bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet‐induced neutrophil activation: platelet‐expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18
Authors: RUF ANDREAS , PATSCHEKE HEINRICH
Published: March 2008

Summary. Mutual contacts and platelet‐expressed fibrinogen seem to be required for the stimulation of neutrophils by activated platelets. The β2‐integrins CDllb/CD18 and CDllc/CD18 are potential receptors for fibrinogen on neutrophils. In order to investigate whether binding of fibrinogen to these integrins is involved, monoclonal antibodies (MoAbs) and Gly‐Pro‐Arg‐Pro (GPRP) peptide that inhibits fibrinogen binding to CDllc/CD18 were checked for their effects on the interaction of activated platelets and neutrophils. The luminol‐amplified chemilumi‐nescence (CL) as a measure for the oxidative burst of neutrophils was recorded simultaneously to the platelet aggregation in mixed cell suspensions. The adhesion of platelets and neutrophils was determined microscopically. The thromboxane A2 mimetic U46619 was used as a potent platelet agonist but that does not stimulate neutrophils. Stimulation of the platelets with U46619‐induced platelet aggregation and a strong CL of neutrophils. The platelet‐induced activation of neutrophils required added fibrinogen which fibronectin or thrombospondin could not substitute for. Cytochalasin D (Cyto D) that blocks actin polymerization totally abrogated the platelet‐induced CI of neutrophils. The MoAb OKM1 against CDllb, which blocks fibrinogen binding to CDllb/CD18 as well as the MoAbs I0T16 and IOT18 directed against CDlla and CD18, respectively, had no effect. In contrast, the MoAb LeuM5 which inhibits the binding of fibrinogen to CDllc/CD18 revealed a strong inhibition. Furthermore, GPRP peptide which CDllc/CD18 recognizes on the Aa‐chain of fibrinogen also strongly inhibited the platelet‐induced CL of neutrophils, whereas control peptides such as Gly‐His‐Arg‐Pro (GHRP) or Gly‐Pro‐Gly‐Gly (GPGG) had no effect. In contrast to the platelet‐induced CL of neutrophils, Cyto D, MoAb against CD11c and GPRP peptide did not inhibit the CL induced by FMLP and PAF in pure neutrophil suspensions. They also did not affect U46619‐induced platelet aggregation. The adhesion of platelets and neutrophils was neither dependent on added fibrinogen nor inhibited by Cyto D, MoAb against CD1 lc and the GPRP‐peptide. Therefore fibrinogen and actin polymerization seem not to be required for the adhesion of neutrophils to platelets. However, the activation of neutrophils depends on the interaction of CDllc/CD18 with the Aa‐chain of platelet‐expressed fibrinogen and the contractile system of neutrophils.
Reverse dot‐blot detection of Thai β‐thalassaemia mutations
Authors: SUTCHARITCHAN PRANEE , SAIKI RANDALL , FUCHAROEN SUTHAT , WINICHAGOON PRANEE , ERLICH HENRY , EMBURY STEPHEN H.
Published: March 2008

Summary. Pending curative therapy, newborn screening and prenatal diagnosis are essential to the management of (3 thalassaemia. Diagnosis using electrophoretic methods is difficult in the presence of composite phenotypes and high Hb F levels. Direct DNA detection of mutant alleles circumvents both problems, but the enormous diversity of β‐thalassaemia mutations poses challenges for this approach. Among PCR‐based tests, the reverse dot‐blot method enables screening several mutations with a single hybridization reaction. Unfortunately it has often been targeted to only the common mutations of a particular ethnic population, necessitating the use of more arduous detection methods for the less common mutations. We developed a reverse dot‐blot strip for the 10 β‐thalassaemia mutations, including the β‐thalassaemic haemoglobino‐pathies Hb E and Hb Malay, that account for 96% of j3 thalassaemia in Thailand, and another strip for six less common Thai mutations. The second strip precludes the need for more technically challenging methods. To avoid problems associated with secondary structure of amplified full‐length target DNA, we amplified and labelled β‐globin DNA as two shorter fragments that encompassed all known Thai mutations. Reverse dot‐blotting is a rapid, accurate method for detecting β‐thalassaemia mutations.
Autotumour reactive T‐cell clones among tumour‐infiltrating T lymphocytes in B‐cell non‐Hodgkin's lymphomas
Authors: SHI ISABELLE , BONNEFOIX THIERRY , VACON FRANCHISE HEUZE‐LE , JACOB MARIE‐CHRISTINE , LEROUX DOMINIQUE , GRESSIN REMY , SOTTO MARIE‐FRANCE , CHAFFANJON PHILIPPE , BENSA JEAN‐CLAUDE , SOTTO JEAN‐JACQUES
Published: March 2008

Summary. Seventy‐three T‐cell clones (TCC) were established from tumour‐infiltrating lymphocytes‐T (TIL‐T) derived from lymph nodes involved by B‐cell non‐Hodgkin's lymphomas (B‐NHL) in nine patients with different histological subtypes and clinical stages. 40 TCC (55%) expressed the CD25 Ag and were also able to proliferate in the presence of irradiated autologous B‐NHL cells. Among them, 23 autotumour (AuTu) proliferative TCC were found not to proliferate to autologous EBV‐transformed B‐cell lines, indicating that the proliferative reactivity of these TCC was preferentially directed at autologous B‐NHL cells. Tested against autologous B‐NHL cells, only three AuTu prolifera‐ tive TCC (CD8 +) showed a significant level of cytotoxicity (specific lysis > 15%). In blocking experiments, the AuTu proliferative reactivity of three TCC from one patient was strongly inhibited by anti‐DR and anti‐DQ mAbs, whereas that of three TCC from another patient was not affected by either anti‐MHC class I or class II (DR., DP, DQ) mAbs. These findings suggest that the recognition of autologous B‐NHL cells by AuTu proliferative TCC may occur through MHC‐restricted as well as MHC‐unrestricted mechanisms.
Theophylline, a new inducer of apoptosis in B‐CLL: role of cyclic nucleotides
Authors: MENTZ FRANK , MERLE‐BERAE HFLENE , OUAAZ FATEH , BINET JACQUES‐LOUIS
Published: March 2008

