bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations of the p53 tumour suppressor gene in haematologic neoplasms
Authors: Mori Naoki , Wada Makio , Yokota Jun , Terada Masaaki , Okada Michiko , Teramura Masanao , Masuda Michihiko , Hoshino Shigeru , Motoji Toshiko , Oshimi Kazuo , Mizoguchi Hideaki
Published: March 2008

Mutations of the p53 tumour suppressor gene have frequently been observed in several types of solid tumours and are believed to be implicated in the development of these tumours. To determine the relevance of p53 mutations in haematologic neoplasms, we performed polymerase chain reaction‐single strand conformation polymorphism analysis on the p53 gene in 45 patients with various types of haematologic neoplasms. In exons 5–8 containing highly conserved regions, mobility shifts indicating sequence alterations were detected in four of the 45 patients, and subsequent sequencing was performed. A point mutation resulting in a novel stop codon was detected at codon 213 in one of 23 cases of chronic myelogenous leukaemia (one of five cases of blast crisis). Point mutations causing amino acid substitutions were detected in one of four cases of myelodysplastic syndrome at codon 195, one of three cases of adult T‐cell leukaemia at codon 281, and one of eight cases of acute lymphoblastic leukaemia at codon 281, and these missense mutations were accompanied by loss of the wild type allele. Patients harbouring these nonsense and missense mutations were in advanced disease stages. These findings suggest that mutational inactivation of the p53 gene is infrequent but is involved in the tumorigenesis of several types of haematologic neoplasms at least in some cases.
Electrophoretic and functional characteristics of the von Willebrand factor in human fetal plasma
Authors: Mazurier Claudine , Daffos Fernand , Forestier Francois
Published: March 2008

Although the haemostatic role of von Willebrand factor (vWF) in adults is well known, there is little information currently available about its possible contribution to in utero haemostasis. We have investigated the distribution of vWF multimers in 27 pure fetal platelet‐poor plasma (FP) samples aged from 20 to > 36 weeks, by using electrophoresis in both low‐and high‐resolution agarose gels in the presence of sodium dodecyl sulphate. Our data confirm that most FP samples contain higher molecular weight (HMW) vWF multimers than those present in normal adult plasma, the proportion of these HMW forms being lower in FP samples aged >36 weeks. However, we found that the multimeric unit and the protomeric form of vWF were similar in normal fetal and adult plasma. Functional assays of vWF were performed on three pooled FP samples. In all cases, fetal vWF was able to interact with factor VIII and to bind to GP Ib platelet receptor in the presence of ristocetin and to types I and III collagen. These results indicate that plasma vWF has already acquired in fetal life the functional activities required for its role in both coagulation and primary haemostasis.
Further characterization of the NB 1 antigen as a variably expressed 56–62 kD GPI‐linked glycoprotein of plasma membranes and specific granules of neutrophils
Authors: Goldschmeding R. , van Dalen C. M. , Faber N. , Calafat J. , Huizinga T. W. J. , van der Schoot C. E. , Clement L. T. , von dem Borne A. E. G. Kr.
Published: March 2008

The human neutrophil‐specific alloantigen NB1 was identified as a glycosyl‐phosphatidylinositol (GPI)‐anchored N‐glycosylated protein of Mr 56–62 kD under reducing conditions. Under non‐reducing conditions its Mr was 49–55 kD. This glycoprotein antigen was found to be expressed by only a subpopulation of normal donor neutrophils, and could not be detected on other blood cells. The allotypic epitope recognized by human anti‐NB1 IgG was also recognized by the mouse monoclonal antibody 1B5. The percentage of neutrophils stained by these antibodies varied greatly among healthy donors (range 0–100%). When 16 donors were repeatedly tested, the NB1‐positive neutrophil fraction appeared to remain remarkably constant over time in most donors, but significant fluctuations were seen in some. NB1 antigen was found to be expressed not only on the plasma membrane, but also intracellularly on the membranes of small vesicles and specific granules. The neutrophils which expressed NB1 antigen on the plasma membrane were the same as those with intracellular expression of this antigen. Crosslinking of NB1 antigen on the plasma membrane with monoclonal antibody 1B5 and goat‐anti‐mouse Ig resulted in internalization of the complex, while in‐vitro stimulation of neutrophils caused an increase of the intensity of plasma membrane staining with anti‐NB1, but only of those cells that were positive already prior to stimulation. The NB1 glycoprotein thus appears to identify a distinct subset of neutrophils, the size of which greatly varies among healthy donors. The function of the NB1 glycoprotein remains unclear, but its behaviour upon crosslinking and chemotactic peptide stimulation suggests a possible role as receptor molecule.
Acute myeloid leukaemia with an unusual phenotype: myeloperoxidase (+), CD13 (‐), CD14 (‐) and CD33 (‐)
Authors: Seshi Beerelli , Kashyap Ashwin , Bennett John M.
Published: March 2008

We herein describe an unusual case of acute myeloid leukaemia (AML) showing strong cytochemical reactivity for myeloperoxidase (MPO) but surprisingly no reactivity using flow cytometry for any of the lineage‐specific cell surface markers, i.e. myelomonocytic antigens CD13, CD14 and CD33: or B‐lymphoid antigens CD19, CD20 and immunoglobulins: or T‐lymphoid antigens CD2, CD3 and CD 5. The strong reactivity for MPO and the complete absence of reactivity for CD13 and CD14 was verified by an independent assay involving alkaline phosphatase‐anti‐alkaline phosphatase (APAAP). Our case is of interest for at least two reasons: First, a poorly differentiated variant of AML (negative for MPO but positive for one or more of the myeloidlineage CD antigens) has been designated FAB Mo. In terms of the expression of phenotypic markers, our case may be considered as an ‘MPO (+), CD antigen (‐) AML’. The CD antigens are known to be expressed very early during myeloid differentiation whereas MPO (in its functional form) is viewed as being expressed relatively late in the process. It is therefore intriguing from a biological standpoint why the supposedly early antigens (CD33 and CD13) remain unexpressed: this may represent an example of ‘asynchronous differentiation’in leukaemia. Second, from a practical standpoint, the use of immunophenotyping as a first‐line diagnosis would fail to detect such cases. This case strengthens the notion that immunophenotyping by flow cytometry does not eliminate the necessity of performing peroxidase cytochemical staining.
Differential action of diffusible molecules in long‐term marrow culture on proliferation of leukaemic and normal haemopoietic cells
Authors: Firkin Frank C. , Birner Raquella , Farag Sherif
Published: March 2008

