bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythroid development of the FDCP‐Mix A4 multipotent cell line is governed by the relative concentrations of erythropoietin and interleukin 3
Authors: Heyworth Clare M. , Alauldin Mazin , Cross Michael A. , Fairbairn Leslie J. , Dexter T. Michael , Whetton Anthony D.
Published: March 2008

Summary .Conditions are described which promote the erythroid development of the FDCP‐Mix A4 (A4) cell line with accompanying proliferation of the cells. The requirements for this development are low concentrations of interleukin 3 (IL‐3) plus the presence of erythropoietin (epo) and haemin. When high concentrations of IL‐3 are added with erythropoietin and haemin the cells do not differentiate and maintain their blast cell morphology. Addition of haemin, in the absence of erythropoietin, does not promote erythroid development, but the presence of haemin with erythropoietin promotes increased proliferation and maturation. The morphological maturation of A4 cells along the erythroid lineage is accompanied by a gradual loss of clonogenic potential, loss of A4 cell multipotency, increased erythropoietin receptor expression, and an increased expression of the β‐globin gene. An initial increase in mitogenic responsiveness to erythropoietin is followed by a decrease as the cells become refractory to all mitogenic stimuli with the acquisition of a postmitotic, mature erythroid cell phenotype.
Degradation of platelet glycoprotein Ib by elastase released from primed neutrophils
Authors: Aziz Khalil A. , Cawley John C. , Kamiguti Aura S. , Zuzel Mirko
Published: March 2008

Summary Mutual interactions between neutrophils (PMN) and platelets are recognized to be important in modulating the respective functions of these two cell types. Here we show that primary granule secretion from appropriately‐stimulated PMN can lead to complete proteolytic removal of GPIb from the platelet surface.

Thus, when the PMN in PMN/platelet mixtures were stimulated by FMLP, platelets lost GPIb as measured by ristocetin‐induced aggregation, flow cytometry and SDS‐PAGE analysis. This loss was most marked when PMN were primed by GM‐CSF, and could be inhibited by a specific elastase inhibitor. As expected, the al‐antiproteinase in plasma inhibited GPIb loss, but when PMN were strongly stimulated by FMLP and GM‐CSF in the presence of platelets this inhibition was incomplete or absent.

It is concluded that joint priming of PMN with GM‐CSF and platelets can cause a previously unrecognized degree of primary granule secretion which, via elastase, leads to platelet GPIb loss. We suggest that this loss is likely to be of physiological and pathophysiological importance.
A solitary plasmacytoma of donor origin arising 14 years after kidney allotransplantation
Authors: Shustik C. , Jamison B. M. , Alfieri C. , Scherer S. , Loertscher R.
Published: March 2008

Summary Allotransplantation of solid organs transfers passenger leucocytes which may give rise to a state of persistent microchimaerism. In this report we describe the case of a patient who developed a solitary plasmacytoma in a transplanted kidney more than 10 years after allografting. The diagnosis was established on the basis of the presence of a monoclonal IgG kappa peak in the serum, and light chain proteinuria, the plasmacytoid features of tumour cells including cell surface expression of IgG, kappa light chains, CD20, CD38 and CD56, the absence of lytic bone lesions and a normal bone marrow biopsy, and the disappearance of the monoclonal IgG peak after graft nephrectomy. A donor origin of the tumour was established by HLA DNA typing of tumour, tumour‐free kidney tissue, and peripheral blood leucocytes, respectively.
Splenic irradiation for chronic autoimmune thrombocytopenic purpura in patients with contra‐indications to splenectomy
Authors: Caulier Marie Thearegse , Darloy Franck , Rose Christian , Camier Ghislaine , Morel Pierre , Bauters Francis , Fenaux Pierre
Published: March 2008

Summary We treated by splenic irradiation eight patients with chronic idiopathic thrombocytopenic purpura (ITP, seven cases) or secondary autoimmune thrombocytopenic purpura (one case) who had contra‐indications to splenectomy. A total dose of 15 Gy was delivered to the spleen, with left kidney protection. One patient had a good durable response (>1 year); two patients had a good transient response (of 3 months duration) but they responded again to a second course of irradiation; two patients had only partial response, but have required no other treatments for 2 years; the three remaining patients had no response. Side‐effects were minor. Therefore splenic irradiation appears to be a therapeutic option in patients with chronic ITP who have contra‐indications to splenectomy.
Homologous restriction of complement‐mediated cell lysis can be markedly enhanced by blocking decay‐accelerating factor
Authors: Zhong Rui‐Kon , Kozii Rostyk , Ball Edward D.
Published: March 2008

