between children and adults. We investigated the frequency and distribution of mature progenitor cells (colony‐forming cells, CFCs) and primitive progenitor cells [CD34+ CD38− and CD34+ Thy‐1+ cells, long‐term culture‐initiating cells (LTC‐ICs)] in children and adults mobilized using granulocyte colony‐stimulating factor alone. We found similar proportions of granulocyte colony‐forming units (CFU‐G) and/or macrophage CFUs (CFU‐M), mixed lineage CFUs (CFU‐Mix) and megakarocyte CFUs (CFU‐Mk), CD34+ CD38− and CD34+ Thy‐1+ cells, and LTC‐ICs (16·5 ± 3·5 vs. 10·65 ± 5 per 104 CD34+ cells), which produced the same number of CFCs (5 ± 1 vs. 6 ± 1 CFCs/LTC‐ICs) in PB CD34+ cells from children and adults. However, we noted a higher proportion of erythroid blast‐forming units (BFU‐E) in PB CD34+ cells from adults (× 1·5, P = 0·003). Using cord blood as a third ageing point, we observed an inverse age‐related propensity for commitment to the monocyte/macrophage lineage that was still found after normalizing the data per body weight and processed blood mass. This ontogeny‐related programming was detected from the LTC‐IC level, which produced 1·7 times more CFU‐M in children than in adults (P = 0·048). These subtle differences in commitment between children and adults, shown here for the first time, are of interest for the in vitro manipulation of PBPCs and, in particular, for application in adoptive immunotherapy in children.
Single site polymorphisms and alternative splicing of the human CD13 gene – different splicing frequencies among patients with acute myeloid leukaemia and healthy individuals
Authors: Dybkær Karen , Kristensen J. S. , Pedersen F. S.
Published: March 2003

Within the haematopoietic system, CD13/aminopeptidase N (APN), a transmembrane glycoprotein, is expressed on the surface of early committed progenitors of granulocytes and monocytes and by all cells of these lineages as they mature. CD13 is expressed on the majority of leukaemic myeloblasts in acute myeloid leukaemia (AML), and on leukaemic lymphoblasts in a small percentage of acute lymphoid leukaemia cases. Thus, anti‐CD13 monoclonal antibodies are used as diagnostic markers in leukaemia typing. By systematically amplifying overlapping reverse transcription polymerase chain reaction (RT‐PCR) amplicons throughout the CD13 mRNA, we identified two splice variants in which exon 3 and exon 14 were lost. Fourteen healthy individuals and 34 patients with AML were screened for these splice variants. All healthy individuals, and the majority of AML patients, had both splice variants but they represented less than 10% of the total RT‐PCR‐amplified CD13 product. Increased expression of both truncated CD13 mRNA forms were observed in 6% of AML patients, whereas no detectable exon 3 or exon 14 splice variants could be generated in 26% and 9% of AML patients respectively. The different splicing frequencies may reflect altered processing of pre‐mRNA or expansion of certain cell types for some AML patients, even though no correlation existed to blast percentage, FAB classification, surface antigens or cytogenetic characteristics. In addition, we identified an intron of 506 bp between exon 1 and exon 2 as well as two sites of single nucleotide polymorphism with a heterozygosity index of about 0·5, making them useful as genetic markers.
The association of vitamin K status with warfarin sensitivity at the onset of treatment
Authors: Cushman Mary , Booth Sarah L. , Possidente Carl J. , Davidson Kenneth W. , Sadowski James A. , Bovill Edwin G.
Published: March 2003

We investigated the association of vitamin K status with warfarin sensitivity among 40 orthopaedic patients beginning perioperative algorithm‐dosed warfarin. Baseline vitamin K status was assessed using plasma vitamin K‐1 and vitamin K‐1 2,3 epoxide concentrations, and a questionnaire‐based estimation of usual vitamin K intake. Warfarin sensitivity was assessed as the increase in the International Normalized Ratio (INR) after two doses of 5 mg of warfarin and as the 4‐d accumulation of under‐γ‐carboxylated prothrombin (PIVKA‐II), adjusted for warfarin dose requirement. Multivariate models were used to assess vitamin K variables as predictors of warfarin sensitivity. The mean INR increase was 0·53 U and the mean PIVKA‐II increase was 771 ng/ml/mg warfarin. Demographic factors were not associated with warfarin response. For each 1 standard deviation (SD) lower value of plasma vitamin K‐1, but not the other vitamin K variables, the INR rose 0·24 U (P ≤ 0·01). A higher usual vitamin K intake and plasma vitamin K‐1, and lower plasma vitamin K‐1 2,3 epoxide, were all associated with a lower PIVKA‐II increase over 4 d. Respective differences in PIVKA‐II accumulation per SD increase of each variable were −165, −218 and 236 ng/ml/mg warfarin (all P ≤ 0·05). We concluded that dietary and biochemical measures of vitamin K status were associated with early warfarin sensitivity.
Pathogenetic analysis of three cases with a bleeding disorder characterized by defective platelet aggregation induced by Ca2+ ionophores
Authors: Fuse Ichiro , Higuchi Wataru , Uesugi Yumiko , Aizawa Yoshifusa
Published: March 2003