Summary. The capacity of diffusible molecules in the fluid phase of long‐term human bone marrow culture (LTMC) to exert preferential adverse effects on leukaemic relative to normal haemopoietic cells has been investigated. Responses of isolated cell populations were assessed in diffusion chamber inserts which permitted contact with fluid phase molecules but not with the adherent stromal cell layer of the LTMC system. Growth of AML cells in diffusion chambers was inhibited during co‐culture with LTMC of autologous leukaemic bone marrow, and the same effect was produced during co‐culture with normal LTMC. No inhibitory action was exerted on growth of normal haemopoietic precursors under the same conditions. Comparable responses were observed with human leukaemic cell lines and patient leukaemic cells, and studies on cell lines demonstrated inhibition of growth was induced by molecules generated in LTMC which caused accumulation in G1 phase of leukaemic cells of both myeloid and lymphoid lineage. The inhibitory effect was not reproduced by TGFβ, IFNγ, IFNα, TNFα, LIF, SCF or II‐6, and was not impaired by inhibitors of nitric oxide or PGE production in the LTMC. These observations suggest the action of diffusible molecules of undefined constitution contributes to the preferential loss of leukaemic cells in LTMC.
Bone marrow transplantation for acute myeloblastic leukaemia: an EBMT Leukaemia Working Party prospective analysis from HLA‐typing
Authors: Ljungman Per , de Witte Theo , Verdonck Leo , Gahrton Gösta , Freycon Fernand , Gravett Peter , McCann Shaun , Morgenstern H. G. , Nikoskelainen Jukka , Powles Ray , Proctor Steven J. , Volin Liisa , Zwaan Ferry , Gratwohl Alois
Published: March 2008

Summary. The optimal post‐remission therapy for patients with acute myeloblastic leukaemia remains controversial. Allogeneic bone marrow transplantation, autologous bone marrow transplantation, and consolidation chemotherapy are the major options. In order to evaluate their respective value the European Group for Bone Marrow Transplantation conducted a prospective registration study. Patients with newly diagnosed acute myeloblastic leukaemia were registered at the time of HLA‐typing and intention to treat in case of presence or absence of an HLA‐identical donor was recorded. 27/79 (34%) patients HLA‐typed at diagnosis had an identical donor identified. The estimated survivals at 3 years from HLA‐typing were 44% and 21% among patients with or without HLA‐identical donor, respectively (P= 0·02). 22/26 (85%) patients for whom allogeneic bone marrow transplantation was intended were transplanted but only 15/47 (32%) patients for whom autologous bone marrow transplantation was intended were indeed transplanted (P < 0·001). The survival was 50%. 29% and 17% (P= 0·004) for patients treated with allogeneic bone marrow transplantation, autologous bone marrow transplantation, or chemotherapy, respectively. 40/68 patients HLA‐typed in first complete remission had an HLA‐identical donor. The estimated 3‐year survival among patients typed in first remission with and without HLA‐identical donors was 42% and 35% (n.s.), respectively. This technique of early patient registration illustrates the problems of patient selection during the course of the disease and might be used as a complement to randomized trials when comparing bone marrow transplantation and other treatment options.
Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia in first chronic phase: correlations with acute graft‐versus‐host disease and relapse
Authors: Cross Nicholas C. P. , Hughes Timothy P. , Feng Lin , O'Shea Paul , Bungey Julie , Marks David I. , Ferrant Augustin , Martiat Philippe , Goldman John M.
Published: March 2008

Summary. We have studied 61 patients who underwent allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML) in first chronic phase. Minimal residual disease was detected by the amplification of the leukaemia‐specific BCR‐ABL fusion mRNA with the polymerase chain reaction (PCR) using a highly sensitive nested primer strategy. As a general pattern, patients often had detectable BCR‐ABL (PCR positive) for up to 6 or 9 months post BMT after which time BCR‐ABL became undetectable (PCR negative). The conversion from PCR positive to PCR negative was not associated with the time at which cyclosporin A treatment was stopped. Six patients (10%) have relapsed during the period of this study, two within 1 year and four more than 1 year after transplant. The relationship between PCR positivity more than 1 year post transplant and relapse was significant (P= 0·036) but 15 patients who were PCR positive beyond 1 year remain in complete clinical and cytogenetic remission. Thus late positivity identifies a group of patients at increased risk of relapse but is of little predictive value for individual patients. Of the four late relapses, two had been persistently PCR positive and two were initially PCR positive, converted to negative and subsequently to positive again. Although all relapses were preceded by PCR positivity, relapse may occur only 12 months after a PCR negative result. The proportion of patients PCR negative at 3/4 months after BMT was found to increase significantly with the severity of acute GVHD (P= 0·002) but no relationship was found between acute GVHD and subsequent PCR results. There was no clear association between severity of chronic GVHD and PCR result.
An MseI RFLP in the 5’flanking region of the factor IX gene: its use for haemophilia B carrier detection in Caucasian and Thai populations
Authors: Winship P. R. , Nichols C. E. , Chuansumrit A. , Peake I. R.
Published: March 2008

