bluuebunny/crossref_metadata_2025 huggingface dataset:
Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X‐irradiation and glucocorticoids
Authors: Amos T. A. S. , Lewis J. L. , Grand F. H. , Gooding R. P. , Goldman J. M. , Gordon M. Y.
Published: March 2008

Summary. Inhibition of apoptosis (genetically programmed active cell death) by p210 BCR‐ABL expression is a mechanism that might contribute to clonal expansion in chronic myeloid leukaemia (CML). Since cell death following exposure to ionizing radiation and many chemotherapeutic agents can occur by the apoptotic pathway, inhibition of apoptosis would be expected to confer a relative resistance to these treatments. Similarly, cells deprived of growth factors in vitro die by apoptosis, and inhibition of apoptosis would therefore be expected to allow cells to survive better in growth factor‐deprived conditions. We found that the survival of normal and CML myeloid progenitors was the same after in vitro incubation in deprived conditions and after treatment with X‐irradiation or glucocorticoids. We also found that mature cells in colonies produced by CML progenitors (CFU‐GM) did not survive better than those produced by normal progenitor cells. Flow cytometric analysis of propidium iodide‐stained cells provided a direct indication that the degree of apoptosis may correspond to the degree of deprivation. These results suggest that inhibition of apoptosis may not be the primary mechanism whereby BCR‐ABL influences the expansion of the malignant clone in CML.
Serum concentrations of E‐selectin, P‐selectin, ICAM‐1 and interleukin 6 in acute leukaemia patients with chemotherapy‐induced leucopenia and bacterial infections
Authors: Bruserud ØYstein , Akselen Per Espen , Bergheim Jann , Nesthus Ingrid
Published: March 2008

Summary. Serum concentrations of E‐selectin (CD62E), P‐selectin (CD62P), ICAM‐1 (CD54) and interleukin 6 were investigated in acute leukaemia patients with chemotherapy‐induced leucopenia and complicating bacterial infections. Serum concentrations of both E‐selectin and P‐selectin were decreased in the leucopenic patients without infections when compared with levels before chemotherapy; and serum concentrations of both E‐selectin and P‐selectin showed a further decrease during complicating bacterial infections. In contrast to the leukaemia patients, previously healthy individuals with meningococcal disease showed markedly elevated serum concentrations of E‐selectin and normal levels of P‐selectin during infection. Serum concentrations of ICAM‐1 and interleukin 6 increased during bacterial infections in the acute leukaemia patients with chemotherapy‐induced leucopenia. The alterations in serum concentrations of soluble adhesion molecules and interleukin 6 reversed when clinical signs of bacterial infections resolved during antibiotic therapy. Our results demonstrate that acute leukaemia patients with chemotherapy‐induced cytopenia show altered levels of both soluble adhesion molecules and interleukin 6 during complicating bacterial infections.
Diagnosis of transfusion‐associated graft‐versus‐host disease by polymerase chain reaction in fludarabine‐treated B‐chronic lymphocytic leukaemia
Authors: Briz Montserrat , Cabrera Rafael , Sanjuan Isabel , Forés Rafael , Díez Jose Luis , Herrero Miguel , Regidor Carmen , Algora Manuel , Fernández Manuel Nicolas
Published: March 2008

Summary. Transfusion‐associated graft‐versus‐host disease (TA‐GVHD), has rarely been reported associated with B‐chronic lymphocytic leukaemia (B‐CLL). We report a patient diagnosed with B‐CLL, previously treated with fludarabine, who developed TA‐GVHD after being transfused during surgery for splenectomy. Diagnosis was confirmed by polymerase chain reaction (PCR) detection of donor DNA in the patient, by amplification of Y‐chromosome sequence and analysis of minisatellite polymorphisms. B‐CLL patients treated with fludarabine appear to be at risk for TA‐GVHD and should be regarded as candidates for transfusions with irradiated blood products. This case illustrates that PCR is a rapid technique for the early diagnosis of TA‐GVHD.
Monoclonal proliferation of CD4+ large granular lymphocytes with cytolytic activity
Authors: Yasukawa Masaki , Utsunomiya Yuji , Inoue Yasuhiro , Kimura Nobuhiro , Fujita Shigeru
Published: March 2008

Summary. A rare case of monoclonal proliferation of CD3+4+8∼ T‐cell receptor‐αβ+ large granular lymphocytes (LGL) is presented. CD4+ LGL in the present case showed spontaneous cytotoxicity against herpes simplex virus‐infected cells and antibody‐ and lectin‐dependent cytotoxicity. Perforin, which is one of the important cytolytic mediators of cytotoxic T cells (CTL) and natural killer cells, was abundantly expressed in CD4+ LGL of this case. The present case suggests that perforin‐positive CD4+ CTL, which have recently been shown in the in vitro studies, certainly exist in vivo.
Thrombopoietin is not responsible for the thrombocytosis observed in patients with acute myeloid leukemias and the 3q21q26 syndrome
Authors: Bouscary Didier , Fontenay‐Roupie Michaela , Chretien Stany , Hardy Anne Claire , Viguié Franck , Picard Françoise , Melle Josianf , Dreyfus François
Published: March 2008

Summary. Patients with acute myeloblastic leukaemia (AML) and chromosomic abnormalities of the 3q21;q26 region have striking dysmegakaryopoiesis and normal or increased platelet counts. Leukaemic cells ectopically express the Evi‐1 gene which maps to human chromosome 3q26;q27. Thrombopoietin (TPO) has been cloned recently and shown to be the major hormone stimulating both megakaryocytopoiesis and thrombopoiesis. The TPO gene maps to human chromosome 3q26. For this report we studied four patients with typical 3q21;q26 syndrome. Karyotype analysis showed inv(3)(q21;q26) in three cases and t(3;3)(q21;q26) in one case. Although high levels of Evi‐1 transcripts could be detected in mRNA isolated from the bone marrow cells of these patients by Northern blot analysis, no TPO transcripts were detectable by RT‐PCR technique on the same mRNA samples. These results demonstrate that TPO gene transcription is not activated in patients with 3q26 chromosomic abnormality, and that abnormal TPO production is not responsible for the observed thrombocytosis.
Minor breakpoint cluster region (m‐BCR) positive chronic myeloid leukaemia with an acute lymphoblastic leukaemia onset: a case report
Authors: Costello Régis T. , Gabert Jean , BRUNEL VÉRONIQUE , Sainty Danielle , Arnoulet Christine , Mozziconacci Marie‐Joëlle , Camerlo Jacques , Perret Caroline , Gastaut Jean‐Albert , Bouabdallah Reda , Lafage‐Pochitaloff Mårina
Published: March 2008