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore‐induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin‐ or thrombin‐induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5‐triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.
Occupational and environmental risk factors of the myelodysplastic syndromes in the North of France
Authors: Nisse Catherine , Haguenoer Jean Marie , Grandbastien Bruno , Preudhomme Claude , Fontaine Bernard , Brillet Jeanne Marie , Lejeune Regis , Fenaux Pierre
Published: March 2003

Aetiological factors of the myelodysplastic syndromes (MDS) are largely unknown, with the exception of alkylating agents, ionizing radiation and benzene. Some other risk factors have been suggested by the few epidemiological studies reported (solvents, ammonia, exhaust gases, metals, pesticides, alcohol). We performed a case–control study to assess the relationship between occupational or environmental factors and MDS. Two hundred and four patients with newly diagnosed MDS, and 204 sex‐ and age‐matched controls were included. Medical history, demographic data, lifetime exposure and hobbies were obtained. Qualitative and quantitative exposure to chemical and physical hazards were evaluated with the patients and reviewed by a group of experts in occupational exposure. The median age was 70 years and 62% of the patients were men. In univariate analyses, we found relationships between MDS and smoking habits, gardening, occupations such as health professionals, technical and sale representatives, machine operators, agricultural workers, textile workers, qualitative occupational exposures (exposed/non‐exposed) to oil, solvents, ammonia, pesticides, fertilizers, cereal dusts, contact with poultry or livestock and infective risk, and lifetime cumulative exposure to solvents, oil, textile dust and infective risk. The main risk factors of MDS determined by multivariate analyses (conditional logistic regression) were, being an agricultural worker [odds ratio (OR) = 3·66; 95% confidence interval (CI) 1·9–7·0], textile operator (OR = 3·66; 95% CI 1·9–7·9), health professional (OR = 10·0; 95% CI 2·1–48·7), commercial and technical sale representative (OR = 4·45; 95% CI 1·4–14·6), machine operator (OR = 2·69; 95% CI 1·2–6·0), living next to an industrial plant (OR = 2·45; 95% CI 1·5–4·1), smoking (OR = 1·74; 95% CI 1·1–2·7) and lifetime cumulative exposure to oil (OR = 1·1; 95% CI 1·0–1·2). Further studies should be performed to assess specific exposures more precisely and it would be of interest to develop a map of haematological malignancies according to industrial background.
Outcome of relapsed or refractory childhood B‐cell acute lymphoblastic leukaemia and B‐cell non‐Hodgkin's lymphoma treated with the UKCCSG 9003/9002 protocols
Authors: Atra A. , Gerrard M. , Hobson R. , Imeson J. D. , Hann I. M. , Pinkerton C. R.
Published: March 2003

Twenty‐six children with B‐cell acute lymphoblastic leukaemia (B‐ALL) or Murphy Stage III or IV B‐cell non‐Hodgkin's lymphoma (B‐NHL) progressed or relapsed after first‐line therapy with a short, intensive multiagent chemotherapy regimen [United Kingdom Childhood Cancer Study Group (UKCCSG) 9003] (n = 62) or a slightly less intensive regimen (UKCCSG 9002) (n = 112). Eight patients (4·6%) never achieved complete remission (CR) and 18 (10·3%) relapsed. Second‐line therapy resulted in remission for eight patients (30%). All patients initially treated with the 9003 protocol died. Three patients (11·5%) in the 9002 group, including one who never achieved CR in the primary site, are alive after second‐line therapy. This study confirms that the prognosis of relapsed or refractory B‐ALL/B‐NHL is poor and exceptionally so if relapse occurred less than 6 months from diagnosis. High‐dose therapy with stem cell rescue was used in only seven patients; its role needs to be studied further.
Bone marrow involvement in Whipple's disease: rarely reported, but really rare?
Authors: Walter Roland , Bachmann Stefano P. , Schaffner Andreas , Rüegg Regula , Schoedon Gabriele
Published: March 2003

Infection with Tropheryma whippelii, the causative agent of Whipple's disease, involves nearly every organ. Involvement of bone marrow may be an overlooked area of Whipple's disease. We report a case of lymphoma‐like Whipple's disease with bone marrow involvement together with a brief review of the literature on this topic. Despite minimal documentation, bone marrow may be commonly involved in Whipple's disease and, although not specific, diastase‐resistant periodic acid‐Schiff (PAS)‐positive macrophages in bone marrow may offer an important clue to diagnosis using PAS histology of upper endoscopic biopsies, polymerase chain reaction or electron microscopy.
Determinants of outcome after intensified therapy of childhood lymphoblastic leukaemia: results from Medical Research Council United Kingdom acute lymphoblastic leukaemia XI protocol
Authors: Hann Ian , Vora Ajay , Harrison Georgina , Harrison Christine , Eden Osborn , Hill Frank , Gibson Brenda , Richards Sue ,   the UK Medical Research Council's Working Party on Childhood Leukaemia
Published: March 2003