Summary. We have identified an MseI restriction fragment length polymorphism (RFLP) in the 5’flanking region of the factor IX gene. RFLPs in the factor IX locus are routinely used as linkage markers to track defective factor IX genes through affected pedigrees, allowing diagnosis of the haemophilia B carrier state in female members of the kindred. Currently, seven intragenic polymorphic loci have been characterized allowing carrier diagnosis in 89% of cases in the Caucasian population. Additional screening with the MseI RFLP reported in this publication increases this figure to 94% of cases. In Asian populations only one of these RFLPs (HhaI) is present at any significant frequency. Hence, carrier detection rates are much reduced in comparison to the corresponding figure of 89% in Caucasians. We report that this MseI RFLP is also present in the Thai population. Indeed, when used in combination with the HhaI polymorphism, the MseI RFLP should significantly improve the carrier detection rate in the Thai population from 11% to 40% of cases.
Clonal haematopoiesis in children with acquired aplastic anaemia
Authors: Tsuge Ikuya , Kojima Seiji , Matsuoka Hiroshi , Abe Tokuichiro , Kamachi Yoshiro , Torii Shinpei , Matsuyama Takaharu
Published: March 2008

Summary. The methylation pattern of three X‐linked genes, phosphoglycerate kinase (PGK), hypoxanthine phosphoribosyl transferase (HPRT) and DXS255 detected by hypervariable M27β probe, was analysed to determine the proportion of aplastic anaemia (AA) with clonal haematopoiesis in Japanese children. Methylation analysis was performed on DNA from separated granulocytes and compared to that of bone marrow derived fibroblasts to exclude selective lyonization in all somatic cells. Of 20 female patients examined, the methylation pattern of at least one gene was informative in granulocyte DNA from 18 patients (90%). Of these, 8/20 patients (40%) were heterozygous for PGK, 8/18 (44%) were heterozygous for HPRT and 17/18 (94%) were heterozygous for DXS255. In 14/18 patients both alleles were equally methylated. Four patients exhibited a unilateral methylation pattern in their granulocytes. The same unilateral pattern was again demonstrated in fibroblasts from two of the four patients suggesting that in the latter one X chromosome was selectively inactivated in all of the somatic cells. The remaining two patients showed a unilateral methylation pattern that was restricted to their granulocytes, suggesting the existence of true clonal haematopoiesis. They responded well to antilymphocyte globulin (ALG) and presently have no evidence of a clonal disorder such as myelodysplastic syndrome (MDS) or paroxysmal nocturnal haemoglobinuria (PNH). Although these results indicate that some children with AA exhibit clonal haematopoiesis, analysis of a greater number of subjects will be required to establish the clinical value of clonal haematopoiesis in patients with AA.
Peripheral blood lymphocytes express the platelet‐type thrombin receptor
Authors: Howells Gareth L. , Macey Marion , Curtis Michael A. , Stone Stuart R.
Published: March 2008

Summary. Northern blot analysis of human mononuclear cells indicated that the platelet thrombin receptor may be expressed by lymphocytes. In order to investigate this, we prepared affinity purified rabbit antibodies against the thrombin receptor which bound platelets and blocked thrombin activation. Using flow cytometry on peripheral blood cells, we found that the vast majority of NK cells (CD16/CD56 positive) and a fraction of CD3/CD4 positive T cells expressed the thrombin receptor. B cells, neutrophils and monocytes were negative. These data suggest that potentially thrombin may play a direct role in regulating NK and T cell function.
Successful childbirth by a patient with congenital factor XI deficiency and an acquired inhibitor
Authors: Ginsberg Steven S. , Clyne Lionel P. , McPhedran Peter , Duffy Thomas P. , Hanson† T.
Published: March 2008

Summary. Acquired inhibitors in factor XI deficiency (FXI) are rare. The presence of an inhibitor during pregnancy poses a potential haemorrhagic risk to the fetus. We report an uncomplicated pregnancy and successful childbirth by a woman with congenital FXI deficiency and an acquired inhibitor, and discuss the persistence of residual FXI activity in the presence of an inhibitor.
The type of BCR/ABL junction does not predict the survival of patients with Ph1‐positive chronic myeloid leukaemia
Authors: Zaccaria Alfonso , Martinelli Giovanni , Buzzi Marina , Testoni Nicoletta , Farabegoli Patrizia , Zuffa Eliana , Zamagni Maria Donatella , Russo Domenico , Baccarani Michele , Ambrosetti Achille , Guerrasio Angelo , Saglio Giuseppe , Tura Sante
Published: March 2008

Summary. The prognostic value of the location of the breakpoint on chromosome 22 in patients with Ph 1+ chronic myeloid leukaemia (CML) is still controversial. We analysed both DNA rearrangement and transcript type in a new continuous series of CML patients. By Southern blotting analysis, we found that, out of 72 patients, 43 had a 5’rearrangement and 29 a 3’one, of the 43 5′‐rearranged patients, 35 carried an a2b2 transcript and eight an a2b3 one, while, of the 29 patients rearranged in the 3’part of the M‐BCR area, 26 had an a2b3 transcript, one had an a2b2 transcript and two carried both types of transcript. Thus, mRNA studies allow to detect an a2b3 transcript in 17·7% of 5’rearranged patients. However, no correlation was observed between type of transcript and survival, as after DNA studies.
Symptomatic type II protein C deficiency caused by a missense mutation (Gly 381 → Ser) in the substrate‐binding pocket
Authors: Marchetti G. , Patracchini P. , Gemmati D. , Castaman G. , Rodeghiero F. , Wacey A. , Cooper D. N. , Tuddenham E. G. D. , Bernardi F.
Published: March 2008

