bluuebunny/crossref_metadata_2025 huggingface dataset:
Potential role of insulin‐like growth factor binding protein‐4 in the uncoupling of bone turnover in multiple myeloma
Authors: Feliers Denis , Woodruff Kathleen , Abboud Sherry
Published: March 2003

Decreased bone formation plays an important role in the development of lytic lesions during the late stage of multiple myeloma (MM). Release of insulin‐like growth factor binding protein‐4 (IGFBP4) by tumour cells adjacent to bone may inhibit IGF‐I‐stimulated osteoblast growth and contribute to decreased bone formation. The present study demonstrates that the human MM cell line, ARH‐77, expresses IGFBP4 and, to a lesser extent, IGFBP6 mRNA and protein. IGFBP4 expression in myeloma cells may be modulated by cytokines released by stromal cells and T cells in the microenvironment. We tested the effect of recombinant interferon‐gamma (INF) on IGFBP4 expression in ARH‐77. INF increased IGFBP4 mRNA and protein levels at 12 h, with a decline to baseline by 24 h. In contrast, IGFBP4 was not regulated in response to IL‐6, TNF‐alpha, PDGF BB, bFGF, TGF‐beta or the cAMP agonist, forskolin. In other systems, IGFBP4 may also be regulated post‐transcriptionally by a protease that is activated by IGF‐I or ‐II. Conditioned medium from ARH‐77 cultures incubated with IGF‐I or ‐II for up to 24 h failed to demonstrate proteolytic activity. Proteolysis was also not observed when conditioned medium containing exogenous rhIGFBP4 was incubated with IGF‐I or ‐II under cell‐free conditions. To determine if human myeloma tumours also express IGFBP4, total RNA was isolated from four tumour biopsies. All samples expressed detectable levels of IGFBP4 mRNA. These findings indicate that interferon‐gamma may indirectly modulate bone formation via the the release of tumour‐derived IGFBP4, suggesting that the immune system may influence bone turnover in MM. Failure of myeloma cells to release protease activity may promote IGFBP4 accumulation in the microenvironment during tumour growth.
Molecular evolution of acute myeloid leukaemia in relapse: unstable N‐ras and FLT3 genes compared with p53 gene
Authors: Nakano Yasuyuki , Kiyoi Hitoshi , Miyawaki Shuichi , Asou Norio , Ohno Ryuzo , Saito Hidehiko , Naoe Tomoki
Published: March 2003

Relapse is a major cause of treatment failure in acute myeloid leukaemia (AML), and is usually accompanied by resistance to chemotherapy. To study whether relapse is accompanied by genetic alterations, we compared N‐ras, p53 and FLT3 gene mutations in paired samples obtained at initial diagnosis and first relapse. 28 patients with relapsed AML were studied, and their duration of complete remission ranged from 133 to 989 d (mean 318 d). Karyotype changes were observed at relapse in 11 patients. Point mutations of the N‐ras gene were positive at both stages (+/+) in three patients, positive at initial diagnosis and negative at relapse (+/−) in three patients, and negative at initial diagnosis and positive at relapse (−/+) in two patients. Internal tandem duplications of the FLT3 gene (FLT3/ITD) were +/+ in five patients, +/− in one patient, and −/+ in six patients. The p53 gene mutations were +/+ in two patients, +/− in one patient, and −/− in 25 patients. FLT3/ITD and mutant p53 at relapse were associated with short survival after relapse. These results indicate that relapse is frequently accompanied by molecular alterations that include the loss and/or acquisition of mutations. Thus relapse can be understood as clonal shift or collateral succession rather than clonal progression.
Erythroid gene expression is differentially regulated by erythropoietin, haemin and delta‐aminolaevulinic acid in UT‐7 cells
Authors: McHale Cliona M. , Winter Paul C. , Lappin Terence R. J.
Published: March 2003

Erythropoietin (Epo) is essential for the later stages of erythropoiesis, acting to promote cell survival and proliferation, but its role in differentiation remains to be defined. The UT‐7 cell line exhibits both erythroid and megakaryocytic characteristics and can be induced to differentiate along the erythroid pathway by Epo or the megakaryocytic pathway by phorbol myristic acetate. We have compared the effects of Epo and the chemical inducers, δ‐aminolaevulinic acid (δ‐ALA) and haemin on the differentiation capacity of UT‐7 cells. Epo alone promoted relatively early events in erythroid maturation, without significant changes in haemoglobin production or morphology. GATA‐2 and c‐myb were down‐regulated by Epo, and GATA‐2 was further down‐modulated by the inducers. Conversely, SCL expression was up‐regulated by Epo and further increased by haemin and δ‐ALA. Epo caused an increase in the proportion of cells expressing cell surface glycophorin A (GPA) and up‐regulated β‐ and γ‐globin by several fold. Both haemin and δ‐ALA caused a de novo increase in α‐globin expression as well as enhancing Epo‐induced β‐globin expression, leading to a marked increase in haemoglobin production. These results suggest that haemoglobin production in UT‐7 cells is limited by a deficiency of erythroid‐specific aminolaevulinic acid synthase (ALAS‐E) activity or globin synthesis as a consequence of their immaturity as a multipotential cell line.
Pregnancy‐associated autoimmune neonatal thrombocytopenia: role of maternal HLA genotype
Authors: Gandemer V. , Kaplan C. , Quelvennec E. , Poulain P. , Laurent M. C. , Semana G. , Renouard J. , Le Gall E.
Published: March 2003