The single most important prognostic determinant in childhood acute lymphoblastic leukaemia (ALL) is effective therapy and changes in therapy may influence the significance of other risk factors. The effect of intensified therapy on the importance of currently recognized phenotypic and genotypic determinants of outcome was assessed in 2090 children enrolled on the Medical Research Council United Kingdom acute lymphoblastic leukaemia XI (MRC UKALL XI) protocol. Treatment allocation was not determined by risk factors. Multivariate analysis confirmed the dominant influence on prognosis of age, sex and presenting white cell count (WCC). After allowing for these features, blast karyotype, d 8 marrow blast percentage and remission status at the end of induction therapy were the only remaining significant predictors of outcome. Organomegaly, haemoglobin concentration, French–American–British type, body mass index, presence of central nervous system disease at diagnosis, immunophenotype and presence of TEL/AML1 fusion gene (examined in a subset of 659 patients) either had no significant effect on outcome or were significant only in univariate analysis. Among karyotype abnormalities with an independent influence on prognosis, high hyperdiploidy (> 50 chromosomes) was shown to be favourable, whereas near haploidy (23–29 chromosomes), presence of the Philadelphia chromosome, t(4;11) and abnormalities affecting the short arm of chromosome 9 [abn (9p)] were adverse risk factors. Early responders to therapy, determined by residual marrow infiltration after 8 d of induction therapy, had a good outcome, while the small proportion of patients who did not achieve a complete remission by the end of induction therapy had a poor outcome. A third block of late intensification was shown to improve event‐free survival by 8% at 5 years. The effect of these risk factors was not significantly different between those randomized to the third intensification block and those not randomized to a third block.
Intravenous immunoglobulin preparations induce mild activation of neutrophils in vivo via triggering of macrophages – studies in a rat model
Authors: Teeling Jessica L. , Bleeker Wim K. , Rigter Gemma M. M. , Van Rooijen Nico , Kuijpers Taco W. , Erik Hack C.
Published: March 2003

Despite widespread use in various immune disorders, the in vivo mechanisms of action of intravenous immunoglobulin (IVIG) preparations are not well known. We previously reported that human neutrophils degranulate after incubation with IVIG in vitro as a result of interaction with FcγRII. The purpose of this study was to determine whether IVIG might stimulate neutrophils in vivo. Anaesthetized rats received a bolus intravenous injection of IVIG preparations, containing either high (aged IVIG) or low (fresh IVIG) amounts or IgG dimers at a dose of 250 mg/kg. Administration of aged IVIG induced neutrophil activation in vivo, whereas no effect was observed after infusion of fresh IVIG. Histological examination of lung tissue demonstrated mild influx of neutrophils into the pulmonary tissue after aged IVIG administration, though gross damage did not occur. Macrophage‐depleted rats no longer showed activation of neutrophils after infusion of aged IVIG, suggesting that neutrophils become activated via an indirect macrophage dependent way. We conclude that IVIG induces a mild activation of neutrophils in vivo via triggering of macrophages depending on the amount of IgG dimers. For this reason, IVIG preparations with a high content of dimers may not always be as harmless as generally believed and may be responsible for some of the side‐effects observed during IVIG infusions.
Clinical spectrum of hepatitis C‐related liver disease and response to treatment with interferon and ribavirin in haemophilia or von Willebrand disease
Authors: Lethagen Stefan , Widell Anders , Berntorp Erik , Verbaan Hans , Lindgren Stefan
Published: March 2003

Our aim was to evaluate the severity of liver disease resulting from chronic hepatitis C in haemophilia or von Willebrand disease and the efficacy of 6 months treatment with interferon alpha and ribavirin. Fifty‐five liver biopsies were performed in 43 patients without any bleeding complications, as seen with ultrasound immediately after the biopsy and 48 h thereafter. Histological changes were mild, with low scores for both inflammation and fibrosis, in spite of long exposure to blood products (mean 27 years). Two patients had compensated cirrhosis. Thirty‐five out of 39 included patients completed study treatment. Hepatitis C virus (HCV)‐RNA was negative in 77% (30/39) of patients at the end of treatment, and 36% (14/39) achieved a complete sustained response at follow‐up 6 months after treatment. Treatment failure was more frequent in patients with virus genotype 1 compared with non‐1 (P = 0·0003). The response rate correlated well with that of non‐haemophilic patients. In summary: (1) liver biopsy was safe with our regimen; (2) liver disease in our patients was usually mild and had a slow progress; (3) only HCV genotype 1 predicted treatment failure; (4) our treatment results agreed with those from non‐haemophilic patients.
Association between the SDF1‐3′A allele and high levels of CD34+ progenitor cells mobilized into peripheral blood in humans
Authors: Benboubker Lotfi , Watier Hervé , Carion Alexandra , Georget Marie‐Therese , Desbois Isabelle , Colombat Philippe , Bardos Pierre , Binet Christian , Domenech Jorge
Published: March 2003