Summary. A patient with recurrent deep vein thrombosis and heterozygous type II deficiency, characterized by reduced protein C activity in both amidolytic and clotting functional assays, was investigated by direct sequencing of PCR fragments derived from the coding portion of the protein C gene. A G (8856) to A transition was noted in the patient which was not present in healthy controls. This mutation is predicted to cause the substitution of Ser for Gly 381, an evolutionarily conserved residue in the substrate binding pocket of serine‐proteases (Gly 216, chymotrypsin numbering). A computer model of the structure of the serine‐protease domain indicates that the properties of the altered protein C molecule can be explained on the basis of steric hindrance between the substituted serine and the substrate arginine side chains.
The clinical significance of serum transferrin receptor levels in sickle cell disease
Authors: Singhal Atul , Cook James D. , Skikne Barry S. , Thomas Peter , Serjeant Beryl , Serjeant Graham
Published: March 2008

Summary. Serum transferrin receptor (TfR) levels were measured in 182 children with homozygous sickle cell (SS) disease, 47 with sickle cell–haemoglobin C (SC) disease and 41 normal (AA) controls on their eighth birthday. Highly significant elevations occurred in SS compared to SC disease and in SC disease compared to AA controls. Females had higher levels than males in controls but lower levels than males in SS and SC disease. In SS disease, serum TfR levels tended to rise with age from 2 to 8 years, the change within individuals correlating with a change in reticulocyte count (r,=0·38, P=0·017) and fall in fetal haemoglobin levels (r,=−0·51, P= 0·004). Serum TfR levels did not change with infection or painful crisis but were markedly elevated in hypersplenism and fell following splenectomy in these subjects. In the aplastic crisis, serum TfR levels tended to rise following clinical presentation and then fall, reflecting the reticulocyte counts. These observations are consistent with serum TfR levels being a useful indicator of the degree of erythropoietic expansion in sickle cell disease.
Usefulness of the rearrangement of the bcr/abl gene in extramedullary (lymph nodes) blast crisis diagnosed in chronic myeloid leukaemia
Authors: González F. A. , Villegas A. , Ferro M. T. , Cabello P. , Morales D. , Perez J. , Martinez R.
Published: March 2008

Summary. The introduction of molecular biological techniques in the study of chronic myeloid leukaemia (CML) allows us to show the bcr/abl gene rearrangement produced by the translocation between the c‐abl proto‐oncogene located in chromosome 9 and the bcr region located in chromosome 22, which constitutes the molecular alteration of Philadelphia chromosome in CML. We present the usefulness of the bcr/abl gene rearrangement study in the diagnosis of a blast crisis initially located in lymph nodes of a patient with CML. The DNA analysis allows demonstration that the lymph node neoplastic cells originate from the clone responsible for the CML, while obtaining metaphases from a lymph node for the cytogenetic study gives rise to enormous difficulties and is practically impossible if the problem is studied retrospectively based on frozen or fixed samples.
Interactions between purified human cord blood haemopoietic progenitor cells and accessory cells
Authors: Cicuttini F. M. , Loudovaris M. , Boyd A. W.
Published: March 2008

Summary. The role of accessory cells in haemopoiesis remains confused. This appears in large part to reflect the use of impure populations of accessory cells and progenitor cells in previous studies. In this study, cell sorter purified populations of both accessory cells and haemopoietic progenitor cells were used to examine interactions between these cell types. We used a double culture protocol in which purified CD34+ cells were cultured with purified NK, T or monocytic cells in the first liquid culture phase after which the cells were transferred to secondary agar cultures to determine the number of colony forming cells (CFC). NK cells co‐cultured with CD34+ cells resulted in an increased number of erythroid progenitors with no effect on the number of non‐erythroid progenitors. In contrast, there were increased numbers of erythroid and non‐erythroid CFC when the CD34+ cells were co‐cultured with either purified T cells or monocytes. CD34+ cells cultured with cell‐conditioned media derived from NK cells, T cells or monocytes in transwells where the CD34+ cells and the accessory cells were separated by a 0·2 μm membrane, showed no enhancement in CFC. These results suggest that intimate cell–cell contact is required for these events.
The resistance of macrophage‐like tumour cell lines to growth inhibition by lipopolysaccharide and pertussis toxin
Authors: Xie Yue , von Gavel Stephanie , Cassady A. Ian , Stacey Katryn J. , Dunn Timothy L. , Hume David A.
Published: March 2008

Summary. The process of tumorigenesis is frequently associated with resistance to growth inhibition by physiological regulators of normal cells. Murine macrophage‐like cell lines BAC1.2F5, RAW264, J774.1A and PU5/1.8 were resistant to growth inhibition by bacterial lipopolysaccharide (LPS) and pertussis toxin, agents that blocked growth of primary bone marrow‐derived macrophages (BMDM) in the presence of macrophage colony‐stimulating factor (CSF‐1). The resistance of the CSF‐1‐dependent cell line BAC1.2F5 to growth inhibition by pertussis toxin argues against the possibility that pertussis toxin‐sensitive G proteins are essential for the pathway of growth stimulation by CSF‐1. Conversely, these data add further weight to the argument that LPS mediates some of its biological activities by mimicking the action of pertussis toxin and inhibiting G protein function. The resistance of cell lines to LPS and pertussis toxin was not correlated with any alteration in the expression of mRNA encoding any of three pertussis‐toxin sensitive G protein α subunits. The pattern of G protein expression was consistent between primary cells and tumour cells, suggesting that this is a differentiation marker. In particular, Giα2 mRNA was expressed at remarkably high levels in all of the cells. The specificity of LPS resistance was investigated by studying down‐regulation of CSF‐1 binding and induction of protooncogene c‐fos and tumour necrosis factor (TNF) mRNA. BAC1.2F5 cells were LPS‐resistant in each of these assays. In CSF‐1 binding. RAW264 and J774.1A responded in the same way as bone marrow‐derived macrophages but required higher doses of LPS, whereas c‐fos and TNF mRNA were induced in these cells at concentrations that did not inhibit growth.