Summary. We report a case of indolent B‐chronic lymphocytic leukaemia (B‐CLL) in a stage A patient, treated for 10 years only by theophylline for bronchial asthma. As suggested by the spontaneous apoptosis in the patient's blood (10%), theophylline at 50μg/ml increased spontaneous apoptosis after 72 h in culture by a mean percentage of 90% (range 79‐98%) in six B‐CLL cases studied in vitro. This effect was partially reversed with Rp‐cAMP. a cAMP antagonist, which implies a potent role for this second messenger. We describe a new property of theophylline. which might be an alternative treatment in B‐CLL.
Soluble c‐kit molecule in serum from healthy individuals and patients with haemopoietic disorders
Authors: Kawakita Makoto , Yonemura Ydji , Miyake Hirosada , Ohkubo Toshiya , Asou Norio , Hayakawa Kayoko , Nakamura Mitsurd , Kitoh Takashi , Osawa Hisao , Takatsuki Kiyoshi , Suda Toshio
Published: March 2008

Summary The proto‐oncogeae, c‐kit, encodes a transmembrane tyrosine kinase receptor (KIT) and plays an important role in haemopoiesis. We have identified a 95kD soluble form of KIT (S‐KIT) in culture supernatant of human megakaryoblastic cell line, CMK. To study the physiological significance of S‐KIT, we have established a sensitive sandwich ELISA system. Serum samples from healthy individuals contained detectable amounts of S‐KIT. Next, we determined a total of 220 samples from 134 patients with haemopoietic disorders. A considerable number of patients with acute myeloid leukaemia (AML), especially those with more immature phenotypes (MO, Ml or M2) had elevated levels of serum S‐KIT. Those levels decreased to the normal range after effective chemotherapy. In chronic myeloid leukaemia, patients with myeloid blastic crisis showed markedly elevated levels of serum S‐KIT. In contrast, S‐KIT levels decreased in cases with either acute or chronic lymphoid leukaemia. There was a tendency for patients with severe aplastic anaemia to show decreased levels, but it was not significant. In myelodysplastic syndrome, S‐KIT levels appeared to vary by subsets, with higher concentration in more advanced forms of the disease. Although the functional role of S‐KIT is not yet elucidated, these results suggest that the serum S‐KIT levels may reflect the pathological states of various haematological disorders.
Changes in c‐Kit expression and effects of SCF during differentiation of human erythroid progenitor cells
Authors: Uoshima Nobuhiko , Ozawa Masaru , Kimura Shinya , Tanaka Koji , Wada Katsuya , Kobayashi Yutaka , Kondo Motoharu
Published: March 2008

Summary We analysed c‐Kit expression during erythroid differentiation using immunocytochemical staining and flow cytometric analysis. Burst‐forming units‐erythroid (BFU‐E)‐derived cell aggregates were identified in methylcellulose cultures containing human umbilical cord blood CD34+ cells and were stained by the indirect immunoalkaline phosphatase method. To investigate the changes in levels of cell‐surface c‐Kit expression, we subjected progenitor cells in liquid culture to flow cytometric analysis. In addition, the effects of stem cell factor (SCF) on cell‐surface c‐Kit expression were analysed in these two culture systems and the effects of SCF on erythroid colony formation were studied in a methylcellulose culture. c‐Kit was expressed on the cell surface from BFU‐E to erythroid precursors recognized morphologically as basophilic erythroblasts. Flow cytometric analysis showed that c‐Kit expression increased until 6 d in liquid culture, and that decreased expression of c‐Kit was associated with the increased expression of glycophorin A. Moreover, SCF increased the size of erythroid colonies when added at days 0, 4 and 8 in methylcellulose cultures. These results indicate that the c‐Kit/SCF system still plays in proliferation of erythroid progenitor cells at the colony‐forming units‐erythroid stage. Finally, expression of c‐Kit in erythroid progenitor cells cultured without SCF showed a diffuse pattern on the cell surface, whereas we observed positive c‐Kit immuno‐reactivity in the region of the Golgi apparatus of these cells cultured with SCF. Flow cytometric analysis also showed that the levels of cell‐surface c‐Kit expression decreased in the presence of SCF. These results suggest that SCF induced down‐modulation of cell‐surface c‐Kit expression, despite continuous synthesis of c‐Kit protein.
Molecular defect of a phosphoglycerate kinase variant associated with haemolytic anaemia and neurological disorders in a large kindred
Authors: Turner G. , Fletcher J. , Elber J. , Yanagawa Y. , Dave V. , Yoshida A.
Published: March 2008

Summary The X‐chromosome‐linked phosphoglycerate kinase (PGK) deficiency associated with severe chronic and acute haemolytic anaemia and mental disorders was first described in a large Chinese kindred in 1969. The molecular abnormality of this original variant remained to be identified. The red cell PGK activity was only about 5%, but the activity of the patients’lymphoblastoid cells was about 15% of normal. The PGK mRNA content of the patients’lymphoblastoid cells were normal. Analysis of the patients’mRNA showed the existence of a nucleotide transversion A ± T at position 491 (counting from adenine of the initiation codon). The mutation should cause an amino acid substitution Aspval at position 163 of the enzyme. The replacement of the acidic aspartic acid by a hydrophobic valine is expected to induce drastic structural instability resulting in severe enzyme deficiency in the patients’tissues.