Summary. m‐BCR chronic myeloid leukaemia (CML) is a rare entity. We report a patient presenting with Philadelphia (Ph)‐positive, m‐BCR‐positive acute lymphoblastic leukaemia (ALL) who achieved complete remission after induction chemotherapy, but showed a majority of Ph‐positive mitoses during this remission. A diagnosis of m‐BCR CML was established and the patient was given interferon a therapy. This is the first m‐BCR CML presenting as ab initio ALL. This report emphasizes the importance of karyotyping Ph‐positive ALL during remission so as not to misdiagnose CML patients who can benefit from Interferon therapy.
Inter‐individual variation in half‐life of infused recombinant factor VIII is related to pre‐infusion von Willebrand factor antigen levels
Authors: Fijnvandraat Karin , Peters Marjolein , Ten Cate Jan W.
Published: March 2008

Summary. In an attempt to explain the substantial inter‐individual variability in half‐life of infused factor VIII (FVIII) among haemophilia A patients, we studied the correlation of pre‐infusion von Willebrand factor antigen (vWFAg) levels and the half‐life of a recombinant‐DNA‐derived FVIII (r‐VIII SQ, Pharmacia, Sweden). Intra‐individual variation in half‐life was small (range of variation 0.2‐1 h) in 12 severely affected haemophiliacs who received two doses of FVIII concentrate. In contrast, inter‐individual variation in half‐life was large (range of half‐lives 6‐28.8 h). A strong correlation between half‐life and pre‐infusion vWFAg levels was present (r= 0.87, P= 0.0003). We conclude that the half‐life of infused recombinant FVIII is related to pre‐infusion vWFAg levels in severely affected haemophilia A patients.
A simple method for synthesis of B‐cell clonospecific probes
Authors: Saal Russell J. , Gordon Ross B. , Cobcroft Ralph G. , Scott Desley C.
Published: March 2008

Summary. A sensitive PCR‐based method was developed to produce B‐cell clonogenic probes without the need for sequencing and specific oligonucleotide synthesis. Specificity and sensitivity were assessed and found to be comparable to that achieved using established methods. Possible applications include the detection of MRD, bone marrow involvement with lymphoma, and the contamination of autologous bone marrow or peripheral blood progenitor cell harvests with malignant cells carrying IgH rearrangements.
Rapid diagnosis of β‐thalassaemia by mutagenically separated polymerase chain reaction (MS‐PCR) and its application to prenatal diagnosis
Authors: Chang Jan‐Gowth , Lu Jan‐Ming , Huang Jenp‐Ming , Chen Jung‐Te , Liu Hsing‐Jin , Chang Chih‐Peng
Published: March 2008

Summary. We have developed a rapid and simple PCR‐based method which is modified from the mutagenically separated polymerase chain reaction (MS‐PCR) to detect the molecular defects of β‐thalassaemia. We can use this technique to amplify normal and mutant alleles of the β‐globin gene in the same reaction tube, using different‐sized allele‐specific primers. This mutagenesis separates the amplification reactions of alleles performed in the same tube, Subsequent gel electrophoresis shows at least one of the two allelic products at the same locus or at least two of the several allelic products at different loci. Therefore, in addition to simple handling, MS‐PCR provides a within‐assay quality control for the exclusion of false negative results. The five most common mutations of β‐thalassaemia and haemoglobin E which occur in the Taiwanese population were tested, and 14 prenatal samples were checked with accurate results. This method is simple, rapid and accurate, and can be used routinely in prenatal diagnosis. The principle used here can also be applied to other genetic diseases.
A new α chain variant Hb Sallanches [α2 104(G11) Cys→Tyr] associated with HbH disease in one homozygous patient
Authors: Morlé F. , Francina A. , Ducrocq R. , Wajcman H. , Gonnet C. , Philippe N. , Souillet G. , Godet J.
Published: March 2008

Summary. We identified a new α‐chain variant (αSal) associated with haemolytic anaemia and low level of HbH in one homozygous patient. This new mutation is located in codon 104 (TGC → TAC) of the a2 globin gene and results in a Cys→Tyr replacement. In vitro and in vivo biosynthetic studies suggest that the mechanism leading to HbH disease in this homozygous patient is mostly related to a significant instability of αSal:β dimers rather than to the hyper‐instability of the αSal chain itself only.
Hypocellular myelodysplastic syndromes (MDS): new proposals
Authors: Tuzuner Nukhet , Cox Christopher , Rowe Jacob M. , Watrous Dennis , Bennett John M.
Published: March 2008