Some patients unexpectedly fail to mobilize sufficient numbers of haematopoietic progenitor cells (HPCs) into the peripheral blood for autologous transplantation. Considering the important role of the chemokine stromal cell‐derived factor 1 (SDF‐1) in HPC homing, we investigated a possible relationship between SDF1 gene polymorphism and HPC mobilization capacity in 63 patients with malignancy. Some 67% of the good mobilizers (≥ 50 CD34+ cells/µl) and only 36% of the intermediate/poor mobilizers were SDF1‐3′A allele carriers (P = 0·032). In multivariate analysis, the presence of the SDF1‐3′A allele was the only factor predictive of good CD34+ cell mobilization (P = 0·025). This is the first report showing the involvement of genetic factors for HPC mobilization in humans and suggests a significant role for SDF‐1 in this process.
Phenotypic and functional characterization of the host immune compartment of chronic myeloid leukaemia patients in complete haematological remission
Authors: Guarini Anna , Breccia Massimo , Montefusco Enrico , Petti Maria Concetta , Zepparoni Alessia , Vitale Antonella , Foa Robin
Published: March 2003

The role of the host immune compartment in the control of chronic myeloid leukaemia (CML) has been suggested by numerous biological and clinical evidence. In the present study, the phenotypic and functional machinery of both T and cytotoxic lymphocytes was evaluated in a series of CML patients in complete haematological, and frequently also in cytogenetic, remission after treatment with interferon (IFN) α or hydroxyurea, and compared with the profile observed in patients at diagnosis and in normal controls. In particular, the lymphocyte subset distribution, the cytotoxic activity and the intracellular production of tumour necrosis factor (TNF)α and IFNγ by CD4+, CD8+ and CD56+ cells were investigated. CML patients in complete haematological remission showed a normalized CD4/CD8 T‐cell subset distribution, as well as a restored spontaneous and interleukin 2 (IL‐2) induced cytotoxic function compared with the pattern observed at diagnosis. This was associated with a significantly increased proportion of activated CD4+ lymphocytes (CD25+). TNFα and IFNγ production by CD4+, CD8+ and CD56+ lymphocytes was significantly enhanced compared with that of patients at diagnosis. However, the values were lower than those of normal controls. These results indicate that, in contrast to the observations at presentation, CML patients, at the time of the best possible response to treatment, show a normalized T‐cell subset distribution associated with an activated CD4 T‐cell compartment and a restored cytotoxic activity. In addition, they also show a markedly increased intracellular cytokine production by the lymphoid populations that play an important role in the process of specific tumour recognition. The design of therapeutic strategies aimed at stimulating the host immune compartment finds a further rationale for CML patients responsive to treatment with both IFNα and hydroxyurea.
In multiple myeloma, only a single stage of neoplastic plasma cell differentiation can be identified by VLA‐5 and CD45 expression
Authors: Rawstron A. C. , Barrans S. L. , Blythe D. , English A. , Richards S. J. , Fenton J. A. L. , Davies F. E. , Child J. A. , Jack A. S. , Morgan G. J.
Published: March 2003

The nature of the proliferating fraction in myeloma is still not known and understanding the characteristics of this fraction is central to the development of effective novel therapies. However, myeloma plasma cells typically show a very low rate of proliferation and this complicates accurate analysis. Although the level of CD45 and/or VLA‐5 has been reported to identify proliferating ‘precursor’ plasma cells, there are discrepancies between these studies. We have therefore used a rigorous sequential gating strategy to simultaneously analyse cycle status and immunophenotype with respect to CD45, VLA‐5 and a range of other integrin molecules. In 11 presentation myeloma patients, the proliferative fraction was distributed evenly between CD45+ and CD45− cells, however, cycling plasma cells were consistently VLA‐5‐. There was close correlation between the expression of VLA‐5 and a range of other integrin molecules (CD11a, CD11c, CD103), as well as the immunoglobulin‐associated molecules CD79a/b (Spearman, n = 10, P < 0·0001). In short‐term culture, cells that were initially VLA‐5‐showed increasing VLA‐5 expression with time. However, simultaneous analysis of the DNA‐binding dye 7‐amino‐actinomycin D demonstrated that this was not as a result of differentiation, as VLA‐5+ plasma cells were all non‐viable. This was confirmed in freshly explanted plasma cells from nine patients. Discrete stages of plasma cell differentiation could not be distinguished by the level of CD45 or VLA‐5 expression. The results indicate that there is a single stage of plasma cell differentiation, with the phenotype CD38+CD138+VLA‐5−. These findings support the hypothesis that neoplastic bone marrow plasma cells represent an independent, self‐replenishing population.
Identification of novel FLT‐3 Asp835 mutations in adult acute myeloid leukaemia
Authors: Abu‐Duhier F. M. , Goodeve A. C. , Wilson G. A. , Care R. S. , Peake I. R. , Reilly J. T.
Published: March 2003