The genotypes of two affected males, their mothers and 22 females of the family were identified by the PCR‐mediated method using their genomic DNA samples. 13/24 females examined were found to be variant heterozygous. In this large family, affected males over three generations have died at a pre‐adult age. Post‐ and pre‐natal genotyping of the family members may prevent future problems.
β‐Thalassaemia unlinked to the β‐globin gene interacts with sickle‐cell trait in a Portuguese family
Authors: Pacheco P. , Peres M. J. , Faustino P. , Pischedda C. , Gonclalves J. , Carvajales‐Ramos M. , Seixas T. , Martins M. C. , Moi P. , Lavinha J.
Published: March 2008

Summary An autosomally transmitted hypochromic microcytic mild anaemia with elevated haemoglobin (Hb) A2 and globin chain imbalance has been observed in a three‐generation family of Portuguese origin. Extensive DNA analysis of the β‐globin gene cluster, including the complete sequencing of the,β‐globin gene and flanking regions, failed to reveal any genetic alteration. The co‐segregation of sickle‐cell trait in this family enabled us to postulate a defective erythroid trans‐acting factor was playing a role in the down‐regulation of both βA‐ and βs‐globin genes. Among the transcription factors that could possibly have caused the reported phenotype, NF‐E2 is unlikely to be implicated, whereas Nrf1 and Nrf2 cannot be ruled out. Thus, this family carries a novel β‐thalassaemia autosomal determinant unlinked to the β‐globin gene. This observation reinforces the notion of the haemoglobinopathies as single gene disorders under polygenic regulation.
Detection of CBFB/MYH11 transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and remission
Authors: Tobal K. , Johnson P. R. E. , Saunders M. J. , Harrison C. J. , Yin J. A. Liu
Published: March 2008

Summary .The pericentric inversion of chromosome 16 [inv(16)(pl3q22)] and t(16;16)(pl3;q22) are chromosomal rearrangements frequently associated with AML FAB type M4Eo resulting in the production of a fusion gene CBFB/ MYH11. We studied 17 patients with a chromosome 16 abnormality (eight M4Eo, two Ml, one M2, three M4 without abnormal eosinophils, three MDS) for the presence of CBFB/MYH11 transcripts using an RT‐PCR technique. 10 AML patients with inv(16) tested RT‐PCR positive (eight at presentation, one in remission, one in remission and relapse). Three of these patients were originally reported by cytogenetic analysis to have del(16q22) but the positive RT‐PCR results prompted a cytogenetic re‐examination, resulting in the correction of the reports to inv(16). We show that although inv(16) is closely associated with AML M4Eo, it can also be detected in cases of AML M4 without abnormal eosinophils. Three cases of MDS with inv(16) were also RT‐PCR positive. Four patients with other chromosome 16 abnormalities were RT‐PCR negative. Four AML patients with inv(16) were studied in remission. All were RT‐PCR positive, including one patient in remission for 108 months and one 22 months post allogeneic bone marrow transplant. In the latter two remission patients, RT‐PCR evaluation was positive in bone marrow (BM) but not in peripheral blood, suggesting that BM may be the more informative. We conclude that this technique is valuable in the accurate molecular classification of AML, particularly as treatment options may be influenced by such information. Though RT‐PCR is highly sensitive in detecting CBFB/MYH11 fusion transcripts during remission, monitoring of minimal residual disease in patients with inv(16) remains to be established.
Malignant transformation of monoclonal gammopathy of undetermined significance among out‐patients of a community hospital in Southeastern Netherlands
Authors: van de Poel M. H. W. , Coebergh J. W. W. , Hillen H. F. P.
Published: March 2008

Summary Patients with a monoclonal gammopathy without evidence of lymphoproliferative or plasma cell malignancy within a year are still at risk for malignant transformation to multiple myeloma, Waldenstrom's macro‐globulinaemia or non‐Hodgkin's lymphoma. In a prospective study performed at the Mayo Clinic, the cumulative incidence of malignant transformation was 29% in 14 years. We conducted a retrospective study to determine the frequency of malignant transformation among 334 un‐selected out‐patients with monoclonal gammopathy of undetermined significance (MGUS) from a community hospital in Southeastern Netherlands. The cumulative incidence of malignant transformation was 11% in 14 years (95% confidence interval 6–17%). The long‐term survival of patients with MGUS was slightly lower than that of the average regional population. In a nested case‐control study, presence of a kappa light chain was found to be a risk factor for malignant transformation (70% of patients who developed malignant transformation compared to 30% of the control group, P < 001). Likewise, an initial high gamma globulin level was also found to be a risk factor (18–7g/1 v 13–7g/l in the control group, P<001). As neither risk factor has been described before, the significance of these factors for definition of a high‐risk group among patients with monoclonal gammopathy remains to be determined.
Structural integrity of the cyclin‐dependent kinase inhibitor genes, p15, p16 and p18 in myeloid leukaemias
Authors: Nakamaki Tsuyoshi , Kawamata Norihiko , Schwaller Juurg , Tobler Andreas , Fey Martin , Pakkala Seppo , Lee Young Y. , Kim Byoung K. , Fukuchi Kunihiko , Tsuruoka Nobuyoshi , Kahan Jerry , Miller Carl W. , Koeffler H. Phillip
Published: March 2008

Summary The cyclin‐dependent kinase inhibitors known as p15, p16 and p18 have been suggested as candidates for tumour suppressor genes. We examined these genes for their alterations in 46 myeloid leukaemias and 15 myeloid leukaemia cell lines, p16 mRNA expression was studied in 41 myeloid leukaemias. The p15 and p16 genes were either deleted or mutated in myeloid leukaemia lines at a high frequency [6/15 (40%) for p15; 8/15 (53%) for p16] but alterations in primary myeloid leukaemias are much less frequent [2/46 (4%) for p15; 3/46 (6%) for p16]. Alterations of p18 were not found in any of the samples. 13 primary myeloid leukaemia samples had negligible levels of p16 mRNA. In summary, the deletions of p15 and p16 genes identified in the myeloid leukaemia cell lines probably occurred during their in vitro immortalization. Alterations of the pl6 or pl5 gene only occurred in primary acute myeloid leukaemia samples that were of mixed myeloid/ lymphoid lineage (CD19/CD20‐positive acute myeloid leukaemia [AML], CD2/CD19‐positive AML, and lymphoid blastic crisis of chronic myeloid leukaemia). Further studies are required to determine if the absence of mRNA expression results from inactivation of the p16 gene.
Reactivity of autoantibodies from chronic ITP patients with recombinant glycoprotein Ilia peptides
Authors: Bowditch R. D. , Tani P. , McMillan R.
Published: March 2008