In a prospective study between 1993 and 1998, data was collected from 46 pregnant women and subsequently from their babies. 25 pregnant women with active autoimmune thrombocytopenic purpura (AITP) or a history of AITP (group A) and 21 pregnant women with isolated thrombocytopenia and identification of specific platelet autoantibodies detected by monoclonal antibody‐specific immobilization of platelet antigens (MAIPA) assay (group  B) were evaluated for platelet‐associated immunoglobulin‐G (PAIgG), MAIPA assay and HLA genotype. Neonatal platelet counts were performed at least three times in the first week. 11 neonates were thrombocytopenic (23.9%). No severe haemorrhage occurred. There were no significant differences regarding the values of PAIgG or positive MAIPA tests between mothers of thrombocytopenic or healthy newborns. A significant difference, however, regarding the HLA DRB3* allele was found, with a high incidence in the subgroup of mothers of healthy newborns (P = 0.005). A similar trend was found among mothers with anti‐GPIIbIIIa antibodies (P = 0.06). In contrast, HLA DRB5* allele appeared to be present especially in mothers of thrombocytopenic newborns (not significant). Our data suggest that mothers with AITP who have the HLA DRB3* genotype are unlikely to give birth to a thrombocytopenic baby. This study provides a preliminary report on a noninvasive test to identify infants who are likely to be affected.
Survival of human leukaemic B‐cell precursors is supported by stromal cells and cytokines: association with the expression of bcl‐2 protein
Authors: Nishii Kazuhiro , Katayama Naoyuki , Miwa Hiroshi , Shikami Masato , Masuya Masahiro , Shiku Hiroshi , Kita Kenkichi
Published: March 2003

We searched for cytokines with the potential to support the survival of human B‐cell precursor acute lymphoblastic leukaemia (pre‐B ALL) cells. 47 patients with pre‐B ALL were classified into four stages: stage I, CD19+CD10−CD20−; stage II, CD19+CD10+CD20−; stage III, CD19+CD10+CD20+cytoplasmic μ‐heavy chain (cμ)−; stage IV, CD19+CD10+CD20+cμ+. Interleukin (IL)‐3 receptor α chain (IL‐3Rα) was expressed in all stages, whereas the expressions of IL‐7Rα and IL‐2Rα were pronounced in stages IV and II, respectively. Neither IL‐3, IL‐7 nor IL‐2 supported the survival of pre‐B ALL cells. When pre‐B ALL cells were layered on stromal, MS‐10, cells, viability of the pre‐B ALL cells increased. Addition of IL‐3 to culture containing MS‐10 cells enhanced the survival of pre‐B ALL cells in all cases, whereas addition of IL‐7 augmented the survival of pre‐B ALL cells of some cases of stage III and all cases of stage IV. The survival of pre‐B ALL cells was also supported by the conditioned media of MS‐10 cells. Stromal‐cell‐derived factor 1 (SDF‐1) supported the survival of pre‐B ALL cells. Effects of the conditioned media of MS‐10 cells were abrogated by an anti‐SDF‐1 neutralizing antibody. The extent of survival of pre‐B ALL cells supported by stromal cells and IL‐3 and IL‐7, correlated with the expression level of bcl‐2 protein. The effects of stromal cells may be in part related to SDF‐1.
Increased sensitivity of CLL‐derived lymphocytes to apoptotic death activation by the proteasome‐specific inhibitor lactacystin
Authors: Masdehors Peggy , Omura Satoshi , Merle‐Béral Hélène , Mentz Frank , Cosset Jean‐Marc , Dumont Jeanine , Magdelénat Henri , Delic Jozo
Published: March 2003

Ubiquitin‐proteasome‐dependent protein processing appears to be an essential component in the control of radiation‐induced apoptosis in human lymphocytes. This control is altered in chronic lymphocytic leukaemia (CLL), compared to that of normal human lymphocytes which mainly showed high apoptotic values after irradiation, but in some cases no sensitivity was observed. Interestingly, lactacystin activated the apoptotic pathway in both radio‐resistant and sensitive CLL cells, at doses which had no effect in normal cells where significantly higher concentrations were required. Therefore the resistance of some CLL cells to apoptosis initiation by radiation does not correlate to observed increased sensitivity to lactacystin. The nuclear level of the transcription factor NF‐κB or the cytoplasmic level of IκBα remained unaltered upon irradiation or lactacystin CLL cells treatment, suggesting that the activity of the other factors involved in apoptotic death control were altered through proteasomal inhibition. These results strongly suggest an essential role of the ubiquitin system in apoptotic cell death control in CLL lymphocytes. The inhibition of proteasome‐ubiquitin‐dependent processing could be a discriminatory apoptotic stimulus between normal versus malignant lymphocytes and therefore might potentially be of use in this specific human pathology.
Plasminogen activation in human acute leukaemias
Authors: Scherrer A. , Wohlwend A. , Kruithof E. K. O. , Vassalli J.‐D. , Sappino A.‐P.
Published: March 2003

Plasminogen activation is implicated in solid tumour growth, invasion and metatastic spread. However, little is known about its role in leukaemia. We investigated the production by leukaemic cells of plasminogen activators [urokinase (uPA) and tissue‐type PA (tPA)], cell surface receptor for uPA (uPAR) and PA inhibitors (PAI‐1 and PAI‐2). Leukaemic cells from 37 patients [26 with acute myeloid leukaemia (AML) and 11 with acute lymphoid leukaemia (ALL)] were analysed for mRNA content and enzymatic activities. High levels of uPA mRNA were found in M1, M2, M3 and M4–M5 AMLs, whereas tPA mRNA was not detected in any of the analysed cases. uPAR mRNA was confined to subtypes M4–M5. PAI‐1 mRNA was detected in M3 and M4–M5. PAI‐2 mRNA was found predominantly in M2 and M4–M5. SDS‐PAGE/zymography analyses of cell extracts and supernatants after 24 and 48 h of culture confirmed the production of active uPA by AML cells (mainly M4–M5), but not by ALL. The finding of uPA, uPAR, PAI‐1 and PAI‐2 synthesized by leukaemic cells suggests that plasminogen activation may contribute to the invasive behaviour of these cells, the fibrinolytic imbalance observed in leukaemic patients and the differentiation and proliferation of M4–M5 by interaction of uPA with uPAR.
Activation of endothelium by immunotherapy with interleukin‐2 in patients with malignant disorders
Authors: Locker Gottfried J. , Kapiotis Stylianos , Veitl Marvio , Mader Robert M. , Stoiser Brigitte , Kofler Julia , Sieder Anna E. , Rainer Hugo , Steger Günther G. , Mannhalter Christine , Wagner Oswald F.
Published: March 2003