Summary. To determine whether hypocellular MDS differs from normo/hypercellular MDS, we attempted to identify hypocellular MDS cases either by correcting the bone marrow (BM) cellularity by age (28 patients) or by using a single arbitrary value of BM cellularity (25 patients) and compared these two groups of hypocellular cases to the normo/hypercellular MDS cases (72 patients). 18 patients were common to both hypocellular groups. Patients with hypocellular MDS in both of these selected groups have similar features with regard to age and sex distribution, peripheral blood and bone marrow parameters, FAB subtypes, karyotypes, leukaemic transformation, and survival. However, the median age of patients in < 30% BM cellularity group was higher than those patients in the age‐corrected group (69 years v 62 years). The selection of < 30% cellularity excluded 10 cases in the age group < 70 years but included another seven patients in the age group of > 70 years. However, correction of BM cellularity by age revealed that those included patients (selected for < 30% cellularity) who had normocellular BM by their age. Therefore we recommend the age‐correcting grouping to ensure comparable series for comparison, for response to treatment, and survival. Finally, BM cellularity does not appear to be an important factor on prognosis in MDS, because patients with hypocellular MDS in both selected groups have similar prognosis to those with normo/hypercellular MDS patients.
Involvement of the cyclin‐dependent kinase‐4 inhibitor (CDKN2) gene in the pathogenesis of lymphoid blast crisis of chronic myelogenous leukaemia
Authors: Serra Anna , Gottardi Enrico , Ragione Fulvio Della , Saglio Giuseppe , Iolascon Achille
Published: March 2008

Summary. Recent data suggest that homozygous deletion of the cyclin‐dependent kinase 4 inhibitor gene (CDKN2), a putative tumour suppressor gene located on chromosome 9p21, represents a common genetic event in human cancer. As the molecular basis of the evolution of chronic myelogenous leukaemia (CML) into blast crisis remains largely unknown, we decided to investigate if the occurrence of similar deletions could represent one of the mechanisms underlying the disease progression. Whereas none of 22 chronic phase CML cases examined showed alterations, we found that 3/17 total blast crisis examined (18%) showed a homozygous deletion of the CDKN2 gene. The deletions were restricted to cases of lymphoid blast crisis, being present in 3/8 (40%) of the lymphoid and in none of the nine myeloid cases examined. The fact that the chronic phase DNA obtained at diagnosis in one of the cases lacks the homozygous deletion observed in blast crisis, suggests that the final deletion event took place concomitantly with the progression of the disease. Furthermore, the analysis of polymorphic regions on chromosome 9p21 flanking at both sides the CDKN2 gene, showed that deletions at 9p21 differ between cases and are characterized by a wide range of extensions. A concomitant search for a possible involvement of the p53 tumour suppressor gene in the same series of patients showed mutations of the gene and loss of heterozygosity at 17p only in myeloid blast crisis, suggesting the presence of distinct molecular pathways in the pathogenesis of lymphoid and myeloid blast crisis.
Decreased potency of MDR‐modulators under serum conditions determined by a functional assay
Authors: Ludescher Christof , Eisterer Wolfgang , Hilbe Wolfgang , Hofmann Johann , Thaler Josef
Published: March 2008

Summary. A variety of agents are capable of overcoming P‐glycoprotein‐mediated multidrug resistance (MDR) in vitro. However, the clinical potential of these compounds is often limited due to high plasma protein binding. We compared the efficacy of several MDR‐reversing compounds in serum‐free culture medium and under serum conditions by means of a functional assay. Using flow cytometry the efflux of the fluorescent dye rhodamine 123 (Rhl23) was measured from normal peripheral blood CD8+ T‐lymphocytes which express low levels of P‐glycoprotein. Inhibition of Rhl23 efflux by R‐verapamil, dexnigludipine‐HCl, cyclosporin A, SDZ PSC833 and the protein kinase C (PKC) inhibitor CGP 41251 was determined in serum‐free medium and in serum at concentrations from 0.1 to 50μmol/l. With the exception of SDZ PSC833 all MDR modulators showed an insufficient or suboptimal modulation of P‐glycoprotein under serum conditions at concentrations achievable in vivo. The highest potency under serum conditions demonstrated SDZ PSC833: even at a concentration of 0.5μxmol/1 a sufficient inhibitory effect was observed. Subsequently this approach was applied to patients suffering from B‐cell chronic lymphocytic leukaemia (B‐CLL; n= 3) and acute myeloid leukaemia (AML; n= 2) which were positive in the Rh l23 efflux assay. As for normal CD8+ T‐lymphocytes, much higher drug concentrations were required under serum conditions to effectively inhibit Rhl23 efflux from the leukaemic cells. Thus the interpretation of results of clinical‘modulator’trials should consider the decreased bioavailability of MDR‐reversing agents.
Experience with liposomal Amphotericin‐B in 60 patients undergoing high‐dose therapy and bone marrow or peripheral blood stem cell transplantation
Authors: Krüger William , Stockschläder Marcus , Rüssmann Bettina , Berger Carolina , Hoffknecht Matthias , Sobottka Ingo , Kohlschütter Brigitte , Kroschke Gerd , Kröger Nicolaus , Horstmann Martin , Kabisch Hartmut , Zander Axel R.
Published: March 2008

Summary. 60 patients undergoing bone marrow or stem cell transplantation were treated with liposomal Amphotericin‐B for documented or suspected mycosis. 34 patients had a prior course of conventional Amphotericin‐B with the following adverse effects: increasing creatinine above l‐4mg/dl (n=17), increasing creatinine below l‐5mg/dl (n= 9), no response (n= 6), and clinical side‐effects (n= 4). Liposomal Amphotericin‐B failed in 6/7 patients with culture‐proven mycosis who died from infection withAspergillus (n= 2) andCandida (n = 4), respectively. One patient withCandida lambica sepsis was cured. No patient with clinically or serologically suspected or diagnosed infection died from mycosis. Liposomal Amphotericin‐B was well tolerated in 57 patients, even after side‐effects of the conventional formulation. Adverse effects occurred in three cases, requiring the withdrawal of the drug in one patient. Due to toxic side‐effects of the high‐dose therapy and transplant‐related complications, it was difficult to evaluate the influence of liposomal Amphotericin‐B on laboratory parameters. Eight patients showed a decrease of creatinine levels, which had increased above normal values under preceding therapy with conventional Amphotericin‐B. Liposomal Amphotericin‐B is well tolerated in patients undergoing high‐dose therapy and bone marrow transplantation. The efficacy of liposomal Amphotericin‐B needs to be investigated in randomized studies in comparison with conventional Amphotericin‐B.
Hepatitis C virus genotypes and severity of chronic liver disease in haemophiliacs
Authors: Tagariello Giuseppe , Pontisso Patrizia , Davoli Pier Giorgio , Ruvoletto Maria Grazia , Traldi Agostino , Alberti Alfredo
Published: March 2008