Summary. Regulation of complement (C′) dependent lysis of cells is attributed to certain membrane proteins. One of these is decay‐accelerating factor (DAF), CD55, a 70 kD glycosylated protein which functions to protect host cells from damage by autologous C′. We hypothesized that blockade of DAF function by a monoclonal antibody (mAb) could augment C′‐dependent lysis mediated by another mAb to a cell surface antigen expressed on leukaemia cells. Thus, we tested the effects of the anti‐DAF mAb 1C6 on the ability of both rabbit and human C’to lyse human leukaemia cells through activation by complement‐fixing murine mAb. DAF antigen was highly expressed on most leukaemia cell lines and primary acute leukaemia blast cells tested. Murine mAb to CD15 (PM‐81) and to gp 120 (AML‐1‐99), both IgM, also bound to the majority of myeloid and lymphoid leukaemia cells. Using human serum as a source of C′, the addition of mAb 1C6 reduced by an additional 85‐94% the numbers of clonogenic HL‐60 (myeloid leukaemia) cells lysed by mAb PM‐81 alone. Similarly, the addition of mAb 1C6 reduced by an additional 87% the numbers of HL‐60 colonies eliminated by mAb AML‐1‐99 alone. Similar results were observed in an experimental purging model using the myeloid leukaemia cell lines HL‐60, U937 or NB4 cells as targets. These results show that mAb 1C6 can effectively block the actions of DAF. In the presence of mAb 1C6, the cytotoxic activity mediated by human C’was similar to that of rabbit C′. These results show that increased tumour cell killing can be achieved through DAF blockade. This finding has relevance to clinical trials using C′‐fixing mAb for purging bone marrow of occult tumour cells prior to autologous transplantation.
Is there a role for interleukin‐3 in Diamond‐Blackfan anaemia? Results of a European multicentre study
Authors: Ball S. E. , Tchernia G. , Wranne L. , Bastion Y. , Bekassy N. A. , Bordigoni P. , Debre M. , Elinder G. , Kamps W. A. , Lanning M. , Leblanc T. , Makipernaa A. ,   (Diamond‐Blackfan anaemia working groups for the European Society for Paediatric Haematology and Immunology and the Sociétéď Hematologie et ď Immunologie Pédiatrique)
Published: March 2008

Summary. Forty patients (nine adults aged 20‐54; 31 children aged 1‐17) with Diamond‐Blackfan anaemia (DBA) were treated with recombinant human interleukin‐3 (IL‐3) in a European multicentre compassionate‐need study. IL‐3 was given as a daily subcutaneous injection at a starting dose of 2.5 μg/kg, escalating at day 21 to 5 μg/kg, and then to 10 μg/kg if there was no response, for a total duration of 12 weeks. Three children achieved a significant response, achieving sustained remissions off all therapy. At the time of entry, one was steroid‐responsive and transfusion‐independent, and two were transfusion‐dependent. Two adults had a transient reduction in transfusion requirements, but could not tolerate the complete course of therapy. Eosinophilia was common; neutrophil and platelet counts were unaffected except in three patients in whom previously noted mild thrombocytopenia was transiently exacerbated. Clinical response to IL‐3 did not correlate with in vitro culture results. A comparison of individual patient characteristics of our study with previously reported series confirms earlier impressions that patients who have never achieved significant in vivo erythropoiesis in response to steroids or during a spontaneous remission are highly unlikely to respond to IL‐3. In contrast, there may be a 50% chance of a sustained remission, off steroids, in children who are steroid‐dependent and transfusion‐independent at the time of IL‐3 therapy, suggesting a possible role for a short course of IL‐3 earlier in the treatment of children with steroid‐responsive DBA.
Busulfan and melphalan as conditioning regimen for autologous peripheral blood stem cell transplantation in multiple myeloma
Authors: Alegre A. , Lamana M. , Arranz R. , Fernández‐Villalta M. J. , Tomás J. F. , Figuera A. , Cámara R. , Steegman J. L. , Casado F. , Requena M. J. , Planas J. L. , Vazquez L. , Granda A. , Fernández‐Rañada J. M.
Published: March 2008

Summary. Twenty‐four patients with multiple myeloma (MM), three (12‐5%) in complete remission (CR) and 21 (87‐5%) in partial remission (PR) were treated with high‐dose chemotherapy (HDCT) (busulfan 12 mg/kg‐l‐melphalan 140mg/m2) as preparative regimen for autologous peripheral blood stem cell (PBSC) transplantation. These cells were previously collected by leukapheresis after mobilization by high‐dose cyclophosphamide (HD Cy) + rhGM‐CSF (18 patients) or rhG‐CSF alone (six patients). Considering 23 evaluable patients following HDCT, the CR rate was 58% (14 patients) and the PR rate was 38% (nine patients). One transplant‐related death occurred following this regimen (4%). With a median follow‐up of 20 months (range 4‐34) after transplantation, 21 patients are alive (87%). Disease progression after transplantation was observed in four patients. Overall and relapse‐free actuarial survival at 24 months was 91% and 74%, respectively. 12 patients (50%) remain in CR 15 months (4‐34) post transplant. The major toxicity was mucositis. Busulfan + melphalan is a safe and feasible conditioning regimen for APBSCT in MM with acceptable toxicity and a high objective response rate, which may result in prolonged survival.
Evidence for a polyclonal nature of the cell infiltrate in sinus histiocytosis with massive lymphadenopathy (Rosai‐Dorfman disease)
Authors: Paulli M. , Bergamaschi G. , Tonon L. , Viglio A. , Rosso R. , Facchetti F. , Geerts M. L. , Magrini U. , Cazzola M.
Published: March 2008