Treatment with intravenous recombinant human interleukin‐2 (rh IL‐2) is frequently accompanied by the capillary leak syndrome and disturbances of the coagulation system. Although the exact mechanisms are still not fully understood, the involvement of the endothelium is proven. This investigation aimed to elucidate more precisely the role of the endothelium in the generation of IL‐2‐based side‐effects. In nine tumour patients receiving intravenous rh IL‐2, parameters characterizing endothelial cell activation as well as activation of the coagulation system were evaluated. A significant increase of the circulating endothelial leucocyte adhesion molecule‐1 (cELAM‐1) and the vasoconstrictor peptide endothelin‐1 (ET‐1) was observed (P < 0.05), indicating activation of endothelial cells. The simultaneous increase of tissue‐plasminogen activator and plasminogen activator inhibitor type‐1 during therapy (P < 0.05) corroborated this observation. A decrease in platelet count parallelled by an increase of fibrin degradation products, the prolongation of partial thromboplastin time, and the decrease of fibrinogen (P < 0.05) suggested the development of disseminated intravascular coagulation (DIC), induced by activated endothelium and intensified by transient hepatic failure. We concluded that activation of the endothelium mediated by IL‐2 was accompanied by a loss of endothelial integrity and capillary leak. The activated endothelium can trigger DIC via activation of the coagulation cascade. The increased ET‐1 might act as an endogenous counter‐regulator of the disadvantageous haemodynamic side‐effects induced by IL‐2.
Myeloablative radiochemotherapy followed by reinfusion of purged autologous stem cells for Waldenström's macroglobulinaemia
Authors: Dreger Peter , Glass Bertram , Kuse Rolf , Sonnen Ruth , Von Neuhoff Nils , Bolouri Human , Kneba Michael , Schmitz Norbert
Published: March 2003

Waldenström's macroglobulinaemia (WM) is an incurable lymphoproliferative disorder. The purpose of this study was to investigate the role of autologous peripheral blood stem cell transplantation (ASCT) for the treatment of WM. Seven patients (untreated or after first‐line therapy) with symptomatic WM underwent two or three cycles of Dexa‐BEAM chemotherapy + G‐CSF with stem cell harvesting and proceeded to total body irradiation and high‐dose cyclophosphamide followed by reinfusion of ex‐vivo B‐cell‐depleted stem cells. Engraftment was prompt, and procedure‐related deaths did not occur. A strong reduction or normalization of BM infiltration and serum IgM levels occurred in all evaluable patients, but immunofixation electrophoresis revealed persistent paraproteinaemia in five of them. With 3–30 months of follow‐up, all patients are alive without clinical or serological signs of disease progression. This pilot trial shows for the first time that high‐dose radiochemotherapy with purged stem cells is effective and may improve the course of patients with WM. In the majority of cases, however, complete eradication of the disease does not appear to be possible with ASCT alone.
FICTION‐TSA analysis of the B‐cell compartment in myeloma shows no significant expansion of myeloma precursor cells
Authors: Davies Faith E. , Rawstron Andrew C. , Pratt Guy , O'Connor Sheila , Su'Ut Lela , Blythe David , Fenton James , Claydon David , Child J. Anthony , Jack Andrew S. , Morgan Gareth J.
Published: March 2003

Studies utilizing flow cytometry and PCR have shown that the B‐cell compartment in myeloma contains cells which are clonally related to the myelomatous plasma cells. Current data, however, remains inconclusive regarding the extent of this involvement. By combining fluorescent immunophenotyping, tyramine signal amplification and fluorescence in‐situ hybridization (FICTION‐TSA), we have used the presence of numerical chromosomal abnormalities within plasma cells as a clonal marker to examine the CD20+ B‐cell compartment for the presence of aneuploidy. A series of 54 cases of myeloma were screened for the presence of numerical abnormalities of chromosomes 3 and 11. FICTION‐TSA was performed on 13 cases with either trisomy 3 or 11 and on a control group of six cases known to be disomic for the two chromosomes. B‐cell numbers were reduced in the myeloma cases compared to the normal controls (median 1.8% v 3.0%, P = 0.05). In the cases with a chromosomal marker, three signals were seen in a median of 1.88% of CD20+ B cells compared to 2.58% within the control group. Comparison of the two groups using a Wilcoxon‐Mann‐Whitney U test showed no statistical significant difference. Using this data set, it was possible to exclude a 3.03% expansion of clonally related B cells (95% confidence level). We conclude that the B‐cell compartment in myeloma does not represent the major site of clonal expansion, and if clonally related cells are present then the numbers are few.
Early diagnosis of central nervous system aspergillosis using polymerase chain reaction, latex agglutination test, and enzyme‐linked immunosorbent assay
Authors: Kami Masahiro , Ogawa Seishi , Kanda Yoshinobu , Tanaka Yuji , Machida Utako , Matsumura Tomoko , Sakamaki Hisashi , Hirai Hisamaru
Published: March 2003