Summary. We studied the activity and stage of chronic liver disease in 45 HCV‐seropositive/HIV‐seronegative patients with severe haemophilia followed for at least 10 years. HCV‐RNA was detected in serum in 36 patients (80%) Viraemic cases were further analysed for HCV genotypes: 10 (28%) were infected by type la, 10 (28%) by type lb, seven (19%) by type 2, four (11%) by type 3, four (11%) had mixed infections (one by la + lb, one by la 4‐ 2, one by type 2+3, and one by la + 2 + 3). ALT levels were within the normal range in 55% of the HCV‐RNA negative patients but in only 11% of the viraemic cases. Results show a trend for higher levels of ALT in HCV‐RNA‐positive patients compared with those without viraemia (98 ± 56 v 60 ± 61), and particularly with patients with type 3 HCV infection (148 ± 44). We suggest that a slow progression of chronic liver disease occurs in haemophilic HCV‐positive/HiV‐negative patients and conclude that presence of HCV‐RNA in serum correlates well with cytolitic damage but, in the time‐scale of our follow‐up period, commonly used clinical‐laboratory parameters cannot predict the chronic evolution of liver infection or identify differences in disease progression in relation to specific HCV subtypes.
Common inhibitory effects of human anti‐C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor
Authors: Shima Midori , Nakai Hiroaki , Scandella Dorothea , Tanaka Ichiro , Sawamoto Yoshikatu , Kamisue Seiki , Morichika Shougo , Murakami Tomohiko , Yoshioka Akira
Published: March 2008

Summary. Factor VIII (FVIII) Inhibitor alloantibodies obtained from seven severe haemophilia A patients were examined for their binding regions and their effects on FVHI binding to von Willebrand factor (vWF). Immunoblotting analysis with a panel of recombinant fragments demonstrated that the binding regions of antibodies in cases 1‐5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognized an epitope within residues 2248‐2312, completely inhibited FVIII/ vWF binding in an FXISA (IC50: 5‐0 and 9‐0μg/ml, respectively). Antibodies from case 3 recognizing 2170‐2312 and case 5 recognizing 2170‐2327 also inhibited FVIII/vWF binding (IC50:110 and 400μg/mI, respectively). Case 4 antibodies recognizing 2218‐2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognizing the 44 kD heavy chain, did not inhibit. Our results demonstrate that all anti‐C2 alloantibodies with epitopes that extend to the residue 2312 inhibit vWF binding and that an overlap of the inhibitor epitope with residues 2308‐2312 is critical for maximal inhibition of vWF binding. Prevention of FVIII/vWF binding appears to be a common property of anti‐C2 domain inhibitor alloantibodies.
Molecular basis of inherited factor XIII deficiency: identification of multiple mutations provides insights into protein function
Authors: Anwar Rashida , Stewart Alistair D. , Miloszewski Krzysztof J. A. , Losowsky Monty S. , Markham Alexander F.
Published: March 2008

Summary. Factor XIII (FXIII) is a zymogen essential for normal haemostasis. In inherited FXIII deficiency the majority of cases show absence of the FXHIa subunit. Molecular analysis of PCR‐amplified FXIIIa subunit exonic regions, and of RT‐PCR amplified cDNA from six patients with FXIIIa subunit deficiency, from five unrelated families, has revealed 10 sequence changes: three mutations resulting in abnormal splicing of pre‐mRNA, one nonsense mutation, one deletion/insertion change, three point mutations producing Val34Leu, Asn60Lys and Arg408Gln changes, and two silent mutations. In three families the patients are homozygous for a specific deficiency causing mutation, and patients from the remaining two families are compound heterozygotes. Understanding the molecular pathology of the disorder provides insights into the structure‐function relationships of the various domains within the FXm protein. From a clinical point of view, it enables direct diagnosis at the DNA level and may aid the development of FXIII analogues to promote wound healing.
The prenatal identification of fetal compatibility in neonatal alloimmune thrombocytopenia using amniotic fluid and variable number of tandem repeat (VNTR) analysis
Authors: Denomme Gregory A. , Waye John S. , Borrows Robert F. , Hayward Catherine P. M. , Warkentin Theodore E. , Horsewood Peter , Smith James W. , Jelsema Russel D. , Zuidema Laura J. , Kelton John G.
Published: March 2008

Summary. Most severe episodes of neonatal alloimmune thrombocytopenic purpura (NATP) are caused by antiplatelet alloantibodies against the HPA‐la (PlA1) antigen. However, half of subsequent fetuses produced from a HPA‐la/b father (genotypic frequency 28%) will result in a child who is not affected. Some investigators manage NATP by confirming the fetal platelet phenotype using percutaneous umbilical cord sampling, a procedure that carries a low but real risk of fetal morbidity and mortality. More recently, physicians determine the fetal platelet antigen genotype using DNA derived from amniotic fluid or chorionic villus samples. All therapy is withdrawn for a fetus who genotypes as HPA‐lb/b. However, since the fetus is the same genotype as the mother, there can be uncertainty about the origin of the genetic material and thus the validity of the fetal genotype. The inappropriate withdrawal of therapy for a erroneously genotyped fetus could be fatal, and consequently many physicians advocate fetal HPA‐1 phenotyping with confirmation using percutaneous umbilical blood sampling.