In PU5/1.8 cells, CSF‐1 binding was already very low and was not further down‐regulated, but c‐fos and TNF mRNA was inducible by LPS. By contrast to primary macrophages, the cell lines did not respond to LPS with down‐regulation of c‐fms mRNA, which encodes the CSF‐1 receptor. Hence, the resistance of macrophage‐like tumour cells to LPS and pertussis toxin was specific to the pathways controlling growth, and was correlated with altered regulation of the CSF‐1 receptor.
Peripheral blood progenitor cells mobilized by chemotherapy plus granulocyte‐colony stimulating factor accelerate both neutrophil and platelet recovery after high‐dose VP16, ifosfamide and cisplatin
Authors: Brugger Wolfram , Birken Rainer , Bertz Hartmut , Hecht Thomas , Pressler Klaus , Frisch Jürgen , Schulz Gregor , Mertelsmann Roland , Kanz Lothar
Published: March 2008

Summary. We report on the chemotherapy plus granulocyte colony‐stimulating factor (G‐CSF) induced mobilization of peripheral blood progenitor cells (PBPCs) and their impact on haematopoietic recovery following high‐dose chemotherapy. Twenty‐four patients with advanced solid tumours or lymphomas received standard‐dose chemotherapy with VP16, ifosfamide and cisplatin (VIP) followed by filgrastim (G‐CSF; 5 μg/kg s.c. daily for 14 d) for the prevention of chemotherapy induced neutropenia and for the simultaneous mobilization of PBPCs. Maximal numbers of progenitors of different lineages were reached at day 11 (range 9–14) after VIP chemotherapy. A median of 0·415 × 109/1 CD34+ cells (range 0·11–1·98), 9000 CFU‐GM/ml (range 2800–17700). 3500 BFU‐E/ml (range 400–10800) and 200 CFU‐GEMM/ml (range 0–4400) were recruited. One single apheresis yielded a median of 1·6 × 108 mononuclear cells/kg (range 0·2–5·4) or 5·4 × 106 CD34+ cells/kg body weight (range 0·2–24·2).

Fourteen patients who showed at least a partial remission after two cycles of the standard‐dose chemotherapy regimen were subjected to high‐dose VIP chemotherapy (cumulative doses of 1500 mg/m2 VP16, 12 g/m2 ifosfamide and 150 mg/m2 cisplatin) with or without PBPC support. The first six patients were treated with growth factors only (IL‐3/GM‐CSF) and did not receive PBPCs, whereas the following eight patients were supported with PBPCs in addition to IL‐3 and GM‐CSF. Neutrophil recovery as well as platelet recovery were significantly faster in patients receiving PBPCs with a median of 6·5 d below 0·1 × 109 neutrophils/1 and 3 d below 20 × 109 platelets/1 as compared to 10·5 d and 8 d in control patients receiving growth factors only. The accelerated platelet recovery in patients supported with PBPCs might be explained—in the absence of detectable colony‐forming units megakaryocyte—by the presence of glycoprotein IIb/IIIa+, non‐proliferating endomitotic megakaryocytic precursor cells within G‐CSF mobilized PBPCs. Our data demonstrate that chemotherapy plus G‐CSF mobilized PBPCs accelerate both neutrophil and platelet recovery after high‐dose VIP chemotherapy in patients with solid tumours or lymphomas.
Differential expression of terminal transferase (TdT) in acute lymphocytic leukaemia expressing myeloid antigens and TdT positive acute myeloid leukaemia as compared to myeloid antigen negative acute lymphocytic leukaemia
Authors: Paietta Elisabeth , Racevskis Janis , Bennett John M. , Wiernik Peter H.
Published: March 2008

Summary. We examined whether the allegedly aberrant expression of the lymphoid lineage associated DNA polymerase, terminal deoxynucleotidyl transferase (TdT), in acute myeloid leukaemia (AML) is associated with alterations of the enzyme at the cellular, biochemical or transcriptional level when compared to lymphoid leukaemia (ALL), either lacking or expressing myeloid antigens. By flowcytometric analysis, the intensity of TdT staining with monoclonal anti‐TdT antibody was considerably weaker in TdT+AML and myeloid+ ALL (M+ALL) than in myeloid− ALL (M−ALL). TdT enzyme activity in TdT+AML was on an average 10%, and in M+ALL 25% of that measured in M− ALL. Anti‐TdT antibodies precipitated a major specific protein of identical relative molecular mass (58 kD) from metabolically labelled TdT+ myeloblasts and lymphoblasts. By Northern blot analysis and ribonuclease protection assay, TdT transcript levels were significantly lower in TdT+ myeloblasts and M+lymphoblasts than in M− ALL (P <0·0001). The level of TdT transcription in AML was independent of the simultaneous expression of lymphoid‐specific antigens, such as CD2 and CD19. Our data demonstrate that TdT expression is downregulated in association with myeloid features, not only in AML but also in ALL. This observation may provide the molecular basis for the differential therapeutic responsiveness, particularly to glucocorticoids, in these various leukaemia subtypes.
Occurrence of point mutations in p53 gene is not increased in patients with acute myeloid leukaemia carrying an activating N‐ras mutation
Authors: Büheer‐Leclerc Michel , Gratwohl Alois , Senn Hans‐Peter
Published: March 2008