To investigate the usefulness of examination of cerebrospinal fluids (CSF) in the diagnosis of central nervous system (CNS) aspergillosis, we examined five patients with either brain abscesses or cerebral infarctions and 11 control patients. CSF samples were subjected to enzyme‐linked immunosorbent assay (EIA), latex agglutination test (LA) and polymerase chain reaction (PCR). Cultures of CSF samples were negative in all the patients, but PCR, EIA and LA were positive in five, four and four patients with CNS aspergillosis, respectively. None of these tests were positive in the control patients. CSF examination may be beneficial in the diagnosis of CNS aspergillosis.
Angiogenic growth factors and endostatin in non‐Hodgkin's lymphoma
Authors: Bertolini Francesco , Paolucci Mara , Peccatori Fedro , Cinieri Saverio , Agazzi Alberto , Ferrucci Pier Francesco , Cocorocchio Emilia , Goldhirsch Aron , Martinelli Giovanni
Published: March 2003

A number of clinical studies have demonstrated the prognostic significance of angiogenesis and angiogenic growth factors in solid tumours; however, very little is known about the relevance of these parameters in haematological malignancies. We evaluated circulating levels of angiogenic growth factors and endostatin in 36 non‐Hodgkin's lymphoma (NHL) patients. Baseline vascular endothelial growth factor (VEGF) levels of patients in complete remission (CR) after a median follow‐up of 21 months were significantly lower than those of patients with progressive disease (P = 0.016). Event‐free survival (EFS) rate was significantly higher in patients who had baseline VEGF and basic‐fibroblast growth factor (b.FGF) levels below the median values of 147 and 19.5 pg/ml (P = 0.018 and 0.039 by log‐rank test, respectively). Conversely, the levels of endostatin, angiogenin and leptin were not different in CR patients compared to relapsed patients and did not correlate with EFS. Our data suggest that b‐FGF and, particularly, VEGF might be considered prognostic factors in NHL staging and management.
Treatment‐related deaths during induction and first remission of acute myeloid leukaemia in children treated on the Tenth Medical Research Council Acute Myeloid Leukaemia Trial (MRC AML10)
Authors: Riley Lynne C. , Hann Ian M. , Wheatley Keith , Stevens R. F.
Published: March 2003

Between 1988 and 1995, 341 children with acute myeloid leukaemia (AML) were treated on the Medical Research Council Acute Myeloid Leukaemia Trial (MRC AML10). The 5‐year overall survival was 57%, much improved on previous trials. However, there were 47 deaths (13.8%), 11 of which were associated with bone marrow transplantation (BMT). The treatment‐related mortality was significant at 13.8%, but decreased in the latter half of the trial from 17.8% in 1998–91 to 9.6% in 1992–95 (P = 0.03%). The main causes of death were infection (65.9%), haemorrhage (19.1%) and cardiac failure (19.1%). Fungal infection was a significant problem, causing 23% of all infective deaths. Haemorrhage occurred early in treatment, in children with initial white cell counts >100 × 109/l (P = 0.001), and was more common in those with M4 and M5 morphology. Cardiac failure only occurred from the third course of chemotherapy onwards, with 78% (7/9) in conjunction with sepsis as a terminal event. Some deaths could be prevented by identifying those most at risk, and with prompt recognition and aggressive management of complications of treatment. Future options include the prophylactic use of antifungal agents, and the use of cardioprotectants or alternatives to conventional anthracyclines to decrease cardiac toxicity.
Haemopoietic reconstitution after sublethal irradiation: comparison of the effects of different haemopoietic cytokines on murine lymphocytes and bone marrow cells
Authors: Frasca Daniela , Guidi Francesco , Barattini Paola , Doria Gino
Published: March 2003

Sublethally irradiated mice were injected with recombinant cytokines to stimulate haemopoietic reconstitution. Interleukin (IL)‐11 and IL‐6 were able to significantly accelerate the recovery of thymus, spleen and bone marrow cells when used in combination with IL‐3, but not alone. Stem cell factor (SCF) also displayed detectable effects when used with IL‐3. Conversely, the IL‐6 superagonist K‐7/D‐6 was able, even when used alone, to induce recovery of thymus, spleen and bone marrow cells up to the level of unirradiated controls. Together, these results indicate that it is possible to attain complete recovery of lymphoid organs and tissues as early as 7 d after irradiation by use of haemopoietic cytokines.
The cyclin‐dependent kinase inhibitors p18INK4c and p19INK4d are highly expressed in CD34+ progenitor and acute myeloid leukaemic cells but not in normal differentiated myeloid cells
Authors: Tschan M. P. , Peters U. R. , Cajot J. F. , Betticher D. C. , Fey M. F. , Tobler A.
Published: March 2003

Cyclin‐dependent kinase inhibitors (CKIs) are important for the differentiation of cells in various tissues. In acute myeloid leukaemia (AML) the cells accumulate at particular stages of myeloid maturation. We therefore analysed the expression pattern of different CKIs in fresh samples of AML patients and compared it with that in CD34+ progenitor and normal differentiated myeloid cells. Competitive RT‐PCR and Western analysis revealed a significantly higher expression of p18INK4c and p19INK4d in leukaemic and CD34+ progenitor cells than in granulocytes and monocytes. A different pattern was seen for p27Kip1 and p57Kip2 expression being low in leukaemic cells but high in normal immature and differentiated cells. No marked differences were found in p15INK4b and p21Cip1 mRNA expression between leukaemic and CD34+ progenitor or mature myeloid cells. Our findings therefore indicate that high expression of p18INK4c and p19INK4d in haemopoietic progenitor and leukaemic blast cells may contribute to the premature differentiation block seen in AML.
Leucocyte transfusions from rhG‐CSF or prednisolone stimulated donors for treatment of severe infections in immunocompromised neutropenic patients
Authors: Peters C. , Minkov M. , Matthes‐Martin S. , Pötschger U. , Witt V. , Mann G. , Höcker P. , Worel N. , Stary J. , Klingebiel T. , Gadner H.
Published: March 2003