In this report we describe the management of two pregnancies with previously affected infants due to anti‐HP A‐la alloantibodies. Both husbands were HPA‐la/b. For the current pregnancies, amniotic fluid was collected at 20 or 29 weeks of gestation, and the platelet genotype indicated that the fetuses were HPA‐lb/b. The fetal origin of the amniotic fluid derived DNA was confirmed by the forensic technique of DNA profiling using variable number of tandem repeat (VNTR) analysis. All therapy was withdrawn, percutaneous umbilical blood sampling was not performed, and both women vaginally delivered healthy non‐thrombocytopenic infants. The application of platelet alloantigen genotyping using DNA from amniotic fluid cells identified the HPA‐lb/b fetus, and VNTR analysis confirmed that the tissue was fetal derived, thus avoiding the necessity for percutaneous umbilical blood sampling. The use of this approach in patients at risk will avoid additional investigation and treatment in approximately one‐seventh of all NATP pregnancies involving the HPA‐la antigen.
Expression of granulocyte colony‐stimulating factor receptor increases with differentiation in myeloid cells by a newly‐devised quantitative flow‐cytometric assay
Authors: Shinjo Kaori , Takeshita Akihiro , Ohnishi Kazunori , Ohno Ryuzo
Published: March 2008

Summary. In order to develop a non‐isotopic quantitative assay of granulocyte colony‐stimulating factor (G‐CSF) receptors on human or murine cells, we devised a flow‐cytometric assay using cells stained with biotin‐labelled G‐CSF (b‐G‐CSF) and a streptavidin‐RED670 conjugate. For quantification, we applied the Kolmogorov‐Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two fluorescence profiles according to the increase of fluorescence intensity due to the specific binding of b‐G‐CSF to G‐CSF receptors. A good correlation was observed between the number of G‐CSF receptors obtained by the radioisotopic binding assay and the number calculated from the D value by the flow‐cytometric assay. Then, expression of G‐CSF receptors on human bone marrow cells, peripheral blood granulocytes and blast cells from patients with acute myeloid leukaemia (AML) were studied. G‐CSF receptors was expressed on CD34+CD33−, CD34+CD33+ and CD34−CD33+ cells in the following order: CD34 CD33+CD34+CD33+CD34+CD33 cells, indicating that the receptors increased with maturation. The receptor levels of CD34−CD33+ cells in bone marrow were apparently lower than those of CD34−CD33+ cells in peripheral blood granulocytes. On the other hand, an abnormal expression pattern of G‐CSF receptors was noted in AML blast cells.
A novel factor‐dependent human myelodysplastic cell line, MDS92, contains haemopoietic cells of several lineages
Authors: Tohyama Kaoru , Tohyama Yumi , Nakayama Takashi , Ueda Takanorf , Nakamura Toru , Yoshida Yataro
Published: March 2008

Summary. A novel long‐term cultured interleukin(IL)‐3‐dependent human myelodysplastic cell line, MDS92, was shown to contain several myeloid‐lineage cells such as neutrophils, macrophages, eosinophils, and a small number of megakaryocyte‐lineage cells. Therefore this cell line possesses at least bipotential characteristics of myeloid‐ and megakaryocyte‐lineages. Granulocyte colony‐stimulating factor clearly promoted the neutrophil alkaline phosphatase activity of MDS92 cells. To the contrary, the incidence and growth of CD41‐positive cells were hardly affected by the addition of IL‐6, IL‐11, c‐mpl ligand (thrombopoietin, TPO) or erythropoietin. TPO slightly supported the growth of CD34‐positive cell fraction, but not CD41‐positive cell fraction of MDS92 cells in combination with IL‐3 or Steel factor. This cell line will be a useful tool for the study of MDS stem cells, but the mechanism of commitment of differentiation in MDS stem cells remains unknown.
Liver iron stores in patients with secondary haemosiderosis under iron chelation therapy with deferoxamine or deferiprone
Authors: Nielsen P. , Fischer R. , Engelhardt R. , Tondüry P. , Gabbe E. E. , Janka G. E.
Published: March 2008

Summary. Total body iron stores including liver and spleen iron were assessed by non‐invasive SQUID biomagnetometry. The liver iron concentration was measured in groups of patients with β‐thalassaemia major or other post‐transfusional siderosis under treatment with the oral iron chelator deferiprone (n = 19) and/or with parenteral deferoxamine (n = 33). An interquartile range for liver iron concentrations of 1680‐4470μg/g liver was found in these patients. In both groups a poor correlation between liver iron and serum ferritin values was observed.

Repeated measurements of liver and spleen iron concentrations as well as determination of liver and spleen volume by sonography were performed in six patients under continuous deferiprone treatment for 3‐15 months. In this group detailed information was obtained on the whole body iron store (5‐36 g) and the iron excretion rates (14‐34mg/d) for each patient. As indicated by decreasing liver iron concentrations, five out of six subjects showed a negative iron balance (2‐13 mg/d). Conventional measurements of both serum ferritin and urine iron excretion gave fluctuating results, thus being only of limited use in the control of iron depletion therapy.

The non‐invasive biomagnetic liver iron quantification is a precise and clinically verified technique which offers more direct information on the long‐term efficacy of an iron depletion therapy than the hitherto used methods. This technique may be of use in the clinical evaluation of new oral iron chelators.
CDKN2 gene deletion is not found in chronic lymphoid leukaemias of B‐ and T‐cell origin but is frequent in acute lymphoblastic leukaemia
Authors: Schröder Martin , Mathieu Ulrike , Dreyling Martin H. , Bohlander Stefan K. , Hagemeijer Anne , Beverloo Berna H. , Olopade Olufunmilayo I. , Stilgenbauer Stephan , Fischer Konstanze , Bentz Martin , Lichter Peter , Döhner Hartmut
Published: March 2008