Summary. Sinus histiocytosis with massive lymphadenopathy (SHML), or Rosai‐Dorfman disease, is rare histiocytic disorder of known origin which shares several cell markers with Langerhans’cell histiocytosis (LCH). Although Rosai‐Dorfman cells exhibit an aberrant immunophenotype, the indolent clinical course of SHML suggests a reactive disorder rather than a neoplastic process. Until recently this was prevailing opinion concerning LCH also, but recent studies have detected clonal histiocytes in all forms of this latter condition, which is therefore considered a clonal neoplastic disorder with highly variable biologial behaviour. To determine whether the histiocytic proliferation in SHML is polyclonal or clonal we used X‐linked polymorphic loci to assess clonality in lesional tissues in two women. Polymorpic regions of the human androgen receptor (HUMARA) locus were amplified by polymerase chain reaction (PCR) analysis. The HUMARA locus was informative in both cases and, following digestion with methylation‐sensitive enzymes, typical polyclonal X‐inactivation patterns were observed. Since abnormal cells accounted for >90% lesional tissue cells, we conclude that Rosai‐Dorfman histiocytic proliferation was polyclonal in the women studied.
CD34+ cell immunoselection from G‐CSF‐alone‐primed peripheral blood in children with low body mass
Authors: Kanold J. , Berger M. , Rapatel C. , Lumley L. de , Lutz P. , Plantaz D. , Vannier J. P. , Bergeron C. , Mechinaud F. , Favrot M. , Bonhomme J. , Travade P. , CHRU F. Demeocq
Published: March 2008

Summary. We report the data of CD34+ cell immunoselection from peripheral blood after G‐CSF‐alone mobilization (lOμg/kg/d s.c.) in nine children with neuroblastoma (median age 4 5 years (2‐8), median body weight 16kg (10‐20). Leukaphereses were carried out on a Cobe Spectra separator and two consecutive harvests (4 blood volumes processed) were used for immunoselection on a Ceprate(tm) column. The yield of CD34+ cells in the purified fraction was 50% (23‐80), with a median number of 2.8xl06 CD34+ cells/kg (1‐9‐4). All patients were reinfused with selected CD34+ cells after busulfan 600 mg/m2+ melphalan 180mg/m2 and achieved successful haemopoietic recovery.
Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors
Authors: Gachet Christian , Cattaneo Marco , Ohlmann Philippe , Hechler Béatrice , Lecchi Anna , Chevalier Jérôme , Cassel Dominique , Mannucci Pier Mannuccio , Cazenave Jean‐Pierre
Published: March 2008

Summary. Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP‐induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP‐induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca2+ influx. Binding studies, using the non‐ hydrolysable ligand [33P]2MeSADP, have shown that human platelets contain about 600 high‐affinity binding sites for 2MeSADP (Kd∼ 5 niw). These sites present pharmacological characteristics of a P2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP‐induced platelet aggregation but normal shape change display very low levels of [33P]2MeSADP binding sites.The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca2+ influx and the other a Gi protein‐coupled receptor responsible for Ca2+ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.
Quantitation of reticulated platelets: methodology and clinical application
Authors: Richards E. M. , Baglin T. P.
Published: March 2008

Summary. A practical technique for the preparation and analysis of reticulated platelets is described. The use of fixed platelet‐rich plasma has given optimal results and sample stability. This technique has detected consumptive causes of a platelet count <80 × 109/1 with a positive predictive value of 96% and negative predictive value of 100%. The course of the reticulated platelet percentage (RP%) in patients with resolving thrombocytopenic states is described, contrasting the elevation seen in consumptive disorders with the fall in aplastic states. In patients undergoing stem cell transplant procedures a rise in the RP% precedes the rise in the platelet count, indicating that the reticulated platelets represent cells that are recently released from the bone marrow. The RP% peak in patients receiving autologous peripheral blood progenitor cell transplants is higher and more closely temporally related to the recovery of platelet count than it is in patients receiving autologous bone marrow transplants.
Factor XIIIA calgary: a candidate missense mutation (Leu667Pro) in the beta barrel 2 domain of the factor XIIIA subunit
Authors: Aslam Shazia , Poon Man‐Chiu , Yee Vivien C. , Bowen Derrick J. , Standen Graham R.
Published: March 2008

Summary. Molecular analysis performed on a Canadian family with congenital factor XIII deficiency revealed a homozygous missense mutation (Leu667Pro) in exon 14 of the A subunit gene in three affected siblings. The mutation results from a T‐to‐C transition at nucleotide position 2087 and generates a new Msp1 restriction site. Digestion of an amplified fragment containing exon 14 with this restriction enzyme enabled the heterozygous allele to be identified in both parents (who were third cousins) and three other family members. SSCP analysis detected no additional mutations in the coding or consensus splice sequences of the A subunit gene. The mutant nucleotide substitution was absent in 60 normal alleles and 10 unrelated patients with XIIIA deficiency. Leu667 is located in the carboxyl terminal beta barrel 2 domain of the A subunit molecule. Computer modelling based on 3D crystallographic data predicts that the mutant protein has aberrant folding and is likely to be rapidly degraded following translation.
β spectrinPRAGUE: a truncated β spectrin producing spectrin deficiency, defective spectrin heterodimer self‐association and a phenotype of spherocytic elliptocytosis
Authors: Jarolim Petr , Wichterle Hynek , Hanspal Manjit , Murray James , Robin Hillard L. , Palek Jiri
Published: March 2008