Summary. The frequency of simultaneously detecting N‐ras and p53 gene mutations was studied in leukaemia cells of patients with acute myeloid leukaemia (AML) or with myelodysplastic syndrome (MDS). Using in vitro DNA amplification followed by oligonucleotide hybridization analysis, 45 AML and six MDS patients were screened for activating mutations in codons 12, 13 and 61 of N‐ras. Ten of them (eight AML and two MDS) were found positive. These 10 patients and 10 others without activating N‐ras mutation were further analysed by direct sequencing of the amplified exons for p53 mutations and for atypical N‐ras mutations. Beside the activating mutations in the N‐ras gene, no additional transforming or nontransforming mutations could be detected in the N‐ras. However, exon 7 of p53 was mutated in two AML patients without activating N‐ras mutation. These data show that p53 mutations occurred with half the frequency of N‐ras mutations in AML and that no positive correlation could be found between the onset of mutations in N‐ras and p53 genes.
Improvement of mouse β thalassaemia by hydroxyurea
Authors: Sauvage Claire , Rouyer‐Fessard Philippe , Beuzard Yves
Published: March 2008

Summary. The present report provides evidence that hydroxyurea (HU) improves the β thalassaemic phenotype in mice receiving 200 mg/kg/d for 30 d. The haematocrit rose from 29·3% at day 0 to 37·4% at day 30 (P<0·05), despite myelosuppression and decreased reticulocyte counts. The β minor/α ratio of globin chain synthesis increased from 0·78 at day 0 to 0·97 at day 30 (P<0·001). Membrane defects improved: the proportion of bound α chains decreased, the proportion of spectrin and ankyrin increased and red cell deformability also increased.
Long‐term survival and cure after marrow transplantation for congenital hypoplastic anaemia (Diamond‐Blackfan syndrome)
Authors: Greinix Hildegard T. , Storb Rainer , Sanders Jean E. , Deeg H. Joachim , Doney Kris C. , Sullivan Keith M. , Witherspoon Robert P.
Published: March 2008

Summary. Four patients with Diamond‐Blackfan syndrome (congenital hypoplastic anaemia) whose disease was resistant to corticosteroid treatment and who were red blood cell transfusion‐dependent, were given marrow grafts from allogeneic human‐leucocyte‐antigen (HLA)‐identical siblings. The patients were conditioned with regimens including cyclophosphamide and busulfan. Three of four patients had sustained and complete marrow engraftment. One patient showed early signs of haematopoietic recovery but died on day 35 of pulmonary toxicity. The three surviving patients are well with normal haematopoiesis and Karnofsky performance scores of 100%, 3·0, 7·4 and 10·6 years after transplantation. Congenital hypoplastic anaemia can be treated successfully by allogeneic marrow grafts.
Familial MDS with 5q — abnormality
Authors: Grimwade D. J. , Stephenson J. , De Silva C. , Dalton R. G. , Mufti G. J.
Published: March 2008

Summary. Abnormalities of chromosome 5 and 7 are frequently found in primary MDS. Cases with familial monosomy 7 are well recognized, but there are no reports of familial MDS with deletion of 5q. We describe two sisters, aged 38 and 36 years, both of whom had MDS and interstitial deletion of 5q. The occurrence of this chromosomal abnormality reinforces the concept of tumour suppressor gene hypothesis in some cases with familial MDS.
Granulocyte colony‐stimulating factor (G‐CSF)‐induced mobilization of circulating haemopoietic stem cells
Authors: Teshima Takanori , Harada Mine , Takamatsu Yasushi , Makino Kazuyoshi , Inaba Shoichi , Akashi Koichi , Kondo Seiji , Tanaka Takeshi , Ishii Eiichi , Niho Yoshiyuki
Published: March 2008

Summary. We studied the utility of G‐CSF for harvesting circulating haematopoietic stem cells in patients with leukaemia or lymphoma based on a comparative study in a single patient. Two successive cycles of leukapheresis following cytotoxic chemotherapy were performed in 22 patients as follows: the first cycle was performed with cytotoxic mobilization in all patients while the second cycle was randomized into two groups: cytotoxic (n= 10) and cytotoxic plus G‐CSF (cytotoxic/G‐CSF) (n= 12) mobilization. Repetitive cytotoxic mobilization did not alter the yields of mononuclear cells (MNC), myeloid (CFU‐GM), and erythroid (BFU‐E) progenitors. In contrast, cytotoxic/G‐CSF mobilization produced significantly higher yields of MNC (2·6‐fold), CFU‐GM (5·5‐fold), and BFU‐E (3·9‐fold) than did cytotoxic mobilization alone (P<0·01). The ratio of CFU‐GM to BFU‐E was not affected by G‐CSF. Furthermore, G‐CSF led to an earlier peak of CFU‐GM following chemotherapy. G‐CSF is thus effective in expanding the pool of circulating haematopoietic progenitors.
Whole blood platelet aggregation and coagulation factors in patients with systemic sclerosis
Authors: Goodfield M. J. D. , Orchard M. A. , Rowell N. R.
Published: March 2008

Summary. It is unclear whether the changes in platelet function which are observed in systemic sclerosis are a primary characteristic of this disease or whether they occur secondary to vascular changes. Whole blood platelet aggregation was studied in 26 patients with systemic sclerosis, normal subjects matched for age, sex and secondary characteristics, 19 patients with Raynaud's disease and 19 patients with systemic lupus erythematosus. Plasma levels of fibrinogen, von Willebrand factor antigen and factor VIII:C were also measured.

Systemic sclerosis was associated with a significant (P> 0·001) enhancement of the sensitivity of platelets to collagen. In contrast, significant enhancement of the response to either ADP or adrenaline was not observed. Enhanced sensitivity to collagen was not associated with the presence of either Raynaud's disease or systemic lupus erythematosus.