Genomic DNA from 97 cases of adult de novo acute myeloid leukaemia (AML) was screened using polymerase chain reaction (PCR) and conformation‐sensitive gel electrophoresis (CSGE) for FLT3 exon 20 mutations. Initial sequencing of four cases, representing the spectrum of CSGE abnormalities, revealed changes affecting codon Asp835 in three cases and also an intron 20 A to G change. In order to identify all possible Asp835 alterations, as well as the frequency of the intronic change nucleotide 2541 + 57 A→G, the patient PCR products were digested with EcoRV and NlaIII respectively. Seven cases (7·2%) possessed a mutation affecting Asp835; these were identified, following DNA sequencing, as Asp835Tyr (n = 5), Asp835His (n = 1) and Asp835del (n = 1). Alterations affecting Asp835 were not found in 80 normal control DNA samples. In contrast, the nucleotide 2541 + 57 A→G change was shown to be a polymorphism, with an allelic frequency of 0·24 for the G and 0·76 for the A allele. This study reports, for the first time, point mutations in the human FLT3 gene that, because of their homology with other class III receptor tyrosine kinase mutations, probably result in constitutive activation of the receptor.
Severe and long‐lasting disruption of T‐cell receptor diversity in human myeloma after high‐dose chemotherapy and autologous peripheral blood progenitor cell infusion
Authors: Mariani Sara , Coscia Marta , Even Jos , Peola Silvia , Foglietta Myriam , Boccadoro Mario , Sbaiz Luca , Restagno Gabriella , Pileri Alessandro , Massaia Massimo
Published: March 2003

Vaccine‐based strategies are currently under investigation as a means of inducing tumour‐specific immune responses and improving the clinical outcome of multiple myeloma (MM) patients in remission after high‐dose chemotherapy and peripheral blood progenitor cell (PBPC) infusion. The immune competence of these patients was investigated by determining the overall diversity of the T‐cell receptor (TCR) repertoire in the peripheral blood (PB) and bone marrow (BM). The average time after transplantation was 13 months. The clonality and reciprocal usage of BV gene segments (TCRBV repertoire) was estimated at the cDNA level and membrane protein expression. The TCRBV repertoire of MM was severely disrupted compared with age‐matched normal donors. On average, one‐third of the total repertoire in both the PB and the BM consisted of T cells expressing oligoclonal TCRβ transcripts. Flow cytometry showed an increased frequency of abnormally expanded BV subfamilies at both sites. BV expansions were predominantly CD8+ and had the phenotype of antigen‐experienced memory T cells as well as T cells with the naive phenotype. Oligoclonality was not restricted to phenotypically expanded BV subfamilies, but also involved normally represented BV subfamilies. The TCR repertoire of MM in remission was then compared with monoclonal gammopathy of undetermined significance (MGUS) and MM patients at diagnosis. The degree of TCR diversity was similar in age‐matched normal donors and MGUS, but progressively decreased from MGUS to MM at diagnosis and then to MM in remission. These data indicate that: (1) there is a long‐lasting and severe disruption of TCR diversity after high‐dose chemotherapy and PBPC infusion, and (2) the extent of TCR disruption may affect the clinical outcome of vaccine‐based strategies delivered at the stage of minimal residual disease.
99mTc‐HMPAO (Ceretec) is stored in and released from the granules of eosinophil granulocytes
Authors: Moberg Lena , Karawajczyk Malgorzata , Venge Per
Published: March 2003