Summary. Spherocytic elliptocytosis is a phenotypic hybrid between hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) characterized by the presence of spheroovalocytes and spherocytes which exhibit increased osmotic fragility, indicating a deficiency of surface area. Both the spherocytic red cell morphology and the increased osmotic fragility distinguish this clinical entity from common HE. In contrast to common HE, the molecular basis of spherocytic elliptocytosis is unknown. Here we describe two members of a family who both have the characteristic features of spherocytic HE. We show that the underlying defect involves a G to C transversion at the ‐1 position of the acceptor splice site upstream of exon X of β spectrin leading to skipping of exon X from the mutant β spectrin mRNA allele. The mutant mRNA is present in reticulocytes in similar amounts as the normal mRNA. Pulse‐labelling of erythroblasts prepared from peripheral blood in a two‐phase liquid‐culture system reveals a decreased synthesis of the truncated β spectrin, a finding which is likely to underlie the moderately severe spectrin deficiency in the two patients. In addition, this mutant spectrin, similar to the previously reported spectrins, is defective in spectrin heterodimer self‐association. The spectrin deficiency, which represents a common finding in the majority of patients with HS, together with weakened spectrin heterodimer self‐association, as found in the majority of patients with common HE, provides a molecular explanation for the phenotype of spherocytic elliptocytosis in this kindred and, most likely, in other patients carrying similar β spectrin mutations.
Tumour necrosis factor alpha‐induced oxidative burst in neutrophils adherent to fibronectin: effects of cyclic AMP‐elevating agents
Authors: Ottonello L. , Morone M. P. , Dapino P. , Dallegri F.
Published: March 2008

Summary. Human neutrophils, plated on flbronectin‐coated polystyrene wells, were found to exhibit a prolonged production of superoxide anion (O−2) in response to tumour necrosis factor‐alpha (TNF). The TNF‐triggered O−2 production was significantly reduced by 10μM prostaglandin E2 (PGE2), which was ineffective at lower doses. Moreover, the O−2 production was slightly reduced by the phosphodiesterase type IV (PDEIV) inhibitor RO 20‐1724. When PGE2 and RO 20‐1724 were added together to TNF‐triggered neutrophils they caused a marked synergistic inhibition of O−2 production. The action of PGE2 could be mimicked by forskolin (FK), a well‐known direct activator of adenylate cyclase. These results suggest that cyclic AMP (cAMP)‐elevating agents (PGE2, FK, RO 20‐1724) down‐regulate the capacity of adherent neutrophils to mount the respiratory burst in response to TNF. Consistent with this interpretation, PGE2 and RO 20‐1724 increased the intracellular levels of cAMP displaying synergistic activity. Moreover, the membrane‐permeable analogue of cAMP, dibutyryl cAMP, was found to inhibit the TNF‐induced O−2 production in a dose‐dependent manner. As all the aforementioned cAMP‐elevating agents did not affect the O−2 production in response to phorbol myristate acetate, they appear to act by interfering with the assembly of the O−2‐generating NADPH oxidase complex rather than by directly inhibiting the activity of already working oxidase complex. In conclusion, taking into account the TNF capacity to promote PGE2 formation at sites of inflammation, our observations suggest the existence of a negative PGE2‐dependent feed‐back, potentially capable of controlling the neutrophil response to TNF and susceptible to amplification by PDE IV‐inhibiting compounds.
Increased platelet CD36 constitutes a common marker in myeloproliferative disorders
Authors: Thibert V. , Bellucci S. , Cristofari M. , Gluckman E. , Legrand C.
Published: March 2008

Summary. The distribution of the major platelet membrane glycoproteins (GP), Ib, IX, IIb‐IIIa and IV (or CD36), which play important roles as receptors for adhesive molecules in haemostasis and thrombosis, was studied in 34 patients with myeloproliferative disorders (MPD): 13 had essential thrombocythaemia (ET), 12 had polycythaemia vera (PV) and nine had chronic myelogenous leukaemia (CML). Only occasionally were modifications of the numbers of GPIb or GPIIb‐IIIa measured using the binding of specific radiolabeled antibodies to platelets. In contrast, 2‐3‐fold increases of the total CD36 content and the surface CD36 expression were measured in almost all patients studied, using a radioimmunoassay and the direct binding of the radiolabelled antibody, FA6‐152, to the platelet surface, respectively. These results indicate that the abnormality affected both the external and internal CD36 pools. Therefore platelet CD36 may be a useful tool for the diagnosis and the follow‐up of MPD patients.

Surface CD36 has been proposed as a platelet receptor for thrombospondin, an adhesive glycoprotein that is released from platelets upon activation and promotes aggregate formation. Despite a 2‐fold increase of CD36 molecules, resting and thrombin‐activated platelets from ET patients expressed the same amount of thrombospondin as normal platelets, suggesting that there is not a direct correlation between the CD36 expression and thrombospondin binding either spontaneously or after activation.
High frequency of homozygous deletions of CDK4I gene in childhood acute lymphoblastic leukaemia
Authors: Iolascon Achille , Faienza Maria Felicia , Coppola Brigida , Ragione Fulvio deila , Santoro Nicola , Schettini Francesco
Published: March 2008