Summary Chronic immune thrombocytopenia is an autoimmune disorder characterized by destructive thrombocytopenia due to the formation of autoantibodies against platelet‐associated antigens. Most antiplatelet autoantibodies react with either the platelet glycoprotein IIb/IIIa or Ib/IX complex, whereas some plasma autoantibodies react with glycoprotein IIIa. Previous studies from our laboratory suggested that most platelet‐associated autoantibodies to platelet GPIIb/IIIa, which bind to the intact complex, bind much less avidly to the EDTA‐dissociated complex, suggesting that the epitopes were complex‐dependent. To evaluate this further we have studied the binding of platelet‐associated autoantibody and plasma auto‐ and alloantibody eluates to large recombinant GPIIIa peptides: peptide 1 (GPIIIa Gly1‐Val200); peptide 2 (GPIIIa Arg150‐Glu400); peptide 3 (GPIIIa Lys350‐Asp550); peptide 4 (GPIIIa Asn450‐Val700) and peptide 5 (GPIIIa Trp715‐Thr762, cytoplasmic fragment). Of the 33 platelet‐associated antibody eluates tested, all bound avidly to the GPIIb/IIIa complex, but only one showed significant binding (>3 SD above control values) to one of the immobilized peptides (peptide 3). Conversely, antibodies known to bind to specific regions of GPIIIa (murine monoclonal antibody, anti‐LIBS2; plasma autoantibody against the GPIIIa cytoplasmic fragment and anti‐P1A1 antibody) all bound avidly to the GPIIIa peptide containing the appropriate epitope. Based on these and our previous results, we conclude that platelet‐associated antibodies from chronic ITP patients rarely bind to epitopes localized to GPIIIa alone.
Prevention of donor‐specific T‐cell unresponsiveness after buffy‐coat‐depleted blood transfusion
Authors: van Prooijen H. C. , Aarts‐Riemens M. I. , van Oostendorp W. R. , Hene R. J. , Gmelig‐Meyling F. H. J. , de Weger R. A.
Published: March 2008

SummaryThe immunosuppressive effect of blood transfusions has been demonstrated in several clinical studies. The effect is probably mediated by HLA‐class‐H‐bearing donor leucocytes, because results from laboratory tests show specific down‐regulation of the recipient's T‐cell response after administration of blood from donors sharing one HLA haplotype with the recipient.

In the present study we evaluated the immunosuppressive potential of buffy‐coat‐depleted red cell transfusions in patients waiting for renal transplantation, by measuring the frequency of cytotoxic precursor T cells before and after transfusion. The buffy coat was removed from whole blood by the Optipress system and resulted in ±97% depletion of lymphocytes and monocytes. A single transfusion of HLA‐haplotype‐matched buffy‐coat‐depleted red cells induced donor‐specific down‐regulation of T‐cell responses in only two of 14 patients. Since HLA‐class‐H‐bearing cells are also involved in the induction of anti‐HLA antibodies, we evaluated retrospectively the incident of HLA alloimmunization after a single transfusion of buffy‐coat‐depleted red cells. No anti‐HLA antibodies were found in 140 patients at risk for primary immunization.

We conclude that the poor immunological responses found after a single transfusion of HLA‐haplotype‐matched buffy‐coat‐depleted red cells is due to the small number of residual HLA‐class‐II‐bearing donor cells. This blood component should not be used for induction of immunosuppression.
Effect of interleukin‐3 pretreatment on granulocyte/macrophage colony‐stimulating factor induced mobilization of circulating haemopoietic progenitor cells
Authors: Geissler Klaus , Peschel Christian , Niederwieser Dietger , Goldschmitt Jutta , Hladik Florian , Fritz Angelika , ÖHler Leopold , Bettelheim Peter , Huber Cristoph , Lechner Klaus , Hocker Paul , Kolbe Karin
Published: March 2008

Summary. Recombinant human colony stimulating factors (CSFs) as single agents are increasingly used for mobilizing peripheral blood progenitor cells (PBPCs) for stem cell transplantation. We have shown in rhesus monkeys that interleukin‐3 (IL‐3) pretreatment markedly potentiated the increase in PBPC numbers of subsequent administration of granulocyte/macrophage‐CSF (GM‐CSF). Here we studied the effect of IL‐3 pretreatment on GM‐CSF‐induced mobilization of PB progenitors in patients who were potential candidates for autologous stem cell transplantation (n=16). Patients were treated with GM‐CSF at a dose of 5 μg/kg/d for 5 d and after a treatment free interval received another cycle of GM‐CSF immediately following pretreatment with IL‐3 at different doses and duration: 2.5 μg/kg/d (n = 4), 5μg/kg/d (n = 3) and 10μg/kg/d (n = 3) for 3d, 5μg/kg/d for 7d (n = 4) and 5μg/kg/d for 14 d (n = 2), respectively. Only 7d pretreatment with IL‐3 showed consistent effects. Although IL‐3 did not mobilize by itself, pretreatment with 5μg/kg/d of IL‐3 for 7d significantly potentiated GM‐CSF‐induced mobilization of PB CFU‐GM numbers, leading to a mean increase in PB CFU‐GM numbers over baseline by 18.5 +5.2 (SEM) fold by IL‐3/ GM‐CSF as compared to a 4.7 + 1.7‐fold increase by GM‐CSF alone. A significant enhancement by the 7d IL‐3 pretreatment was also observed for erythroid (BFU‐E) and multi‐potential progenitor cells (CFU‐mix) which were 3.3 + 1.3‐and 3.4 + 0.9‐fold, respectively, mobilized by GM‐CSF alone, as compared to 8.5 + 2.3‐ and 19.2 + 3.4‐fold, respectively, by the IL‐3/GM‐CSF combination. Our results suggest that 7 d pretreatment with IL‐3 may be a useful mean to augment mobilization of circulating progenitors by more lineage‐restricted CSFs. These findings may be important for the design of mobilization strategies that use growth factors without preceding chemotherapy.
Recombinant human interleukin‐3 in refractory severe aplastic anaemia: a phase I/II trial
Authors: Bargetzi Mario J. , Gldckman Eliane , Tichelli Andre , Devergie Agnes , Esperou Helene , Kabata Janusz , Wodnar‐Filipowicz Alexandra , Nissen Catherine , Speck Bruno , Gratwohl Alois
Published: March 2008