Summary. Homozygous deletions of the cyclin‐dependent kinase 4 (CDK4) inhibitor gene CDKN2 (pi6, MTS1) have been demonstrated to occur frequently in human cancer cell lines of different origin. However, in most primary tumours the frequencies of CDKN2 deletions are not well defined. We studied primary samples of 100 patients with lymphoid leukaemias [B‐lineage acute lymphoblastic leukaemia (ALL), n = 23; T‐ALL, n= 7; B‐cell chronic lymphocytic (B‐CLL) or prolymphocytic (B‐PLL) leukaemia, =50; T‐CLL/T‐PLL, n= 20] using fluorescence in situ hybridization (FISH) with eight overlapping cosmid clones covering the region on chromosome band 9p21 containing CDKN2. We did not observe any CDKN2 deletions in the 70 patients with chronic lymphoid leukaemias of B‐ or T‐cell origin. Of the 23 patients with B‐lineage ALL, one (4%) exhibited a CDKN2 deletion: in this patient, two clones were detected, one exhibiting a hemizygous and the other a homozygous deletion. On chromosome banding analysis, four patients with B‐lineage ALL had a 9p aberration, whereas all CDKN2 copies were retained. In contrast, six of the seven (86%) patients with T‐ALL exhibited CDKN2 deletions (homozygous, n = 4; hemizygous, n = 2). We conclude that hemizygous or homozygous deletions of the CDKN2 gene occur at high frequency in T‐ALL and at low frequency in B‐lineage ALL, supporting the role of this gene as a tumour suppressor, especially in T‐ALL. However, from our data there is no evidence that CDKN2 is involved in the pathogenesis of chronic lymphoid leukaemias of B‐ or T‐cell origin.
Combined cytogenetic, FISH and molecular analysis in acute promyelocytic leukaemia at diagnosis and in complete remission
Authors: Mancini Marco , Nanni Mauro , Cedrone Michele , Diverio Daniela , Avvisati Giuseppe , Riccioni Roberta , Rosaria Maria , Cuia De , Fenu Susanna , Alimena Giuliana
Published: March 2008

Summary. This study reports the results of a simultaneous application of cytogenetic fluorescence in situ hybridization (FISH) and molecular analysis (RT‐PCR) in 28 APL cases (23 M3 and five M3v; 26 studied at diagnosis and two at relapse). FISH on metaphases identified the t(15;17) in all cases who were positive for the PML/RAR a transcript by RT‐PCR. Conventional cytogenetics revealed the t(15; 17) in only 68% of cases. However, it enabled the detection of additional chromosome changes in five cases, three of whom were M3v. 11 patients were also investigated during complete remission (CR) by both FISH and RT‐PCR, in order to evaluate residual disease; the duration of CR at the time of analysis ranged between 1 and 16 months, with three patients being studied twice. Comparison of RT‐PCR and FISH results showed a very good correlation. In fact, of the 10 samples which were RT‐PCR positive for residual disease, all were also recognized by interphase FISH, and eight were positive by metaphase FISH. Of the three samples negative at RT‐PCR, all were also negative at the interphase FISH. The results of this study indicate that: (a) the t(15;17) is present in all cases positive for the PML/RAR a rearrangement, thus in virtually all true APLs; (b) standard cytogenetics, capable of unravelling the t(15;17) in only 68% of cases, enables recognition of additional chromosome changes of potential clinical and prognostic significance; (c) FISH on interphase nuclei is a reliable tool for the monitoring of residual disease, with a sensitivity greater than that of FISH on metaphase cells and superimposable to that of RT‐PCR.
Effects of homo‐aza‐steroids on acute non‐lymphocytic leukaemia cell proliferation in vitro
Authors: Trafalis Dimitrios T. P. , Sambani Constantina , Kapsimali Violeta , Economidou Joanna , Politis George , Catsoulacos Panayotis
Published: March 2008

Summary. Homo‐aza‐steroids (modified steroid molecules) in their esterified forms have been used extensively as carrier molecules of alkylating agents against several neoplastic malignancies in vivo and in vitro. We studied the effects of two homo‐aza‐steroid carrier molecules alone, namely 3β‐hydroxy‐13α‐amino‐13,17‐seco‐5α‐androstan‐17‐oic‐13, 17‐lactam (compound 1) and 13α‐amino‐13,17‐seco‐1,3,5‐estratrien‐17‐oic‐13,17‐lactam (compound 2), on human acute non‐lymphocytic leukaemia cell proliferation in vitro.

We used peripheral blood samples from 27 untreated ANLL patients (eight M1, four M2, two M3, six M4, three M5a, two M5b and two M6, according to FAB criteria). Proliferative activity was estimated by using thymidine uptake and the percentage of cells in metaphase in 24, 48 and 72 h of culture. Exposure of human leukaemic blasts with either of the two compounds resulted in enhanced cell proliferation in M1, M2, M4, M6 and M5a (only by compound 2) cases, whereas there was no significant effect in the M3 and M5b cases.

Our results indicate that the two compounds tested exhibit stimulatory effect on cell proliferation, particularly in blast cells possessing a relatively smaller degree of differentiation (Ml and M6 cases exhibiting CD34 and CD7). Further research is needed to study the cell growth effect and the therapeutic potential of these steroid molecules in human blood malignancies in vitro and in vivo.
Preferred usage of specific immunoglobulin gene segments in chronic lymphocytic leukaemia cells of three HLA‐identical sisters
Authors: Hakim I. , Amariglio N. , Brok‐Simoni F. , Berkowitz M. , Rosner E. , Kneller A. , Hulu N. , Ramot B. , Ben‐Bassat I. , Silverman G. J. , Rechavi G.
Published: March 2008

Summary. We report three family members, including a set of identical twins, who developed CD5 positive B‐CLL. The patients are female Ashkenazi Jews sharing an identical HLA phenotype. Two of the HLA loci (B35 and Cw4) were common with those already described as being shared by Ashkenazi Jews with an increased incidence of CLL. The rearranged immunoglobulin genes of the malignant cells of all three patients were found to express genetically related VH regions belonging to the VH3 subgroup, and chromosomal studies suggest a process of clonal evolution in one of the twins.
Distribution of Kaposi's sarcoma herpesvirus sequences among lymphoid malignancies in Italy and Spain
Authors: Pastore Cristina , Gloghini Annunziata , Volpe Gisella , Nomdedeu Josep , Leonardo Eugenio , Mazza Umberto , Saglio Giuseppe , Carbonb Antonino , Gaidano Gianluca
Published: March 2008