Summary. To determine the incidence of homozygous deletions of the newly identified tumour suppressor gene, CDK4I, molecular genomic DNA analyses by PCR technique were performed on primary neoplastic cells from 22 childhood acute leukaemias obtained at presentation. The blast cells derived in all the analysed cases from bone marrow. We found that none of acute myeloblastic leukaemias (four cases) showed the CDK4I alteration, whereas 6/13 (46%) common acute lymphoblastic leukaemias (ALLs) displayed homozygous deletions. Moreover, and even more important, all the blasts purified from ALLs derived from early lymphoid precursors (three early‐T ALLs and two pre‐B ALLs) showed the absence of CDK4I gene. When the entire coding sequence of the CDK4I gene from samples without homozygous deletions was analysed by the single‐strand conformational polymorphism method, no point mutations were identified. These results demonstrate that CDK4I gene deletions are very frequent and probably early events in childhood acute leukaemias of lymphoid origin and especially in early‐T and pre‐B ALLs. Moreover, the molecular mechanism of the loss of function of the gene is correlated, at least in childhood ALLs, almost exclusively to deletions and not to point mutations.
The value of magnetic resonance imaging in screening myeloma lesions of the lumbar spine
Authors: Tertti Risto , Alanen Anu , Remes Kari
Published: March 2008

Summary. Screening of the skeleton by plain radiography was compared to magnetic resonance imaging (MRI) of the lumbar spine in 41 patients with multiple myeloma. In the lumbar spine, myeloma lesions were detected in 15 patients with radiography and in 28 patients with MRI. Radiography of the lumbar spine was not positive in any of the 13 cases with negative MRI, but in two of them radiography was positive elsewhere in the skeleton. We suggest that plain radiography is the most suitable method for screening all bone areas in multiple myeloma, but MRI of the lumbar spine is needed to study patients with normal radiographs.
Severe arterial thrombosis in a congenitally factor VII deficient patient
Authors: Escoffre M. , Zini J. M. , Schuamser L. , Mazoyer E. , Soria C. , Tobelem G. , Dupuy E.
Published: March 2008

Summary. We report a patient with congenital factor VII deficiency who developed severe arterial thrombosis. A 63‐year‐old‐woman presented low factor VII clotting activity, amidolytic activity and antigen level < 4%. Activated factor VII plasmatic level was < 0.03 ng/ml compared to 4 ng/ml for the control value. She developed severe aorto‐iliac thrombosis. 7d before the thrombotic event, factor VII replacement therapy had been infused. Successful low molecular weight heparin therapy led to total disappearance of the aortoiliac thrombus without bleeding complications. This suggests that factor VII infusion might have a thrombogenic effect in vivo and might be responsible for thrombosis.
Antigen‐independent reactivation of anti‐E, years after allogeneic bone marrow transplantation: a case report
Authors: Wernet Dorothee , Weiss Burkhard , Schmidt Helmut , Northoff Hinnak
Published: March 2008

Summary. Nine years after allogeneic bone marrow transplantation a strong anti‐E was observed in a patient transplanted with bone marrow from his HLA‐identical brother. This IgG anti‐E, with a titre of 4000, was detected together with autoantibodies to red blood cells and platelets and was not induced by transfusion with E+ cells. The hypothesis is proposed that the anti‐E represents either antigen‐independent desuppression of the donor immune system sensitized at the time of bone marrow transplantation by E antigens in the recipient, or antigen‐independent memory B‐cell activation by Epstein‐Barr virus infection.
Accessory cells do not contribute to G‐CSF or IL‐6 production nor to rapid haematological recovery following peripheral blood stem cell transplantation
Authors: Watts M. J. , Jones H. M. , Sullivan A. M. , Langabeer S. E. , Jamieson E. , Fielding A. , Williams C. , Berenson R. J. , Goldstone A. H. , Linch D. C.
Published: March 2008

Summary. Haemopoietic recovery is more rapid after peripheral blood stem cell (PBSC) transplantation than after autologous bone marrow transplantation, and the aim of this study was to assess the role of the large number of lymphocytes and monocytes (accessory cells) in a PBSC leukapheresis product in this rapid regeneration. Haematological recovery was therefore assessed in 10 PBSC recipients with lymphoma or myeloma in whom monocytes and T cells were depleted by a median of 2.3 and 3.3 logs by CD34+ cell selection using the CEPRATE® SC stem cell concentration system and compared with recovery in 59 recipients who received whole PBSC. After allowing for the number of progenitor cells reinfused, there was no significant delay in engraftment induced by accessory cell depletion. Plasma levels of granulocyte‐colony stimulating factor (G‐CSF), granulocyte/monocyte‐colony stimulating factor (GM‐CSF), interleukin‐6 (IL‐6), stem cell factor (SCF) and macrophage‐inhibition factor‐alpha (MIP‐1‐alpha) during the transplant procedure were similar whether or not accessory cells were given. The G‐CSF and IL‐6 levels rose between days 5 and 14 post transplantation to approximately 1 ng/ml and 50pg/ml respectively. This study indicates that accessory cells reinfused with PBSC collections are not responsible for the subsequent cytokine profile or rapid haematological recovery.
Detection of membrane and soluble interleukin‐6 receptor in lymphoid malignancies
Authors: Lavabre‐Bertrand Thierry , Exbrayat Carole , Liautard Jeannie , Gaillard Jean‐Philippe , Baskevitch Pierre‐Paul , Poujol Nicolas , Duperray Christophe , Bourqoard Pascal , Brochier Jean
Published: March 2008