99mTc‐HMPAO (Ceretec) labelling of leucocytes is used clinically for the detection of inflammatory processes in the body. This study investigated the mechanisms by which 99mTc‐HMPAO is taken up by eosinophils and neutrophils. Blood cells were labelled with 99mTc‐HMPAO and the cells separated by means of their densities in Percoll gradients. For other purposes, eosinophils and neutrophils were purified by means of the MACS system and, after labelling these pure cellular preparations, the cells were ultrasonicated and the organelles separated on sucrose density gradients by means of ultracentrifugation. Organelles were characterized by their morphology on electron microscopy. Granulocytes were stimulated to secrete their granule constituents by means of exposure to complement‐coated particles. ECP (eosinophil cationic protein) and MPO (myeloperoxidase) were measured using specific immunoassays. The uptake of 99mTc‐HMPAO was 15–25‐fold higher in eosinophils than in other leucocytes. 99mTc‐HMPAO was predominantly stored in the secretory granules of eosinophils and released from the eosinophil, upon activation, together with ECP. A second storage compartment was a very light density organelle of unknown nature. These results indicated that, among leucocytes, 99mTc‐HMPAO is preferentially taken up by eosinophils and stored in the secretory granules, which has to be taken into consideration when evaluating images based on this technique. Our findings suggest that 99mTc‐HMPAO (Ceretec) may be used as a tool to follow eosinophil turnover and activity in disease.
In vivo expansion of the endogenous B‐cell compartment stimulated by radiation and serial bone marrow transplantation induces B‐cell leukaemia in mice
Authors: Holyoake Tessa L. , Freshney Mary G. , Samuel Kay , Ansell John , Watson Gwyneth E. , Wright Eric G. , Graham Gerard J. , Pragnell Ian B.
Published: March 2003

Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells. This B‐cell lineage is established during ontogeny and replenished by the process of self‐renewal. Spontaneous and induced leukaemias that frequently affect this lineage are thought to arise as a result of the frequent cell division required to maintain the population throughout adulthood and in response to repeated exposure to environmental antigens. In a series of bone marrow transplant (BMT) experiments performed in B6D2F1 mice, B‐cell leukaemia occurred in recipients of serially transplanted syngeneic bone marrow. This study was therefore designed to determine the frequency and phenotype of the observed leukaemia. Male donor cells were initially transplanted into lethally irradiated female hosts and secondary (2°) BMT was performed at 3 months. At 1, 2, 3 and 16 months following primary (1°) BMT, and when 2° BMT recipients developed leukaemia, animals were sacrificed and their tissues extensively examined. These analyses confirmed a host‐derived CD5+ transplantable B‐cell leukaemia that was initiated in 50% of 1° BMT recipients. With serial passage, the leukaemia became more aggressive and lost CD5 expression, suggesting transformation to a high‐grade leukaemia/lymphoma. This previously unreported observation suggests that the combination of radiation and subsequent serial transplantation induces a proliferative stress to the host B‐cell compartment that is causative in leukaemic transformation.
Significance of angiogenin plasma concentrations in patients with acute myeloid leukaemia and advanced myelodysplastic syndrome
Authors: Verstovsek Srdan , Kantarjian Hagop , Aguayo Alvaro , Manshouri Taghi , Freireich Emil , Keating Michael , Estey Elihu , Albitar Maher
Published: March 2003

Human angiogenin is a potent inducer of angiogenesis. The association between angiogenin and cancer progression and poor outcome in solid tumours has been documented, but its significance in leukaemias has not been evaluated. We evaluated plasma angiogenin levels in 101 previously untreated patients with acute myeloid leukaemia (AML) (59 patients) and advanced myelodysplastic syndrome (MDS) (42 patients). Angiogenin levels were significantly higher in AML and advanced MDS patients than in healthy individuals (P < 0·00001). Angiogenin levels were also significantly higher in advanced MDS than in AML (P = 0·001). Higher levels of angiogenin correlated with prolonged survival periods in both AML and advanced MDS patients (P = 0·02 and 0·01 respectively). We found no correlation between angiogenin plasma level and various patient characteristics, including age, performance status, antecedent haematological disorder, haemoglobin, white blood cell and platelet counts, and poor prognosis cytogenetics. There was no significant correlation between angiogenin level and complete remission rate and duration in AML or advanced MDS patients. In multivariate analysis, angiogenin concentration retained its significance as a prognostic factor in AML (P = 0·03), together with age (P = 0·00007) and haemoglobin (P = 0·03).
Quantification of TEL–AML1 transcript for minimal residual disease assessment in childhood acute lymphoblastic leukaemia
Authors: Drunat Séverine , Olivi Martine , Brunie Ghislaine , Grandchamp Bernard , Vilmer Etienne , Bièche Ivan , Cavé Hélène
Published: March 2003