Systemic sclerosis was associated with significantly raised levels of von Willebrand factor antigen and fibrinogen. On an individual patient basis, von Willebrand factor antigen was related to the severity of the disease whereas platelet sensitivity to collagen was not. In conclusion, this study suggests that the enhanced sensitivity to collagen which occurs in systemic sclerosis is due to a primary change in the platelet and that this change can combine with elevated levels of adhesive proteins.
Vitamin K metabolism and vitamin K1 status in human liver samples: a search for inter‐individual differences in warfarin sensitivity
Authors: Thijssen H. H. W. , Drittij‐Reijnders M. J.
Published: March 2008

Summary. Vitamin K‐dependent parameters in human liver samples were investigated to find a clue to the inter‐individual differences in sensitivity for oral anticoagulants. Vitamin K epoxide reductase and vitamin K‐dependent carboxylase activity differed 2–3‐fold between the samples. Microsomal warfarin binding correlated significantly with the reductase activity. Microsomal vitamin K epoxide reductase of the different samples showed equal sensitivity for warfarin inhibition, 150 about 0·1 μm. Vitamin K epoxide reductase activity stimulated by NADH/lipoamide and microsomal lipoamide dehydrogenase activity showed higher inter‐subject variability than the reductase activity by itself. Liver vitamin K1 levels varied 4–5‐fold. Total and liver microsomal vitamin K1 levels were correlated. One of the liver samples was obtained from a donor anticoagulated with phenprocoumon and additionally treated with vitamin K1. High levels of the vitamin and its epoxide were present. Phenprocoumon was essentially irreversibly bound to the microsomes. In general the results confirm inter‐individual differences in the hepatic vitamin K‐dependent systems; the differences as such were found to be small. However, as the various parameters can work synergistically in the same direction, they may well account for the wide dose range observed in oral anticoagulant therapy.
Serum deoxythymidine kinase: no help in the diagnosis and prognosis of monoclonal gammopathy
Authors: Bäck Hans , Jagenburg Rudolf , Rödjer Stig , Westin Jan
Published: March 2008

Summary. Serum deoxythymidine kinase activity (S‐TK) was determined in the diagnostic evaluation and follow‐up of patients with monoclonal gammopathy of undetermined significance (MGUS). During a 4‐year period 35 patients were included in the study. Normal concentrations of S‐TK were found in 33. Patients with non‐progressive monoclonal gammopathy kept a stable, and for the individual, a characteristic level of S‐TK during several years of follow‐up. A moderately elevated level of S‐TK during observation did not indicate progression. Even in four out of five patients developing symptomatic multiple myeloma S‐TK remained unchanged. Determination of S‐TK does not give diagnostic or prognostic information in monoclonal gammopathy.
Clonal chronic lymphocytic leukaemia‐like B lymphocytes in the blood of patients with cutaneous T‐cell disorders
Authors: Vader S. Daenen. P. C. vanVoorst , Pietens N. Blom. J. , Hollema H. , Smit J. W.
Published: March 2008

Summary A population of B cells with characteristics of chronic lymphocytic leukaemia was found in the peripheral blood of four patients who presented with cutaneous infiltration of atypical CD4+ T cells with cerebriform nuclei. The B cells had a low density of immunoglobulin on their surface membrane, expressed CD5‐positivity, and showed monoclonality based on the restriction to either kappa or lambda light chains. In one patient with tumourous pleiomorphic CD4 + CU30 ‐ T‐cell lymphoma of the skin, it was the first manifestation of a concomitant B‐cell non‐Hodgkin's lym phoma of low‐grade malignancy. In three other patients with reactive atypical T‐cell erythroderma, there was no evidence for the coexistence of a B‐cell malignancy. The number of CD5+ B cells decreased in two erythroderma patients with clinical remission of the cutaneous lesions. It is speculated that the presence of a monoclonal B cell population in patients with T‐cell disorders of the skin is due either to a reactive process possibly conferring some protective effect, or a response to an unknown stimulus produced by the T cells.
Megakaryocyte colony stimulating activity in allogeneic bone marrow recipients prepared with busulfan and cyclophosphamide
Authors: Olson Thomas A. , Kapoor Neena , Wing Claudia , Tutschka Peter
Published: March 2008

Summary Increased megakaryocyte colony stimulating activity (MK‐CSA) has been reported after total body irradiation (TBI) for bone marrow transplant (BMT). We studied the effect of a busulfan (Bu) and cyclophosphamide (Cy) marrow transplant conditioning regimen, without radiation, on MK‐CSA production. Initial screening of MK‐CSA was done on previously collected and banked sera from 14 BMT patients. MK‐CSA was expressed as the ability to stimulate growth of megakaryocyte progenitors (CFU‐MK) in standard plasma clot cultures. In the initial samples, MK‐CSA peaked at day 7. This preliminary data led to a prospective study of MK‐CSA and clinical parameters in seven allogeneic recipients. MK‐CSA activity increased from day −7 pre‐transplant (2.9 ± 1.7 CFU‐MK/105 NATD, mean ± SD) to day O (10.3 ± 4.7 CFU‐MK) and peaked by day 9 post‐transplant (20.6 ± 6.4 CFU‐MK). MK‐CSA activity decreased in all seven patients by day 21 at which time five of seven patients studied had recovery of platelet counts to greater than 100 ± 109/l. MK‐CSA activity rose rapidly in both groups of sera after the initiation of this non‐irradiation. BMT preparative regimen. High MK‐CSA levels, early after transplant, may contribute to the rapid platelet recovery in some patients.
Relapse of aplastic anaemia after immunosuppressive treatment: a report from the European Bone Marrow Transplantation Group SAA Working Party
Authors: Schrezenmeier H. , Marin P. , Raghavachar A. , McCann S. , Hows J. , Gluckman E. , Nissen C. , Veer‐Korthof E. T. van't , Ljungman P. , Hinterberger W. , Lint M. T. Van , Frickhofen N. , Bacigalupo A.
Published: March 2008

Summary This study was designed to determine the incidence of relapse and factors predictive for relapse in 719 patients with severe aplastic anaemia (SAA) after immunosuppressive treatment (IS). Patients developing myelodysplasia or acute leukaemia after IS, and patients receiving a transplant, were excluded from this analysis. Response was defined as reaching complete independence from transfusions, relapse was defined as becoming again transfusion dependent. This criteria was validated by similar figures when using other ‘relapse criteria’ such as drop in neutrophil or platelet counts.