Summary. We studied the membrane expression of the gp80 chain of IL‐6 receptor (IL‐6R) by quantitative flow cytometry in chronic lymphocytic leukaemia (CLL) and leukaemic centrocytic lymphoma using a panel of seven monoclonal antibodies. IL‐6R was detected in 18/26 CLL cases and 4/7 lymphoma cases, with a mean antigen density <3000 molecules/cell. Multiple labelling experiments confirmed the IL‐6R expression by neoplastic cells. Specific mRNA was found by RT‐PCR in neoplastic cells. A specific ELISA test was designed using two anti‐IL‐6 receptor MAbs to measure the serum soluble IL‐6R (sIL‐6R) in CLL (n = 48), B‐cell non‐Hodgkin's lymphoma (NHL; n = 40), and monoclonal gammopathy (MG; n = 32). SIL‐6R was higher in CLL (170±12.6ng/ml) in NHL (160 ± 12ng/ml) and MG patients (183±23ng/ml) than in age‐matched controls (100 ±5.6 ng/ml; P < 0.001) and higher in high‐grade than low‐grade NHL. No correlation was noted with a previous treatment. Among CLL cases the patients classified as stage B according to the Binet's staging of the disease had the highest sIL‐6R values, thus suggesting a link with tumour cell mass.
Fab‐mediated binding of glycoprotein Ib/IX and IIb/IIIa specific antibodies in chronic idiopathic thrombocytopenic purpura
Authors: Hou Ming , Stockelberg Dick , Kutti Jack , Wadenvik Hans
Published: March 2008

Summary. The antibody domain responsible for the interactions between platelet glycoproteins (GP) and serum IgG autoantibodies in patients with chronic idiopathic thrombocytopenic purpura (ITP) was studied. Sera from nine non‐transfused ITP patients and 20 normal controls and a serum containing an anti‐PlA1 antibody were employed. Serum, purified IgG and F(ab′)2 fragments were prepared and their binding to platelet GPIb/IX and GPIIb/IIIa were analysed using a modified MAIPA assay and an antigen capture ELISA. In all experiments most of the autoantibodies studied behaved identically to the anti‐PlA1 antibody in that the IgG‐F(ab′)2 fragments retained their ability to bind to the respective glycoprotein. Substituting the enzyme‐conjugated secondary antibody (Fab specific), in the MAIPA assay, with an Fc specific antibody removed all reactivities observed against platelet GPs, produced by IgG‐F(ab′)2 fragments. Furthermore, in an antigen‐capture ELISA, IgG autoantibodies against platelet GPIb/IX and/or GPIIb/IIIa were blocked preferentially by pre‐incubating the ITP sera with a goat anti‐human IgG (F(ab′)2 specific) antibody, but not with an anti‐Fc antibody. We conclude that these ITP patients produced antibodies specific for platelet GPIb/IX and/or GPIIb/IIIa, and that the autoantibody‐platelet interaction was mediated by the classic Fab binding.
A human monoclonal antibody obtained from EBV‐transformed B cells with specificity for myosin
Authors: Laroche‐Traineau Jeanny , Clofent‐Sanchez Gisele , Daret Daniéle , Bonnaud Evelyne , Barat Jean‐Louis , Ducassou Dominique , Nurden Alan T.
Published: March 2008

Summary. We describe the preparation of a stable human lymphoblastoid cell line obtained during ex vivo studies in which peripheral blood lymphocytes of a Glanzmann's thrombasthenia patient were transformed with Epstein‐Barr virus. Somatic hybrids secreted an IgM monoclonal antibody (B7) that reacted with the myosin heavy chain of human platelets by immunoblotting. Flow cytometry showed that B7 barely recognized unstimulated intact platelets, but bound abundantly after permeabilization of fixed cells with Triton X‐100. The reactivity of the antibody on thin sections of human myocardium and aorta was studied by immunohistochemistry. B7 specifically stained myosin of myocytes, but there was no labelling of aortic smooth muscle cells. The epitope was conserved in cardiac or skeletal myosin prepared from pig or rabbit. Measurement of the dissociation constant in a competitive ELISA showed that B7 bound with high affinity (10−8 M). Purified Fab fragments retained their ability to bind to myosin, suggesting that B7 may be useful in the imaging of myocardial necrosis after myocardial infarction, myocarditis, cardiac drug toxicosis or graft rejection. This work also shows that EBV transformation of B cells may uncover naturally occurring autoantibodies which under normal circumstances are inhibited by the immune surveillance system.
Haemopoietic progenitor cell differentiation: flow cytometric assessment in bone marrow and thymus
Authors: Tjønnfjord Geir E. , Steen Rita , Veiby Ole P. , Morkrid Lars , Egeland Torstein
Published: March 2008