Strategies currently used for residual disease detection in acute lymphoblastic leukaemia (ALL) rely on polymerase chain reaction (PCR) detection of immunoglobulin and T‐cell receptor rearrangements. The TEL–AML1 fusion transcript, which is associated with t(12;21) (p13;q22), is found in 25% of childhood B‐cell precursor ALL, and represents an interesting alternative target. We compared two methods for quantitating TEL–AML1 fusion transcripts: competitive PCR and real‐time PCR. These techniques showed similar sensitivity (5 × 10−5) and reproducibility. Giving highly correlated results, both techniques can be conveniently used for TEL–AML1 transcript quantification. The constancy of TEL–AML1 expression was evaluated by measuring TEL–AML1 transcripts at different steps of the cell cycle, and in 21 cases of ALL at diagnosis. No major variation in TEL–AML1 expression was observed during the cell cycle or in 20/21 of the ALL patients. Residual disease was then determined after completion of induction therapy in 20 patients with a TEL–AML1‐positive ALL. Seven patients out of 20 (35%) were still positive, including two patients with high level of residual blasts (close to or beyond 10−2). When comparison was possible, results obtained using TEL–AML1 quantification were in accordance with those obtained using T‐cell receptor rearrangements analysis.
Persistent polyclonal B‐cell lymphocytosis: further evidence for a genetic disorder associated with B‐cell abnormalities
Authors: Delage R. , Jacques L. , Massinga‐Loembe M. , Poulin J. , Bilodeau D. , Mignault C. , Leblond P. F. , Darveau A.
Published: March 2003

Persistent polyclonal B‐cell lymphocytosis (PPBL) is an intriguing disorder diagnosed predominantly in women, usually cigarette smokers, characterized by an increase in the number of polyclonal B lymphocytes. Abnormality of the B‐cell population is also evidenced by the presence of multiple bcl‐2/Ig gene rearrangements and the finding of an additional long arm chromosome 3q+ (i3)(q10) within a significant proportion of B cells. The physiopathology of PPBL is unknown but its association with the HLA DR7 phenotype suggests a possible genetic disorder. To further determine whether PPBL has a genetic predisposition, we have undertaken an extensive study in a large family of a patient diagnosed with PPBL. Three individuals among the first‐degree relatives presented all the criteria for a diagnosis of PPBL. A slight increase in serum IgM without evidence of B‐cell proliferation was shown in two additional siblings. Multiple bcl‐2/Ig gene rearrangements, a typical feature of PPBL, were identified in 8/10 individuals among first‐degree relatives. A statistically significant association was found between the presence of these rearrangements and of a paternal HLA haplotype. We conclude that PPBL has a familial occurrence suggesting an underlying genetic defect. The development of the complete syndrome probably relies on unidentified additional co‐factors.
Cell cycle analysis and expression of cell cycle regulator genes in myeloma cells overexpressing cyclin D1
Authors: Yata Kenichiro , Sadahira Yoshito , Otsuki Takemi , Sakaguchi Haruko , Isozaki Yumika , Uno Masako , Kurebayashi Junichi , Fujii Tomohiro , Eda Sakura , Ueki Ayako , Yawata Yoshihito , Yamada Osamu , Sugihara Takashi
Published: March 2003

Among the recently discovered myeloma‐specific gene alterations associated with chromosomal translocations, cyclin D1/PRAD1/Bcl‐1 overexpression caused by t(11;14)(q13;q32) is considered to be the most frequent in myeloma patients and cell lines, and may be a prognostic factor clinically. To elucidate the cellular biological role of overexpressed cyclin D1 in myeloma cells, we examined the mRNA expression levels of cell cycle regulators including three cyclin Ds, cyclin‐dependent kinase inhibitors (CDK‐Is) and accelerators. Cyclin D1 overexpression was clearly demonstrated in the lines with abnormal 11q13 and associated with overexpression of S and G2 accelerator genes. The cyclin D1‐overexpressing lines tended to have a shortened G1 phase compared with the non‐expressing lines. In addition, artificial silencing using antisense oligonucleotides for cyclin D1 suppressed the growth rate of some but not all cyclin D1‐overexpressing cells. These results indicate that overexpression of cyclin D1 caused by cytogenetic abnormalities may make cells progress through the cell cycle rapidly, but it seems that other factors such as cyclin D2 and translocation‐related genes affect the cell cycle progression in myeloma cells.
During U937 monocytic differentiation repression of the CD43 gene promoter is mediated by the single‐stranded DNA binding protein Purα
Authors: Shelley C. Simon , Da Silva Nicolas , Teodoridis Jens M.
Published: March 2003

Human CD43 is an abundant, heavily glycosylated molecule expressed exclusively on the surface of leucocytes. When leucocytes are at rest, CD43 acts to prevent intercellular interaction but during leucocyte differentiation such cell–cell interaction is facilitated by CD43. This change in the function of CD43 is mediated in part by a reduction in its level of expression. Previous studies have implicated proteolytic cleavage events at the cell surface in causing such reduction. Here, we report that, in an in vitro model of leucocyte differentiation, CD43 mRNA levels were also subject to reduction. Specifically, we demonstrated that within 48 h of the cell line U937 being induced to differentiate along the monocytic pathway, CD43 mRNA levels were reduced by 69%. This decline coincided with a decrease in the activity of the CD43 gene promoter mediated by the single‐stranded DNA binding protein Purα. Previously, we have demonstrated that Purα mediates induction of the CD11c β2 integrin promoter during U937 differentiation. Consequently, Purα represents a potential means by which the induction of pro‐adhesive molecules and the repression of anti‐adhesive molecules is co‐ordinated during leucocyte differentiation.
High expression of platelet‐derived growth factor and transforming growth factor‐β1 in blast cells from patients with Down Syndrome suffering from transient myeloproliferative disorder and organ fibrosis
Authors: Hattori Hiroyoshi , Matsuzaki Akinobu , Suminoe Aiko , Ihara Kenji , Nakayama Hideki , Hara Toshiro
Published: March 2003