Sepsis in profound neutropenia after chemotherapy is associated with high mortality despite appropriate antibacterial or antifungal treatment. In a prospective phase I/II study we evaluated the feasability and efficacy of leucocyte transfusions (LT) in patients with malignancies or haematological disorders who were suffering from severe bacterial or fungal infection during therapy‐related bone marrow aplasia. 30 patients with severe neutropenia and clinical signs of life‐threatening sepsis not responding to adequate treatment, received LT from rhG‐CSF‐stimulated family donors or from prednisolone‐primed volunteers. A total of 301 LT were administered. The median number of LT per patient was seven (range three to 65), the median duration of LT treatment was 8 d (range 2–35). The white cell count (WBC), absolute neutrophil count (ANC) and lymphocyte count of the concentrates from rhG‐CSF‐stimulated donors were significantly higher than those from prednisolone‐primed volunteers (P = 0.0001). Despite the critical condition of the patients, LT were generally well tolerated. Only 39 (12.9%) LT were associated with adverse reactions. The transfusion of leucocytes collected by continuous flow leukapheresis from both rhG‐CSF and prednisolone stimulated donors resulted in a measurable increment of the peripheral leucocyte and ANC counts in our patients. On day 100 after the first LT, 20/30 patients were alive with complete clearance of the infection.
A novel mutation of δ‐aminolaevulinate dehydratase in a healthy child with 12% erythrocyte enzyme activity
Authors: Akagi Reiko , Yasui Yumiko , Harper Pauline , Sassa Shigeru
Published: March 2003

Cloning, expression and phenotype studies of the defective gene for δ‐aminolaevulinate dehydratase (ALAD) in a family with an asymptomatic girl who had ALAD deficiency were carried out. The proband was identified by neonatal ALAD screening, and had erythrocyte ALAD activity at 12% of the normal control. She was heterozygous for ALAD deficiency, which was inherited from her father. Nucleotide sequence analysis of the cloned ALAD cDNA revealed C36 to G and T168 to C mutations on the same allele. The former mutation resulted in F12L substitution, whereas the latter was a silent mutation. All family members who had decreased ALAD activity had the same mutation. Expression of the mutant ALAD cDNA in Chinese hamster ovary cells produced an ALAD protein without significant enzyme activity. Additionally, the mutant ALAD cDNA which encodes F12L substitution produced an aberrant migration pattern in polyacrylamide gel electrophoresis under denaturing conditions. This finding probably reflects an abnormal folding of the F12L protein, since the mutation occurred in the α1 helix of the N‐terminal arm of the enzyme, which is involved in the extensive quaternary interactions among the subunits. This is also the first report of ALAD gene mutation in an asymptomatic subject.
Competitive repopulation of retrovirally transduced haemopoietic stem cells
Authors: Qin Shenwei , Ward Maureen , Raftopoulos Harry , Tang Hongyu , Bradley Brigid , Hesdorffer Charles , Bank Arthur
Published: March 2003

Gene transfer into haemopoietic stem cells (HSC) may be useful in gene therapy for a variety of inherited and acquired human diseases. Cell division is required for retroviral transduction, and cytokine stimulation is often used to increase mitosis of quiescent HSC. Exposure to cytokines has been shown to have an unfavourable effect on the engraftment of these cells when competed with unmanipulated HSC. We now show that a similar engraftment defect is present when HSC are manipulated and transduced with the human multiple drug resistance (MDR) gene. The extent of the unfavourable competition depended on the relative numbers of cytokine‐treated and fresh cells when the two populations of cells were administered simultaneously into marrow‐ablated isogenic mice. When the manipulated transduced cells were given 2 or 4 d before the unmanipulated cells there was a much greater engraftment of the manipulated cells. The data suggested that the manipulated cells were at a relative disadvantage for marrow engraftment as compared to fresh cells, presumably due to the more efficient homing and engraftment properties of these latter unmanipulated cells. However, the manipulated cells had no intrinsic inability to engraft when they were the predominant donor cell population. In all cases the percent of MDR transduced cells in the engrafting manipulated cells remained relatively constant at about 25–30%. These results have implications for the use of manipulated transduced stem cells in gene therapy, suggesting that administering them before adding fresh cells can overcome their engraftment defect.
Distinct patterns of apoptosis in association with modulation of CD44 induced by thrombopoietin and granulocyte‐colony stimulating factor during ex vivo expansion of human cord blood CD34+ cells
Authors: Seoh Ju‐Young , Woo So‐Youn , Im Seock‐Ah , Kim Young‐Ju , Park Hae‐Young , Lee Seok , Lee Mi‐Ae , Yoo Eun‐Sun , Huh Jung‐Won , Ryu Kyung‐Ha , Lee Soon‐Nam , Chung Wha‐Soon , Seong Chu‐Myong
Published: March 2003