Summary. We have recently shown that expression of any of the lineage‐associated molecules CD2, CD7, CD10, CD19 or CD33 does not ensure lineage‐commitment of CD34+ progenitor cells. Further, normal progenitor cells and leukaemic blast cells have been shown to coexpress molecules associated with more than one haemopoietic lineage. Five‐dimensional flow cytometric analysis of normal bone marrow cells was exploited to investigate the hypothesis of a developmental stage in haemopoiesis comprising CD34+ cells coexpressing CD2, CD5, CD7, CD10, CD19 and CD33 or any combination of these molecules. We report on a subpopulation of CD34+ bone marrow cells constituting < 5% of the CD34+ cells and characterized by extensive coexpression of several molecules associated with the B lymphoid, T lymphoid and myeloid lineages. There is every probability that some cells display the CD34+ CD2+ CD5+ CD7+ CD10+ CD19+ CD33+ phenotype. Studies on postnatal thymocytes suggest that this may be the phenotype or one of a few phenotypes of a candidate thymus‐seeding progenitor cell population. Finally, our findings that CD34+ as well as CD34+ CD5+ thymocytes can be driven into non‐T‐lymphoid differentiation by cytokines, support the notion that the thymus is seeded by uncommitted progenitors.
Role of Epstein‐Barr virus and soluble CD21 in persistent polyclonal B‐cell lymphocytosis
Authors: LARCHER CLARA , FEND FALKO , MITTERER MANFRED , PRANG NADJA , SCHWARZMANN FRITZ , HUEMER HARTWIG P.
Published: March 2008

Summary. The expression of EBV proteins and immunological properties were studied in the first stable cell line (SM) established from a patient presenting with persistent polyclonal B‐cell lymphocytosis (PPBL).

SM cells which represent a small population of EBV‐positive atypical cells found in the peripheral blood of the patient express the KI‐1 antigen (CD30) as well as the proto‐oncogene bcl‐2 product and cell surface markers of mature activated B lymphocytes. The cells harbour an EBV subtype A genome and contain EBNA2 protein. This argues against a transformation‐incompetent virus as the main cause of the chronic active EBV infection observed in our patient. Latent membrane protein (LMP1) was weakly expressed and found predominantly in a perinuclear localization, a location which could lead to decreased immunogenicity in vivo. Similar to the EBV‐transformed marmoset cell line B95‐8, SM cells were in part productively infected as transcription of the immediate early gene BZLF1 could be shown and in some cells high levels of EBV‐genome were detected by in situ hybridization with a BamHl W‐probe. Comparable to the atypical cells in the peripheral blood of the patient, EBV small RNAs were not detected with EBER‐specific probes.

Of interest, we noticed a markedly increased production of soluble CD21 (sCD21) antigen by SM cells as compared to LCL‐type Burkitt's lymphoma cell lines. This could explain the elevated sCD21 levels observed in the serum of our PPBL patient and confirms our previous findings in patients with acute EBV infection. It also suggests a possible role of sCD21 in EBV‐mediated regulation of the immune response and provides a possible explanation for the dysregulation of the humoral immune system observed in PPB1 patients.
Clonal B‐cell expansions in peripheral blood of HCV‐infected patients
Authors: FRANZIN F. , EFREMOV D. G. , POZZATO G. , TULISSI P. , BATISTA F. , BURRONE O. R.
Published: March 2008

Summary. Clonal expansions of IgM‐producing B cells were investigated in 38 patients with a chronic hepatitis C virus infection. Eight patients were affected with type II mixed cryoglobulinaemia (two of whom also had non‐Hodgkin's lymphoma and one had Waldenstrom's disease), one with type III mixed cryoglobulinaemia, one with Waldenstrom's disease, and 28 with chronic liver disease. To detect the clonal B‐cell expansions we used a RT/PCR procedure in which the CDR3/FW4 regions of the IgM heavy chain mRNAs were amplified and resolved in sequencing poly‐acrylamide gels. Clonal Ig gene rearrangements were detected in all patients with type II mixed cryoglobulinaemia and also at a high frequency (24%) in the HCV‐infected patients without cryoglobulinaemia. A polyclonal pattern was present in the patient with type III mixed cryoglobulinaemia and in the 15 normal individuals and 16 age‐related patients with HCV‐negative alcoholic liver disease which were investigated as controls. No association was found between the presence of a clonal B‐cell expansion and age, sex, liver histology, or levels of serum aminotransferase. The serum levels of rheumatoid factor were increased in all patients with a clonal expansion, suggesting that the expanded B‐cell clones belong to the rheumatoid factor producing B‐cell subset.
Hydroxyurea therapy in β‐thalassaemia intermedia: improvement in haematological parameters due to enhanced β‐globin synthesis
Authors: ZENG YI‐TAO , HUANG SHU‐ZHEN , REN ZHAO‐RUI , LU ZHI‐HONG , ZENG FAN‐YI , SCHECHTER ALAN N. , RODGERS GRIFFIN P.
Published: March 2008