Of 358 patients responding to IS, 74 patients relapsed after a mean time of 778 d after treatment. The actuarial incidence of relapse is 35.2% at 14 years after IS. The risk for relapse was higher in patients responding within 120 d from IS (48%) compared to patients responding between 120 and 360 d (40%) and only 20% for slow responders (>360d from IS) (P<0.00001). In multivariate analysis this factor still proved significant (P<0.0001). The mean time between diagnosis and treatment was significantly longer in patients relapsing compared to patients who did not relapse (260 v 134 d, P=0.037). Relapse was not predicted by the severity of the disease, age, and sex. In 39 of the 74 relapsing patients a second response could be achieved. Responses after relapse were associated in univariate analysis with early response to previous IS and early occurrence of relapse. The actuarial survival of patients not relapsing is significantly better than survival of patients relapsing (79.8%v 67.1%, P = 0.0024). However, the actuarial survival of 39 relapsing patients who responded again to IS was similar to patients not relapsing (86%) and significantly better than in 35 patients not reaching a second response after relapse (49.3%, P=0.0015). This study indicates that relapse is a relevant problem in the treatment of aplastic anaemia, and does have an impact on overall survival. Prospective studies of immunosuppressive regimens, looking at responses, should also address this problem in the future.
A trial of recombinant human superoxide dismutase in patients with Fanconi anaemia
Authors: Liu Johnson M. , Auerbach Arleen D. , Anderson Stacie M. , Green Spencer W. , Young Neal S.
Published: March 2008

Summary Fanconi anaemia (FA) is a rare genetic disorder that predisposes to the development of aplastic anaemia and neoplasia. The pathophysiologic hallmark of FA is increased susceptibility to chromosomal breakage. Superoxide metabolism has also been shown to be involved in the cellular pathophysiology of FA. Human SOD (rh‐SOD), an enzyme which dismutates superoxide, has recently been cloned and expressed in yeast. We treated four FA patients with a 2‐week infusion of rh‐SOD (25 mg/kg d daily) to determine whether rh‐SOD had any effect on haemopoietic progenitor cell growth or on the abnormal cellular phenotype. We found that lymphocyte chromosomal aberrations induced by diepoxybutane were decreased during rh‐SOD treatment in two patients and that bone marrow progenitors were increased in one patient.
Fragile X syndrome and myelodysplasia discovered during pregnancy
Authors: Vorst Eljakim J. , Levene Naomi A. , Nisani Rachel , Berrebi Alain
Published: March 2008

Summary We describe a female patient who presented at pregnancy with leucopenia and was found to suffer from both fragile X syndrome [Fra(X)] and myelodysplastic syndrome with cytogenetic abnormalities in bone marrow cells including 4q+ and deletion D13. To date only four cases of Fra(X) syndrome with malignant tumours (one haematological). all in male patients, have been reported. We believe that the occurrence of the myelodysplastic syndrome in this patient could be more than coincidental.
Impaired ability of bone marrow cells from immunodeficient mice to establish long‐term cultures
Authors: Hamburger Anne W. , Chen Rong‐Bing
Published: March 2008

Summary Haemopoiesis is often depressed in patients suffering from acquired immune deficiency syndrome (AIDS). Although several mechanisms have been postulated to be responsible for depressed haemopoiesis in AIDS patients, the aetiology of this disorder is still unknown. We hypothesized that failure of the stromal microenvironment may account for part of the haemopoietic defect observed in patients with AIDS. We therefore studied a murine model of AIDS (MAIDS) caused by infection with LP‐BM5 virus to determine the ability of bone marrow cells from immunodeficient mice to establish long‐term stromal cultures. In addition, normal and MAIDS mice received AZT (2 mg/ml) in their drinking water for up to 1 month to determine the effects of AZT treatment in vivo on the ability of bone marrow cells to support haemopoiesis in long‐term cultures. Decreased numbers of non‐adherent cells were observed in long‐term bone marrow cultures (LTBMC) of MAIDS mice when compared to cultures derived from normal mice. Decreased numbers of non‐adherent cells were observed in cultures of bone marrow cells from AZT‐treated normal mice, when compared to untreated normal controls. Cells from AZT‐treated MAIDS mice produced the smallest number of non‐adherent cells. BFU‐E and CFU‐G/M were decreased in cultures of MAIDS mice when compared to those of normal mice. AZT‐treatment further decreased the number of colony‐forming cells in both MAIDS and normal cultures. Stromal cell function of MAIDS mice was also assessed by inoculating non‐adherent cells from normal mice onto confluent irradiated MAIDS LTBMC. Stroma from MAIDS mice was unable to support haemopoietic function of normal bone marrow cells. Polymerase chain reaction (PCR) analysis of steady state levels of cytokine mRNAs of cells from confluent cultures revealed that levels of interleukin‐6 mRNA were unchanged in MAIDS mice, as compared to normal controls, but the levels of GM‐CSF were decreased in MAIDS mice. These data suggest that LP‐BM5 MuLV infection alters the functioning of the haemopoietic stroma and that one mechanism of this depression in haemopoiesis may be via alterations of cytokine production.