Summary. In a prospective open‐labelled phase I/II trial we tested efficacy and tolerability of recombinant human interleukin‐3 (rhIL‐3) alone in patients with refractory severe aplastic anaemia (SAA). 15 patients with idiopathic (12 patients) or secondary (one post‐hepatitic, one drug induced, one dyskeratosis congenita) SAA, refractory or relapsing after one to three courses of antilymphocyte globulin were included. 14 patients were transfusion dependent (RBC 14, platelet 12). RhIL‐3 was planned for three patients each at five escalating dose levels of 1, 2, 4, 8 and 16pg/kg, given daily as 24 h continuous infusion for 21 d. RhIL‐3 was prematurely withdrawn at days 10 and 11 for adverse events in two patients. 9/15 patients showed an increase in WBC; 2/6 at the 1‐2 μg/kg and 7/9 at the 4‐16 μg/kg level, but no sustained effects were seen. No patient showed a response in platelet counts. Additionally, platelet and RBC transfusion requirements were unchanged pre and post study. All patients experienced one or more adverse event, mainly fever (15 patients), bleeding (nine patients), and headache (six patients). Occurrence of adverse events was dose related and the maximum tolerated dose was reached with 8 μg/kg. Five patients suffered serious adverse events. RhIL‐3 as single growth factor and used alone is of minimal benefit in severe aplastic anaemia.
Band 4.2 Shiga: 317 CGC → TGC in compound heterozygotes with 142 GCT → ACT results in band 4.2 deficiency and microspherocytosis
Authors: Kanzaki Akio , Yasunaga Mutsumi , Okamoto Naoto , Inoue Takafumi , Yawata Ayumi , Wada Hideho , Andoh Akira , Hodohara Keiko , Fujiyama Yoshihide , Bamba Tadao , Harano Teruo , Harano Keiko , Yawata Yoshihito
Published: March 2008

Summary. A novel compound heterozygous mutation of 317 CGC → TGC with 142 GCT → ACT in human red cell band 4.2 deficiency is described. A proband and his son suffered from compensated haemolysis with nearly complete deficiency of red cell band 4.2. Their red cell morphology exhibited microspherocytosis resembling classic hereditary spherocytosis (HS). Sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed band 4.2 to be nearly missing (< 1% of normal controls) with the presence of 74 kU and 72 kD isoforms in trace amounts. Other family members (daughters older and younger than the son) exhibited nearly normal amounts of 72 kD as a wild form of band 4.2 on SDS‐PAGE with the presence of the 74 kD isoform in a trace amount. The proband and his son demonstrated two compound heterozygous mutations in trans: i.e. nucleotide (nt) 949 CGC → TGC (codon 317 Arg → Cys) in exon 7 and nt 424 GCT → ACT (codon 142 Ala → Thr) in exon 3 of the band 4.2 gene. The two daughters demonstrated only the mutation of nt 949 CGC → TGC in exon 7 in heterozygous states, but no 142 mutation. Therefore the proband and his son were compound heterozygotes of these two mutations in trans. It is interesting to note that the 74 kD isoform of band 4.2 protein existed in a trace amount in the two daughters in spite of the absence of the 142 Ala → Thr mutation. In addition, even in the presence of the 142 mutation in one allele in the proband and his son, their red cell morphology demonstrated classic HS with microspherocytosis, although a homozygous state of the 142 mutation known as the Nippon type of band 4.2 deficiency exhibits ovalostomato‐cytosis.
Nasal natural killer (NK) cell lymphoma: report of a case with activated NK cells containing Epstein‐Barr virus and expressing CD21 antigen, and comparative studies of their phenotype and cytotoxicity with normal NK cells
Authors: Kaneko Takako , Fukuda Junko , Yoshihara Toshio , Zheng Heng , Mori Shigeo , Mizoguchi Hideaki , Oshimi Kazuo
Published: March 2008

Summary. Malignant lymphomas arising from the nasal cavity have been considered to be derived from T cells, but recent surface marker studies suggest that more than half of the lymphomas are derived from natural killer (NK) cells. Here we describe a case of nasal lymphoma whose lymphoma cells were identified as NK cells by morphological, phenotypic, immunogenotypic, and functional studies. We believe this is the first study with functional evidence of NK activity. When compared with normal freshly isolated NK cells or activated NK cells, the surface phenotypes and NK activity of the patient tumour cells were those of the activated, but not resting, NK cells. Also, Epstein‐Barr virus (EBV) was detected in the tumour cells and the lymphoma cells were found to be monoclonally expanded. The patient's lymphoma cells also expressed EBV receptor CD21 (CR2) and CD30 (Ki‐1) that have not been described on normal NK cells. We therefore examined highly enriched NK cells of normal donors, and found that some resting and/or activated NK cells express these antigens.
Comparative quantitative expression of bcl‐2 by normal and leukaemic myeloid cells
Authors: Bradbury D. A. , Russell N. H.
Published: March 2008

Summary. Expression of the bcl‐2 oncoprotein by AML blasts has previously been demonstrated to be heterogenous with high levels of bcl‐2 expression being associated with a low complete remission rate and poor survival. We have quantified bcl‐2 expression in AML blasts in relation to expression of the CD34 antigen and in comparison to CD34‐positive cells from normal bone marrow. When expressed as molecules of equivalent soluble fluorochrome (MESF) per cell, AML blast cell bcl‐2 expression varied from 11.1 to 99.9 × lO3 (median 39.4 × 103, n=56) with 28.5% of patients expressing high MESF values (>50 × 103) and 16% of patients expressing low MESF values (<20 × 103), the remainder expressing intermediate values. There was no significant difference between intensity of bcl‐2 expression and FAB classification in the de novo AML cases, and there was no significant differences between de novo and secondary AML cases. Blasts from CD34+ AML patients expressed significantly higher levels of bcl‐2 (mean MESF 43.6 × 103, n =36) than CD34− AML patients (mean MESF 31.7 × 103, n=19).