The insufficient number of haemopoietic stem cells (HSCs) in cord blood (CB) is the major potential limitation to widespread use of CB for marrow replacement. Cytokine‐mediated ex vivo expansion has been proposed as a means of increasing the number of CB HSCs for transplantation. However, the biology of CB HSCs during cytokine‐mediated ex vivo expansion, such as apoptosis or expression of adhesion molecules, has not yet been elucidated. We have investigated the patterns of apoptosis and CD44 expression on human CB CD34+ cells during ex vivo expansion. CD34+ cells isolated from human CB were cultured in a stroma‐free liquid culture system with thrombopoietin (TPO), flt3‐ligand (FL), stem cell factor (SCF), and/or granulocyte‐colony stimulating factor (G‐CSF). During the culture, for up to 5 weeks, apoptosis was measured by staining with 7‐amino‐actinomycin D (7‐AAD) along with concurrent immunophenotyping of CD34 and CD44 with three‐colour flow cytometry. In the cultures with TPO, an apoptotic fraction with down‐regulated CD44 appeared from the fourth day up to the second week. G‐CSF also induced apoptosis but in a different manner; the apoptotic fraction without down‐regulation of CD44 appeared unremittingly for up to 5 weeks. FL did not induce apoptosis or down‐regulation of CD44. These findings show that apoptosis is indeed involved in the regulation of CB CD34+ cells in ex vivo expansion and the patterns of apoptosis are dependent on the type of cytokines used. The distinct patterns of apoptosis suggest different mechanisms of TPO and G‐CSF in inducing apoptosis, which still remains to be elucidated.
The erythrocyte effects of haemoglobin OARAB
Authors: Nagel Ronald L. , Krishnamoorthy Rajagopal , Fattoum Slaheddine , Elion Jacques , Genard Nathalie , Romero Jose , Fabry Mary E.
Published: March 2003

To test the hypothesis that HbOARAB induces an increase in red cell mean corpuscular haemoglobin concentration (MCHC), we studied members of four Tunisian families who were either homo‐ or heterozygous for HbOARAB or were double heterozygotes for HbS and HbOARAB. The α‐gene status was also tested. The findings included: (1) Distinctive variation in red cell density (MCHC) as determined by separation of red cells on isopycnic gradients: (a) All red cells from patients homozygous for HbOARAB were denser than normal red cells, as is observed for homozygous HbC patients. (b) In patients heterozygous for HbOARAB, red cell density was strongly influenced by the presence of α‐thalassaemia. The coexistence of −α/αα resulted in an average red cell density slightly greater than normal (AA) red cells. Patients heterozygous for HbOARAB with a normal complement of four α genes had denser red cells similar to sickle cell disease with some cells of normal density but with most cells very dense. (c) Finally, the double heterozygotes for HbS and HbOARAB had significant haemolytic anaemia and red cells denser than normal with some as dense as the densest cells found in sickle cell anaemia. (2) Reticulocytes in patients homozygous for HbOARAB were found in the densest density fraction of whole blood. (3) Cation transport in patients homozygous for HbOARAB was abnormal, with K:Cl cotransport activity similar to that of HbS‐Oman and only somewhat lower than in sickle cell anaemia red cells. The activity of the Gardos channel was indistinguishable from that found in HbS, HbC and HbS‐Oman cells.

We conclude that the erythrocytic pathogenesis of HbOARAB involves the dehydration of red cells due, at least in part, to the K:Cl cotransport system. The similarity of the charge and consequences of the presence of both HbC and HbOARAB, which are the products of mutations at opposite ends of the β‐chain, raises the possibility that this pathology is the result of a charge‐dependent interaction of these haemoglobins with the red cell membrane and/or its cytoskeleton and that this abnormality is present early in red cell development.
Variable inhibition of placental IgG transfer in vitro with commercial IVgG preparations
Authors: Urbaniak S. J. , Duncan J. I. , Armstrong‐Fisher S. S. , Abramovich D. R. , Page K. R.
Published: March 2003

Maternal administration of high‐dose intravenous immunoglobulin (IVIgG) for treating fetal RhD haemolytic disease and alloimmune thrombocytopenias may be beneficial. Treatment failures, even when IVIgG is used optimally, may result from product differences. Using an in vitro placental perfusion model there was significant inhibition of placental anti‐D IgG transfer with three commercial IVIgG preparations where circulating maternal IgG concentrations were > 20 g/l. One IVIgG product, which was not inhibitory, had lower circulating IgG levels (16.5 ± 0.9 g/l) and significantly reduced placental transfer of total IgG, suggesting that the reduced functional activity of IgG from IVIgG preparations may correlate with poor clinical efficacy.
Flow cytometric detection of CD10 (cALLA) on peripheral blood B lymphocytes of neonates
Authors: Brady Katharine A. , Atwater Susan K. , Lowell Clifford A.
Published: March 2003

CD10 (cALLA) was detected on the surface of CD19‐positive circulating lymphocytes in the peripheral blood of 32/50 neonates tested. These cells are presumed to represent immature B cells, commonly referred to as haematogones, previously undescribed in peripheral blood. The CD10+/CD19+ cells expressed lower levels of CD22 consistent with these cells being immature B lymphocytes. The presence of CD10+/CD19+ cells in the blood was not significantly correlated with a leucoerythroblastic picture, adjusted gestational age, or the presence in blood smears of medium‐to‐large lymphocytes with an immature appearance that morphologically resembled classic bone‐marrow haematogones.
P‐glycoprotein and multidrug resistance‐associated protein, but not lung resistance protein, lower the intracellular daunorubicin accumulation in acute myeloid leukaemic cells
Authors: Borg Anton G. , Burgess Robert , Green Linda M. , Scheper Rik J. , Liu Yin John A.
Published: March 2003