To determine whether platelet‐derived growth factor (PDGF) and transforming growth factor‐β1 (TGF‐β1) are involved in organ fibrosis in patients with transient myeloproliferative disorder (TMD) in Down syndrome, the expression of PDGF and TGF‐β1 mRNA in blast cells of TMD was investigated using real‐time quantitative reverse transcription polymerase chain reaction. Blasts and liver tissue from TMD patients with hepatic fibrosis showed a significantly elevated expression of PDGF gene. The expression of TGF‐β1 gene was higher in TMD and acute megakaryoblastic leukaemia than in the control group. These results suggest that PDGF in combination with TGF‐β1 plays a role in organ fibrosis of TMD.
Cytogenetic and morphological abnormalities in paroxysmal nocturnal haemoglobinuria
Authors: Araten David J. , Swirsky David , Karadimitris Anastasios , Notaro Rosario , Nafa Khedoudja , Bessler Monica , Thaler Howard T. , Castro‐Malaspina Hugo , Childs Barrett H. , Boulad Farid , Weiss Mark , Anagnostopoulos Nikolaos , Kutlar Abdullah , Savage David G. , Maziarz Richard T. , Jhanwar Suresh , Luzzatto Lucio
Published: March 2003

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by the expansion of a haematopoietic stem cell clone with a PIG‐A mutation (the PNH clone) in an environment in which normal stem cells are lost or failing: it has been hypothesized that this abnormal marrow environment provides a relative advantage to the PNH clone. In patients with PNH, generally, the karyotype of bone marrow cells has been reported to be normal, unlike in myelodysplastic syndrome (MDS), another clonal condition in which cytogenetic abnormalities are regarded as diagnostic. In a retrospective review of 46 patients with a PNH clone, we found a karyotypic abnormality in 11 (24%). Upon follow‐up, the proportion of cells with abnormal karyotype decreased significantly in seven of these 11 patients. Abnormal morphological bone marrow features reminiscent of MDS were common in PNH, regardless of the karyotype. However, none of our patients developed excess blasts or leukaemia. We conclude that in patients with PNH cytogenetically abnormal clones are not necessarily malignant and may not be predictive of evolution to leukaemia.
Effect of in vitro fertilization treatment and subsequent pregnancy on the protein C pathway
Authors: Curvers J. , Nap A. W. , Thomassen M. C. L. G. D. , Nienhuis S. J. , Hamulyák K. , Evers J. L. H. , Tans G. , Rosing J.
Published: March 2003

Thirty‐three women who were planned for an in vitro fertilization (IVF) cycle donated blood at four time points during treatment: at baseline, after downregulation, hyperstimulation and luteal support. Levels of progesterone, 17β‐oestradiol and indicators of the protein C pathway, i.e. activated protein C sensitivity ratios (APCsr), protein C, protein C inhibitor and protein S were measured. Compared with baseline, oestradiol decreased twofold at downregulation and increased 40‐fold at hyperstimulation. Progesterone was elevated 2·5‐fold at hyperstimulation and 40‐fold at luteal support. The APCsr increased slightly at downregulation, significantly increased during hyperstimulation and remained high during luteal support. The plasma levels of the anticoagulant proteins did not change or changed moderately during treatment. During downregulation, progesterone correlated negatively with APCsr (r = −0·398, P = 0·024). At hyperstimulation oestradiol correlated with the APCsr (r =0·615, P < 0·0005). Moreover, there was a significant correlation (r = 0·599, P < 0·0005) between the difference in baseline and hyperstimulation values of oestradiol (ΔE2 = 6·6 nmol/l) and the APCsr (ΔAPCsr = 0·30). Six women who participated in this study became pregnant. Compared with baseline, the APCsr was increased 1·9‐fold (ΔAPCsr = 1·48) and free protein S free level decreased 30% at 7 weeks of pregnancy. This study demonstrates that despite the considerable changes in endogenous oestradiol and progesterone during an IVF cycle, changes in plasma levels of anticoagulant proteins are moderate. The significant increase in the APCsr during hyperstimulation indicates that acquired APC resistance observed during sex steroid hormone changes in women is at least partially caused by high oestrogen levels. Our findings demonstrate that IVF treatment is accompanied by the development of a mild prothrombotic condition.