In five cases of CD34+ AML, bcl‐2 expression was determined on purified CD34+ and CD34− blast cell populations. In all cases CD34+ blasts were found to express significantly higher bcl‐2 MESF values compared to the CD34− fraction.

Purified CD34+ cells from normal bone marrow consistently expressed high levels of bcl‐2 (MESF >75 × 103 n = 4), which was comparable to that found on CD34+ AML cells. Our results suggest that the poor prognosis previously associated with AML blasts expressing the CD34 antigen may in part be related to high expression of bcl‐2. Also the ability to measure bcl‐2 in AML blasts quantitatively by flow cytometry and to categorize patients into discrete groups may be of value as a prognostic indicator in AML.
Paraneoplastic autoimmune phenomena in patients with myelodysplastic syndromes: response to immunosuppressive therapy
Authors: Enright Helen , Jacob Harry S. , Vercellotti Gregory , Howe Robert , Belzer Michael , Miller Wesley
Published: March 2008

Summary. We analysed the clinical features, course and response to immunosuppressive therapy in 30 patients with autoimmune disorders associated with myelodysplastic syndromes (MDS). 18 patients with MDS developed acute systemic autoimmune disorders. Common manifestations were skin vasculitis (n=15) and arthritis (n=11). Seven patients had an acute clinical syndrome of vasculitic skin rash, fever and arthritis with peripheral oedema in three and pulmonary infiltrates in five of these seven patients. Other acute manifestations included pericarditis, pleural effusions, skin ulceration, seizures, myositis and peripheral neuropathy. Chronic or isolated autoimmune manifestations (n=11) included glomerulonephritis, polyneuropathy, pyoderma gangrenosum, ulcerative colitis and polyarthritis. Classic connective tissue disorders recognized included relapsing polychondritis, polymyalgia rheumatica, Raynaud's syndrome and Sjogren's syndrome. Autoimmune manifestations responded to immunosuppressive therapy (primarily prednisone) in 26/27 patients treated. Furthermore, cytopenias improved substantially in six patients, including complete normalization of peripheral blood counts in two patients with cytogenetic remission in one. Patients with a haematological response to immunosuppressive therapy had improved survival compared with non‐responding patients. The autoimmune syndrome was implicated as a primary cause of death in 8/17 patients who died. Autoimmune manifestations may be more common than previously recognized in patients with MDS. Aggressive therapy with immunosuppressive agents in selected patients often controls autoimmune phenomena associated with MDS and may lead to haematological responses in some patients.
Trisomy 3 is a consistent chromosome change in malignant lymphoproliferative disorders preceded by cold agglutinin disease
Authors: Michaux L. , Dierlamm J. , Wlodarska I. , Stul M. , Bosly A. , Delannoy A. , Louwagie A. , Mecucci C. , Cassiman J. J. , van den Berghe H. , Michaux J. L.
Published: March 2008

Summary. Cold‐antibody autoimmune haemolytic anaemia is a rare entity that has been associated with a wide variety of pathological processes, including malignant lymphoproliferative disorders. In this retrospective study we recorded, as far as possible, clinical, haematological, immunological, morphological, pathological, cytogenetic and molecular data on 10 patients with cold agglutinin disease (CAD). Cytogenetic anomalies were found in four cases in which an underlying lymphoma could be evidenced. Trisomy 3 was the only recurrent aberration in our series. It was observed in all patients with abnormal karyotype, either as a complete trisomy or as a partial trisomy of the long arm. The importance of this particular karyotypic aberration in the monitoring of CAD is emphasized.
Resistance to activated protein C in healthy women taking oral contraceptives
Authors: Olivieri Oliviero , Friso Simonetta , Manzato Franco , Guella Anna , Bernardi Francesco , Lunghi Barbara , Girelli Domenico , Azzini Margherita , Brocco Giorgio , Russo Carla , Corrocher Roberto
Published: March 2008

Summary. Resistance to activated protein C (APC) is at present considered the most frequent laboratory abnormality in patients with deep‐vein thrombosis. An increased risk for venous thrombosis is associated to the use of oral contraceptives (OC).

We studied APC sensitivity in 50 healthy women taking OC and in 50 healthy controls, matched for age, smoking habit, educational and social levels, and the main biochemical routinary parameters. Subjects with a personal or familial history of thrombosis and also with chronic or acute diseases were excluded. Protein C, protein S, antithrombin III and lupus anticoagulant activity (LAC) were also evaluated. Increased fibrinogen and protein C levels, decreased protein S, and shortened PT and APTT were also observed in women taking OC. APC sensitivity ratio (APC‐SR) was significantly lower in the OC group than in a control group (2.6 ± 0.38 v 2.81 ±0.35, P<0.01). Seven of eight women with APC ratio ≤ 2 (APC resistant) were OC users: the difference of prevalence was statistically significant (chi‐squared test, P<:0′05). Only two out of eight women were found heterozygous for the Leiden factor V mutation. Two APC‐resistant women without the Leiden mutation subsequently discontinued OC and both then normalized their APC‐SR. We conclude that acquired factors, i.e. oral contraceptives, may play an important role in determining plasma APC resistance.
The use of an anti‐β2‐glycoprotein‐I assay for discrimination between anticardiolipin antibodies associated with infection and increased risk of thrombosis
Authors: McNally T. , Purdy G. , Mackie I. J. , Machin S. J. , Isenberg D. A.
Published: March 2008