The in vitro intracellular daunorubicin accumulation (IDA) of blast cells from 69 patients with newly diagnosed acute myeloid leukaemia (AML) was correlated with the expression and functional activity of the multidrug resistance (MDR) proteins, P‐glycoprotein (Pgp), multidrug resistance‐associated protein (MRP) and lung‐resistance protein (LRP). An inverse and significant association was found between IDA and Pgp‐related efflux activity (r = −0.31, P = 0.01) and also MRP (r = −0.25, P = 0.04) but not with LRP (r = −0.13, P = 0.28). Coexpression of the MDR proteins had an additive effect in further lowering of IDA levels, suggesting that the clinical MDR phenotype is dependent on the sum of multiple MDR factors available to the leukaemic cell. Thus, the median IDA of leukaemic cells without any MDR proteins was significantly higher than that of blasts carrying two MDR proteins (0.466 vs. 0.296, P = 0.046). Seven patients with no expression of Pgp, MRP and LRP still had low IDA levels, suggesting the presence of efflux MDR mechanisms other than those studied. The relation of IDA to clinical parameters known to be associated with poor prognosis, such as age, secondary AML, karyotype, peripheral blood blast and CD34 counts, was also studied, but no significance was found on multifactorial analysis. There was a non‐significant trend for earlier relapse in patients with low IDA levels (leukaemia‐free survival of 16.3 months compared with 21.1 months in patients with high IDA levels). Our data suggest that, while the IDA assay is a quick and relatively easy test for the combined efflux MDR phenotype, it is unable to detect other MDR mechanisms, such as LRP, which may be important to the clinical outcome of patients with AML.
Constitutive activation of FLT3 in acute myeloid leukaemia and its consequences for growth of 32D cells
Authors: Fenski  , Flesch  , Serve  , Mizuki  , Oelmann  , Kratz‐Albers  , Kienast  , Leo  , Schwartz  , Berdel  , Serve
Published: March 2003

The receptor tyrosine kinase Flt3 is expressed on leukaemic blasts of most cases with acute myeloid leukaemia (AML). In order to evaluate the presence and significance of constitutive activation of Flt3 for leukaemogenesis, we (1) analysed the expression and activation status of the receptor in AML blasts; and (2) evaluated the functional consequences of constitutively active Flt3 in a myeloid progenitor cell line. Immunoprecipitation studies revealed Flt3 expression in a high proportion of AML cases (27/32) with ligand‐dependent Flt3 autophosphorylation in 18, constitutive autophosphorylation in three and no autophosphorylation in six cases. Only one out of three samples with constitutively active Flt3 but 3/18 samples with ligand‐dependent autophosphorylated Flt3 contained the recently described internal tandem repeat (ITR) mutations. To test the significance of Flt3 activation in myeloid cell function, we also characterized the biochemical and biological effects of the activating mutation D838V of Flt3 (FLt3D838V) on the factor‐dependent myeloid progenitor cell line 32Dcl3: cells transfected with wild‐type Flt3 (32D/Flt3) grew FLt3 ligand (FL) dependent, and the receptor was ligand dependently autophosphorylated. In contrast, the receptor was constitutively autophosphorylated in 32D/Flt3D838V cells, which grew independently of FL. We conclude that, in some AML samples, Flt3 is constitutively activated and that this does not correlate with ITR mutations in the juxtamembrane domain. Furthermore, constitutively active Flt3 confers factor independence to the myeloid progenitor cell line 32D. It remains to be determined whether activation of Flt3 is leukaemogenic in vivo and whether strategies aimed at inhibition of Flt3 activation could inhibit leukaemogenesis.
Splenectomy in haemophagocytic lymphohistiocytosis: report of histopathological changes with CD19+B‐cell depletion and therapeutic results
Authors: Imashuku Shinsaku , Obayashi Mikihisa , Hosoi Gaku , Sako Masahiro , Chen Jiming , Mugishima Hideo , Tsunamoto Kentaro , Hibi Shigeyoshi , Todo Shinjiro
Published: March 2003

The pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in children without a known familial pattern of inheritance is often difficult to establish. Splenic enlargement, one of the main clinical findings in this disorder, has led to the use of splenectomy for uncontrollable coagulopathy, persistent cytopenia or both. This procedure is also thought to be a useful tool in making a differential diagnosis in cases of the immunochemotherapy‐resistant HLH. We report here five cases of splenectomized childhood HLH, in which subsets of mononuclear spleen cells were analysed either by flow cytometry or immunohistochemistry, and the results were compared with those from cases of hereditary spherocytosis (controls). There was a statistically significant depletion of CD19+B cells in the HLH cases (3.8 ± 3.2% vs. 52.6 ± 4.5%,P < 0.0001) associated with an increase of T cells in three cases and of natural killer cells in another. The histopathological findings included atrophic white pulps, B‐cell depletion with fibrosis and haemosiderosis in all five cases. Despite temporary therapeutic benefits, three of the HLH patients had a rapidly deteriorating post‐splenectomy course and all three eventually died. These results demonstrate striking depletion of B cells in the enlarged spleens of children with HLH, which may be an intrinsic feature of HLH pathogenesis. Further study is needed to establish the therapeutic value of splenectomy in this disease.
Extraordinary bone involvement in a Gaucher disease type I patient
Authors: Barone R. , Pavone V. , Nigro F. , Chabàs A. , Fiumara A.
Published: March 2003

We report on a 63‐year‐old patient with Gaucher disease type I who developed severe bone involvement with destructive lesions and huge soft tissue extension in both humeri that appeared to evolve slowly. The clinical course and histopathological findings in our patient suggested a progressive extraosseous extension of the storage cells into the soft tissue, accompanied by a striking increase of fibrotic tissue and resulting in an impressive deformity.