Summary. In this study we have tested the distribution of Kaposi's sarcoma herpesvirus (KSHV) DNA sequences throughout the spectrum of lymphoid neoplasia in Italy and Spain. 180 cases of lymphoid malignancies representative of the major histologic and immunophenotypic categories of B‐ and T‐cell tumours were analysed by means of a polymerase chain reaction‐based assay. KSHV sequences were consistently absent in all categories of lymphoid malignancies studied, with the exception of a subset of B‐cell non‐Hodgkin's lymphomas localizing in the pleural, pericardial or peritoneal cavities, and fulfilling the diagnostic criteria of body‐cavity‐based lymphoma. The selective and consistent association of KSHV sequences with cases of body‐cavity‐based lymphoma throughout the spectrum of lymphoid neoplasms suggests that KSHV may be involved in the pathogenesis of this peculiar type of lymphoid malignancy.
Glycoprotein IIb/IIIa autoantigenic repertoire in chronic idiopathic thrombocytopenic purpura
Authors: Hou Ming , Stockelberg Dick , Kdtti Jack , Wadenvik Hans
Published: March 2008

Summary.The objective of the present study was to further disclose the autoantigenic repertoire carried by the platelet glycoprotein (GP) IIb/IIIa complex. IgG‐F(ab′)2fragments were prepared from two prototype ITP patients, and their ability to block the binding of GPIIb/IIIa reactive antibodies derived from other patients with ITP was evaluated using a modified MAIPA assay; a P1A1alloantiserum and 20 normal sera were included as controls. It was found that the two prototype IgG‐F(ab′)2fragments were each able to significantly block the binding of serum IgG to GPIIb/IIIa in six (55%) and seven (64%) out of 11 patients with chronic ITP, respectively. No significant blocking effect was observed for IgG‐F(ab′)2 fragments prepared from normal subjects. Also, the binding of the P1A1alloantiserum to its epitope on GPIIIa was not affected by any of the blocking IgG‐F(ab′)2 fragments exploited in the study. These data substantiate that in chronic ITP at least half of the GPIIb/IIIa reactive sera bind to homogenous autoepitopes.
A prospective study to determine the frequency and clinical significance of alloimmunization post‐transfusion
Authors: Heddle Nancy M. , Soutar Richard L. , O'Hoski Pamela L. , Singer Joel , McBride John A. , Ali Mohamed A. M. , Kelton John G.
Published: March 2008

Summary. There is debate in the literature about the frequency and importance of delayed transfusion reactions. This uncertainty could reflect the endpoints used (clinical or serological) and the type of study (typically retrospective or case series). In this report we describe a prospective investigation to determine the frequency of alloimmunization post transfusion and whether the alloantibody production is a laboratory event or has clinical relevance.

A total of 2490 patients were transfused 11218 red cell concentrates. One or more blood samples were collected within 7 d post transfusion and screened for serological evidence of alloimmunization. If any antibody was detected the patient's post‐transfusion sample was screened for biochemical evidence of haemolysis and the patient's chart reviewed for documentation of clinical signs of a transfusion reaction. Post transfusion alloimmunization occurred in 2.6% of the patients (95% CI 2.1–3.6%), who had no detectable alloantibody in pre‐transfusion testing. For those 86 patients (3.5%) with alloantibodies detectable pretransfusion, 8.9% (95% CI 3.6–17.4%) developed additional aloantibodies. The most common alloantibodies detected were anti‐Jka, anti‐E and anti‐K. Despite the high frequency of serological evidence of delayed transfusion reactions, only one patient (005%) had clinical evidence of a delayed haemolytic transfusion reaction (95% CI 0.0–0.27%). Serological evidence of a delayed transfusion reaction is common; however, these reactions rarely cause clinical symptoms.
Increased expression of the PRAD‐1/CCND1 gene in hairy cell leukaemia
Authors: Bosch Francesc , Campo Elias , Jares Pedro , Pittaluga Stefania , Muñoz Josep , Nayach Iracema , Piris Migdel Angel , Dewolf‐Peeters Chris , Jaffe Elaine S. , Rozman Ciril , Montserrat Emilio , Cardesa Antonio
Published: March 2008

Summary. The PRAD‐1/CCND1 gene encodes Cyclin Dl, a cyclin involved in cell cycle regulation at the Gi‐S transition. Over‐expression of this gene is a highly specific molecular marker of mantle cell lymphomas (MCLs), but it may also be up‐regulated in some chronic lymphoprolifera‐tive disorders, mainly chronic lymphocytic leukaemia.

We have examined PRAD‐1/CCND1 gene expression by Northern blot and Western blot analysis in a series of 18 hairy cell leukaemias (HCLs), nine other splenic malignant lymphoproliferative disorders, and three normal/reactive spleens. Over‐expression of the mRNA PRAD‐1/CCND1 gene was observed in 16/18 HCLs, including one case of hairy cell leukaemia variant, whereas this molecular alteration was not found in other cases examined. mRNA levels varied from case to case, but they were lower than those observed in MCLs. At the protein level, Western blotting analysis showed Cyclin D1 protein expression in the 11 HCLs analysed. No bcl‐1 rearrangements were seen with the MTC, p94PS and PRAD‐1 (Δ‐P1‐4) probes used, and no PRAD‐1/CCND1 gene amplification was detected in any case. These findings indicate that PRAD‐1/CCND1 is over‐expressed at mRNA and protein levels in a high number of HCLs. However, the levels of expression are much lower than in MCLs, and this expression is not associated with bcl‐1 rearrangements or PRAD‐1/CCND1 gene amplification.
Chronic active Epstein‐Barr virus disease in a case of persistent polyclonal B‐cell lymphocytosis
Authors: MITTERER MANFRED , PESCOSTA NORBERT , FEND FALKO , LARCHER CLARA , PRANG NADJA , SCHWARZMANN FRITZ , COSER PAOLO , HUEMER HARTWIG P.
Published: March 2008

Summary. Persistent polyclonal B‐cell lymphocytosis (PPBL) is a rare haematological disorder. It is characterized by activated and morphologically atypical B lymphocytes and polyclonal IgM production and has been associated with female sex, cigarette smoking, and HLA‐DR7 expression.