Summary. Antiphospholipid antibodies (aPAs), occurring in association with infection, are not generally associated with an increased risk of thrombosis. Anticardiolipin antibodies (aCL) from patients with infection, unlike those from patients with SLE, do not have the β2GPI cofactor requirements. Antibodies to β2GPI (αβ2GPI) are more closely associated with a previous history of thrombosis than aCL in patients with SLE. In the present study we have investigated the reactivity of the αβ2GPI assay for aPAs associated with infection. Serum from 114 patients with infections including syphilis (n= 11), tuberculosis (n= 63) and Klebsiella (n=42) were assayed for αβ2GPI and aCL antibodies. The incidence of aCL in serum of patients with tuberculosis, Klebsiella infection and syphilis was 6.0%. 5.0% and 64.0%. respectively, but all patients were negative for αβ2GPI. These results indicate that the αβ2GPI assay is negative in patients with transiently positive aCL assays associated with infection.
Pulsed high‐dose dexamethasone in refractory chronic idiopathic thrombocytopenic purpura: a report on 10 cases
Authors: Caulier Marie Thérèse , Rose Christian , Roussel Marie Thérèse , Huart Claude , Bauters Francis , Fenaux Pierre
Published: March 2008

Summary. We treated 10 patients who had chronic refractory idiopathic thrombocytopenic purpura (ITP) with high‐dose dexamethasone (DXM, 40mg/d for 4 sequential days every month). The interval from diagnosis ranged from 49 to 300 months, and patients had previously received a median of 5.5 treatments (including splenectomy in nine cases). Median platelet count was 14 × 109/1 (range 6‐26 × 109/1) at the onset of DXM and eight patients had bleeding symptoms. Eight patients received at least three cycles of DXM.

Five patients had a response (i.e. platelet count at least doubled and increased by >20 × 109/1), including one almost complete remission and four minor responses (MR).

Of the MR, one was probably due to concurrent IVIg administration, and all four MR were transient, in spite of further cycles of DXM. In three patients DXM was a failure after three or four cycles. In two patients DXM had to be stopped after one course because of major side‐effects (systemic hypertension with stroke and insulin‐dependent diabetes, respectively).

In our experience, high‐dose DXM had a relatively limited effect in chronic refractory ITP and was associated with severe side‐effects in some cases.
Hepatitis C virus infection in multi‐transfused children with haematological malignancy
Authors: Myers B. , Irving W. , Holungsworth R. , Readett D. , Lilleyman J. S. , Dolan G.
Published: March 2008

Summary. Transmission of hepatitis C virus (HCV) is an important hazard of blood transfusion and may result in chronic liver disease. 98 children from Nottingham and Sheffield with haematological malignancies were studied to determine the prevalence of HCV infection by enzyme immunoassay and RT/PCR techniques. The children had been exposed to up to 184 donors through red cell and platelet transfusion, the majority prior to routine testing for HCV infection in blood donors. Only one sample showed evidence of HCV infection being both FXISA and RT/PCR positive. None of the samples taken since donor screening were positive. This provides reassurance as to the low rate of HCV acquisition in multi‐transfused patients in this part of the U.K. compared to other parts of the world.
Duodenal expression of NF‐E2 in mouse models of altered iron metabolism
Authors: Raja K. B. , Gerard B. , McKie A. T. , Simpson R. J. , Peters T. J. , Grandchamp B. , Beaumont C.
Published: March 2008

Summary. This study investigated the relationship between duodenal mucosal mRNA levels of the transcription factor, NF‐E2, H‐ferritin (a putative NF‐E2 regulated gene) and iron absorption in mice. CD 1‐strain mice with normal and altered iron metabolism (hypoxic, iron‐deficient, iron‐loaded) and animals with genetic defects of iron metabolism (hypotrans‐ferrinaemia, /Mhalassaemia) were studied. Tissue RNA from these mouse models was subjected to reverse transcription and PCR amplification for NF‐E2 and a stable ribosomal protein (SI4) and the products analysed with an automated laser fluorescent sequencer. Duodenal NF‐E2 mRNA levels were generally low and decreased in the hypoxic and iron‐deficient groups, both of which exhibited elevated iron absorption as compared to controls. A modest increase in the NF‐E2 mRNA level was seen in the iron‐loaded mice, whose iron absorption was decreased. In contrast, both the genetic strains showed elevated NF‐E2 mRNA levels in conjunction with raised iron absorption values. Only the iron‐deficient group exhibited an alteration in the duodenal mucosal H/L ferritin ratio. Hence, no relationship was evident between the NF‐E2 mRNA levels and the H/L ferritin ratio. These data indicate that NF‐E2 is not the primary regulator of intestinal iron absorption.
The effect of human fit‐ 3 ligand on committed progenitor cell production from normal, aplastic anaemia and Diamond‐Blackfan anaemia bone marrow
Authors: Scopes John , Daly Stephen , Ball Sarah E. , McGockin Colin P. , Gordon‐Smith Edward C. , Gibson Frances M.
Published: March 2008

Summary. We investigated the effect of the human ligand for flt‐3 (FL) on the committed progenitor colony formation of normal bone marrow (BM) (n = 9) and BM from four aplastic anaemia (AA) and three Diamond‐Blackfan anaemia (DBA) patients. Methylcellulose committed progenitor cell assays were carried out using FL alone and in combinations with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). interleukin‐3 (IL‐3) and c‐kit ligand (KL). FL alone had a limited, though significant, effect on the production of granulocyte‐macrophage colony‐forming unit (CFU‐GM) colonies from normal BM and showed an additive effect with IL‐ 3 and GM‐CSF separately, but not in combination. FL did not increase the stimulation of KL and did not have an effect on the production of erythroid progenitor colonies. FL had no effect on the AA and DBA BMs studied.
Correlation between a sandwich ELISA and an in‐vitro bioassay for erythropoietin in human plasma
Authors: Sakata Susumu , Enoki Yasunori , Shimizu Satoshi , Hattori Masatoshi , Ueda Masatsugu
Published: March 2008