The extraordinary bone involvement in this patient expands our knowledge on the most severe skeletal complications of untreated Gaucher disease.
Proliferative involvement of ENX‐1, a putative human Polycomb group gene, in haematopoietic cells
Authors: Fukuyama Tomofusa , Otsuka Teruhisa , Shigematsu Hirokazu , Uchida Naoyuki , Arima Fumitou , Ohno Yuju , Iwasaki Hiromi , Fukuda Takahiro , Niho Yoshiyuki
Published: March 2003

Homeobox genes have important roles in haematopoiesis and are regulated in an activated state by the trithorax group (trxG) of genes. In a repressed state, they are regulated by the Polycomb group (PcG) of genes. ENX‐1, a putative human PcG gene product, interacts with the proto‐oncogene product Vav. We report an investigation of the role of ENX‐1 in human haematopoiesis. CD34+ cells mobilized to peripheral blood strongly expressed ENX‐1. When stimulated to proliferate, both T and B lymphocytes rapidly up‐regulated ENX‐1. ENX‐1 was expressed in all cell lines of the various lineages examined. When HL‐60 cells were differentiated to mature granulocytes with all‐trans retinoic acid, ENX‐1 was down‐regulated. Moreover, ENX‐1 antisense oligodeoxynucleotide suppressed DNA synthesis in HL‐60 cells. Our data indicate that ENX‐1 is involved in the proliferation of both normal and malignant haematopoietic cells.
Characterization of lineage‐specific chimaerism in patients with acute leukaemia and myelodysplastic syndrome after allogeneic stem cell transplantation before and after relapse
Authors: Bader P. , Stoll K. , Huber S. , Geiselhart A. , Handgretinger R. , Niemeyer C. , Einsele H. , Schlegel P. G. , Niethammer D. , Beck J. , Klingebiel T.
Published: March 2003

Recently, we have shown that patients with acute leukaemias and myelodysplastic syndromes (MDS), who showed increasing mixed chimaerism (MC) upon serial PCR analysis after transplant, have a significantly increased risk of relapse. To determine whether the increasing MC in these patients is caused by the reappearance of normal recipient haematopoiesis or by the reoccurrence of malignant cells, we purified different leucocyte subpopulations and analysed these subfractions with regard to their donor–recipient ratio by a PCR‐based method for the analysis of minisatellite DNA regions. In 14 patients [eight acute lymphoblastic leukaemia (ALL), three acute myelogenous leukaemia (AML) and three MDS] subfractions were analysed when increasing MC was first noted upon serial analysis of the peripheral blood. In seven of these 14 patients (four ALL, two AML and one MDS), subfractions were characterized at the time of frank haematological relapse. In all 14 patients investigated with increasing MC, recipient cells were detected in different mononuclear cell subpopulations. In patients characterized during frank relapse, two distinct distribution patterns were found. Patients who relapsed before day +300 (one ALL, two AML and one MDS) showed recipient‐derived (normal) cells in addition to blast populations in different mononuclear subsets as well as granulocytes. In patients with acute leukaemias who relapsed after day +300 (two ALL and one AML), only leukaemic cells were found that were of recipient origin, whereas all other haematopoietic cell lines were donor derived.

These data show that persistent MC in the early post‐transplant period is caused predominantly by normal recipient haematopoietic cells. This finding further supports the hypothesis that a state of mixed haematopoietic chimaerism may reduce the clinical graft‐versus‐leukaemia (GVL) effect of alloreactive donor‐derived effector cells in patients with acute leukaemias and MDS, and thus facilitate the proliferation of residual malignant cells that may have survived the preparative regimen.
Two types of acquired idiopathic sideroblastic anaemia (AISA): a time‐tested distinction
Authors: Germing U. , Gattermann N. , Aivado M. , Hildebrandt B. , Aul C.
Published: March 2003

In 1982, acquired idiopathic sideroblastic anaemia (AISA) was included by the French–American–British (FAB) Co‐operative Group in their classification of myelodysplastic syndromes (MDS). However, the malignant potentiality of AISA has always been a matter of debate. In different series, median survival and rates of transformation into acute myeloid leukaemia (AML) varied extensively. On cytomorphological grounds, AISA can be divided into pure (dyserythropoietic) sideroblastic anaemia (PSA), in which dysplasia is confined to erythropoietic cells, and a true myelodysplastic form (RARS), which is characterized by additional dysplastic features of granulopoiesis and/or megakaryopoiesis. In a previous study, based on retrospective analysis of 94 patients with AISA, we found that both types of sideroblastic anaemia differed considerably in terms of survival and risk of AML transformation. Almost identical results have now been obtained through a prospective study of 232 new patients with AISA. The difference in survival between PSA and RARS remained significant over the whole period of follow‐up (survival after 3 years being 77% vs. 56%; P = 0.003), and the incidence of AML did not increase with time in the PSA group, even in the long term. This prospective study strongly supported our conclusion that cytomorphological distinction between PSA and RARS provides valuable prognostic information.
Persistent mediastinal mass is not indicative of recurrence after chemotherapy only in paediatric Hodgkin's disease
Authors: Van Den Berg H. , Verhulst L. , Behrendt H. , Staalman C. R.
Published: March 2003

Most patients with Hodgkin's disease are treated with chemotherapy in conjunction with radiotherapy, but at the end of treatment a residual mass is often present. After combined therapy, it has been assumed that no additional treatment is needed. However, for children treated without radiotherapy, no data exist on the relevance of a residual mediastinal mass to risk of relapse. We report on the findings of follow‐up thorax radiographs of a group of 27 children with initial mediastinal involvement, who were treated with chemotherapy only. We conclude that the regression rate of the mediastinal mass was not related to a later recurrence. Regression after chemotherapy without radiotherapy is probably slower than after combined therapy. We consider chest radiograph examinations to be appropriate for the follow‐up of tumour regression. When the data were compared with a group of children with Hodgkin's disease without mediastinal involvement, we found that survival was not related to initial mediastinal involvement.