We report a case of PPBL with intermitting symptoms compatible with a chronic fatigue syndrome, recurrent erythema nodosum and multiforme. Serological findings suggested a chronic active Epstein‐Barr virus (EBV) infection. Messenger RNA of EBV immediate early gene transactivation BZLF1 was detected in peripheral blood lymphocytes by reverse transcriptase PCR indicating a persistent replication of the virus. Over 2 years of observation we detected varying numbers of atypical lymphocytes. These cells hybridized with a probe specific for the EBV internal repeat region (BamHI W) which indicates a productive infection. Of interest, no reaction was observed with a probe specific for the latency‐associated small RNAs (EBERs). The immunological phenotype of the polyclonal B cells was similar to B‐cell lines immortalized by EBV in vitro, expressing a number of activation molecules (CD23, CD25, CD54) and the bcl‐2 protein. In summary, our findings suggest that persistent EBV replication might be crucial in the development of lymphoproliferative disorders such as PPBL.
Ethnic distribution of allele aLELY, a low‐expression allele of red‐cell spectrin a‐gene
Authors: MARECHAL J. , WILMOTTE R. , KANZAKI A. , DHERMY D. , GARBARZ M. , GALAND C. , TANG T. K. , YAWATA Y. , DELAONAY J.
Published: March 2008

Summary. AlleleαLELY low‐expression allele of erythroid spectrin a‐chain. It carries mutations both in exon 40 and intron 45 and is associated with partial skipping of exon 46. Allele αLELY remains asymptomatic by itself. In contrast, it enhances the expression level of deleterious α‐alleles occurring in trans, and as such has clinical importance. The aim of this study was to evaluate the incidence of allele QLBLY mvarjous ethnic groups, i.e. Caucasians, African Blacks, Japanese and Chinese. Allele QLELY occurred in all groups investigated with a fairly uniform frequency: 31%, 21%, 20% and 22%, respectively. Mutations in exon 40 and intron 45 appeared linked to one another without exception. Partial skipping of exon 46 or the low‐expression feature, whenever they could be assessed, were invariably observed. Allele αLELY appears to be an ancient and stable allele.
The myelodysplastic syndromes: an analysis of prognostic factors in 226 cases from a single institution
Authors: CUNNINGHAM I. , MACCALLUM S. J. , NICHOLLS M. D. , BYTH K. , HEWSON J. W. , ARNOLD B. , MOTUM P. I. , MULLIGAN S. P. , CRANE G. G.
Published: March 2008

Summary. Two hundred and twenty‐six patients were diagnosed with myelodysplastic syndrome (MDS), according to the French‐American‐British (FAB) criteria, over a 13‐year period, and studied retrospectively in a single institution in order to study indicators which were prognostically significant.

Analysis of clinical and laboratory data indicated that the FAB classification, the Bournemouth, Dusseldorf, Goasguen, Sanz and FAB Scoring Systems were all good predictors of survival.

We found advancing age, haemoglobin (Hb) ≤ 9g/dl, platelet count ≤ 50 × 109/1, increased peripheral total white cell count (WCC) and monocytosis, increased bone marrow blasts, dysgranulopoiesis, and bone marrow fibrosis were significant adverse prognostic variables.

The commonest complication and cause of death was infection; however, infective episodes were not significantly associated with low neutrophil counts (either ≤ 1‐5 × 109/1 or ≤ 08 × 109/1) and there was also no significant association between neutropenia and survival. These findings indicate that neutrophil dysfunction plays an important role in the clinical progression of patients with MDS. The effect of new therapeutic modalities, such as the haemopoietic growth factors, on reducing infective episodes may be as significant as their effect on increasing neutrophil counts.
CD3‐induced T‐cell activation in the bone marrow of myeloma patients: major role of CD4+ cells
Authors: BIANCHI ALBERTO , MONTACCHINI LAURA , BARRAL PAOLA , BORRIONE PAOLO , ATTLSANO CARMELA , ORSINI ENRICA , BOCCADORO MARIO , PILERI ALESSANDRO , MASSAIA MASSIMO
Published: March 2008

Summary. A large expansion of activated T cells (CD3+CD25+) with the potential to act as anti‐tumour effector cells is inducible in multiple myeloma (MM) patients by culturing bone marrow mononuclear cells (BMMCs) with the anti‐CD3 monoclonal antibody (mAb) OKT3. The aim of this study was to provide a greater characterization of CD3‐activated T cells. On day 6, most T cells coexpressed the CD1 la, CD18, CD54, CD45R0 antigens and consisted of activated (CD25+) CD4+ and CD8+ cells in nearly equal proportions. Kinetics studies showed that CD4+CD25+ cells proliferated more rapidly and peaked earlier than CD8+CD25+ cells. When experiments were performed with purified subpopulations by removing CD4+ cells (resulting in CD8+ BMMCs) or by removing CD8+ cells (resulting in CD4+ BMMCs), T‐cell activation and autologous plasma cell decrease were observed in CD4+ BMMCs only. Transwell cultures showed that CD4 help was necessary to make CD8+ BMMCs susceptible to CD3 stimulation. Relevant amounts of IL‐2 were found in the supernatants of CD4+ BMMCs cultures, whereas no secretion of IL‐4 was detected, indicating a Thl‐like profile of CD3‐activated CD4+ cells.

These data indicate that CD4+ cells proliferate earlier and provide optimal help to induce the subsequent expansion of CD8+ cells after CD3 stimulation of MM BMMCs. Adequate stimulation of CD4+ cells is therefore essential in any strategy aiming to recover T‐cell‐mediated immunity in MM.