Summary. A sandwich‐type enzyme‐linked immunosorbent assay (ELISA) for human erythropoietin (EPO) using two anti‐EPO monoclonal antibodies has been compared with an in‐vitro EPO bioassay based on CFU‐E colony formation in fetal mouse liver cell cultures. In normal subjects and non‐uraemic anaemic patients the plasma EPO values estimated with the ELISA correlated well with those by the bioassay, and also inversely with the values of blood Hb, PCV and RBC counts. Dose‐response curves for plasma and standard recombinant human EPO in the ELISA were parallel to each other. These results further confirm the validity for the ELISA in measuring circulating human EPO.
Alterations of effector cell molecule expression on neutrophils in granulocyte colony‐stimulating factor‐producing tumour
Authors: Ohsaka Akimichi , Saionji Katsu , Endo Katsuyuki , Kikuchi Masahiro , Takahashi Atsushi , Hagiwara Shinji , Itoh Yohjiro , Igari Jun
Published: March 2008

Summary. A 6 8‐year‐old man was diagnosed as having a granulocyte colony‐stimulating factor (G‐CSF)‐producing mediastinal tumour. Mediastinotomy was performed, and thereafter the elevated leucocyte count and serum G‐CSF concentration returned to the normal range. The surface expression of effector cell molecules on neutrophils was serially examined. Before operation, the expression of FcRI and CR1 was increased but the expression of FcRIH and L‐ selectin was reduced in the patient. The altered expression of these molecules returned to the normal levels after operation. These findings suggest that G‐CSF produced by the tumour modulated neutrophil effector cell molecule expression in the patient.
Glucosamine 6‐phosphate deaminase in Plasmodium falciparum
Authors: Weidanz Jon A. , Campbell Patrick , Moore Dwight , DeLucas Lawrence J. , Roden Lennart , Vezza Anne C.
Published: March 2008

Summary. The pathways of glucose utilization for energy production in the malaria parasite, Plasmodium falciparum, have been studied extensively. Little is known, however, about the reactions by which glucose is converted into complex carbohydrates in the parasite, and knowledge of the catabolism of these substances is likewise scanty. The present investigation was undertaken to determine whether the parasites possess a key enzyme of glucosamine catabolism, i.e. glucosamine 6‐phosphate deaminase (EC 5.3.1.40), which catalyses the conversion of the sugar phosphate to fructose 6‐phosphate and ammonia. Lysates of Plasmodium‐infected erythrocytes had substantially higher deaminase activity than control samples from normal erythrocytes, and an even higher specific activity was observed in extracts of isolated parasites, amounting to 20‐40 times that of uninfected cells. Anion exchange chromatography indicated that the parasite deaminase eluted in a retarded position when compared to the elution profile of the erythrocyte enzyme. The charge difference suggested by these findings was established more directly by chromatofocusing, which indicated pi values of 6‐85 and 8‐55 for the parasite and erythrocyte deaminases, respectively. Other differences were also observed, notably a greater thermolability on the part of the parasite enzyme. These results indicated that the parasites synthesize a specific deaminase that is distinct from the normal erythrocyte enzyme. Studies on synchronized parasite cultures further indicated that the parasite deaminase is developmentally regulated, because a dramatic increase in activity levels occurred during the later stages of parasite development.
Hb Arta [β45 (CD4) Phe→Cys]: a new unstable haemoglobin with reduced oxygen affinity in trans with β‐thalassaemia
Authors: Vassilopoulos George , Papassotiriou Ioannis , Voskaridou Ersi , Stamoulakatou Alexandra , Premetis Evangelos , Kister Jean , Marden Michael , Griffon Nathalie , Poyart Claude , Wajcman Henri , Galacteros Frederic , Loukopoulos Dimitris
Published: March 2008

Summary. The interaction of rare Hb variants with β°‐thalassaemia results in a quasihomozygous state where the erythrocytes contain the variant as the only major adult Hb component. Such a situation is a unique model that enables functional studies even in the case of a neutral variant that could not be isolated from Hb A. We report here an unusual patient carrying Hb Arta, a novel Hb variant [β45 (CD4) Phe→Cys], in trans with β°‐thalassaemia gene (β° 39). The aminoacid substitution at the critical CD corner of this Hb molecule renders the molecule unstable. In addition, haem is displaced in a position that favours the deoxy (T) conformation of the variant, but less than in Hb Cheverly [β45 (CD4) Phe‐Ser], and results in a p50 of 43 mmHg (pH 7‐4, 37°C) in the red cells with preservation of cooperativity. Solution studies of the almost pure Hb Arta show a 50% decrease in oxygen affinity and normal cooperativity; the Bohr effect and the interaction with organic phosphates are similar to those of Hb A. Hb Arta retains both normal homo‐ and hetero‐tropic effects allowing a well‐preserved oxygen transport in vivo despite a mild anaemia.
c‐kit point mutation of extracellular domain in patients with myeloproliferative disorders
Authors: Nakata Yoshio , Kimura Akiro , Katoh Osamd , Kawaishi Kuniko , Hyodo Hideo , Abe Kazuhiro , Kuramoto Atsushi , Satow Yukio
Published: March 2008

Summary. c‐kit is a tyrosine kinase receptor whose ligand is stem cell factor (SCF). Gene alteration of the c‐kit extracellular domain was analysed by polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) in 25 patients with myeloproliferative disorders (MPD). In the N‐terminal part of the domain, mobility shifts indicating sequence alteration were detected in three of the patients, two primary myelofibrosis (PMF) and one chronic myelogenous leukaemia (CML). The subsequent sequencing revealed the same point mutations at codon 52 causing amino acid substitution (Asp → Asn). To our knowledge this is the first report with a c‐kit point mutation found in human fresh tumour cells.