Summary. The β‐thalassaemias represent a heterogenous group of diseases resulting from decreased erythroid β‐globin mRNA expression and imbalanced a/β‐globin chain synthesis which are manifest clinically by ineffective erythropoiesis and excessive haemolysis. Increasing levels of haemoglobin F (HbF) by pharmacological agents has been proposed to ameliorate the severity of the disease by improving the balance in globin chain synthesis. Hydroxyurea (HU), as an effective agent with low toxicity for activating 7‐globin gene, has been shown to enhance HbF synthesis in experimental animals and in patients with sickle cell anaemia. However, previous trials of HU in β‐thalassaemia patients are ambiguous, with a small number having increased HbF synthesis. In a recent study of HU effects in Chinese j3‐thalassaemia patients we unexpectedly found that two unrelated patients with β‐thalassaemia intermedia demonstrated an improvement in the effectiveness of erythropoiesis reflected by an increase in haemoglobin concentration (from 4‐1 to 6‐3 g/dl, patient 1; from 6‐5 to 97 g/dl, patient 2) and in red cell volume (from 68 to 104 fl, patient 1; from 68 to 85 fl, patient 2) after a period of excess of 300 d of low‐dosage HU treatment. These effects, however, appear to be due to increased,3‐globin biosynthesis, because the percentage of HbF decreased in each patient as total Hb increased. This was reflected by changes in the β/a ratio (from 0′301 to 0‐581, patient 1; from 0′348 to 0‐487, patient 2) with minimal changes in 7‐globin biosynthesis. We conclude that in addition to its known effects in stimulating 7‐globin production, hydroxyurea may have a more general role in augmenting globin synthesis, including β‐globin in some thalassaemia intermedia patients who maintain the capacity to express normal β‐globin chains.
The detection of clonal proliferation in granular lymphocyte‐proliferative disorders of natural killer cell lineage
Authors: SHIMODAIRA SHIGETAKA , ISHIDA FUMIHIRO , KOBAYASHI HIKARU , MAHBUB BILKIS , KAWA‐HA KEISEI , KITANO KIYOSHI
Published: March 2008

Summary. The clonal proliferation of large granular lymphocytes can be detected in patients with T‐cell‐lineage granular lymphocyte‐proliferative disorders (T‐GLPD) by Southern blotting T‐cell receptor genes. However, this cannot be applied to patients with natural killer‐cell‐lineage GLPD (NK‐GLPD) as it lacks a clonal marker. We therefore investigated the use of two other diagnostic techniques in evaluating clonal proliferation in Japanese patients with NK‐GLPD (n = 4) and T‐GLPD (n=3) by chromosomal analysis of peripheral blood mononuclear cells (PBMC) stimulated with either interleukin‐2 or phytohaemaggluti‐nin, and Epstein‐Barr viral (EBV) genomic DNA analysis. Chromosomal analysis revealed abnormal karyotypes in the PBMC of three of four patients with NK‐GLPD, whereas EBV analysis showed a monoclonal terminal configuration in the PBMC in the fourth patient. Southern blots revealed rearrangements of the TCR genes in all three patients with T‐GLPD but in none of those with NK‐GLPD. It is suggested that these methods may be useful in detecting the abnormal proliferation of large granular lymphocytes in NK‐GLPD.
Confirmation that GP Ib‐IX complexes have a reduced surface distribution on platelets activated by thrombin and TRAP‐14‐mer peptide
Authors: NURDEN ALAN , CAZES ERIC , BIHOUR CLAUDE , HUMBERT MICHEL , COMBRIE ROBERT , PAPONNEAU ANNIE , WINCKLER JOELLE , NURDEN PAQUITA
Published: March 2008

Summary. In 1990 we reported that GP Ib‐IX complexes accumulated within the surface‐connected canalicular system (SCCS) of thrombin‐stimulated platelets. This conclusion was reached following investigations using monoclonal antibodies (MAbs) in flow cytometry and a polyclonal antibody to GP Iba in electron microscopy with immunogold staining performed on ultrathin sections of resin‐embedded platelets. Recent controversy concerning these results has prompted us to perform further studies using 14 anti‐GP Ib‐IX MAbs obtained from the 1993 Boston Workshop on Leukocyte Antigens. Features were the use of the MAbs in mixtures and the fact that immunogold staining was performed on frozen thin sections. Platelets were stimulated with either a‐thrombin or TRAP‐14‐mer peptide. In all cases a decreased density of GP Ib‐IX complexes on exposed areas of the activated platelet surface was accompanied by an increased expression within the SCCS. At the same time we noted that when platelets were stimulated with TRAP‐14‐mer they progressively exhibited a different internal morphology in comparison to that seen with thrombin. In particular, the dense central mass disappeared and large vacuoles were present throughout the cytoplasm. Overall, these studies confirm that changes in the distribution of GP Ib‐IX complexes which follow thrombin‐induced platelet activation (i) are indeed observed when antibody mixtures are used to detect them, and (ii) are mediated through the receptor recognized by the TRAP‐14‐mer peptide.
Absence of incorporation of plasma von Willebrand factor into porcine platelet a‐granules
Authors: ROUSSI JACQUELINE , DROUET LUDOVIC , SIGMAN JEFF , VAIMAN MARCEL , PIGNAUD GEORGES , BONNEAU MICHEL , MASSE JEAN‐MARC , CRAMER ELISABETH MARTIN
Published: March 2008

Summary. In order to study the relationship between plasma and platelet von Willebrand factor (vWF), we used an experimental model of crossed bone marrow transplantation (BMT) between SLA immunocompatible normal and homozygous von Willebrand (vWD) pigs. A normal pig received bone marrow from a vWD pig and a second pig with vWD was engrafted with marrow from a normal pig.

Each recipient, after total irradiation of 10 Grays, received by a central catheter 1010 monocellular bone marrow cells without immunosuppression. The animals were followed for 50 d and no graft rejection or graft‐versus‐host disease was observed. After aplasia occurring 3 weeks after BMT, white blood cells and platelets returned to normal.

Before transplantation, in the vWD pig, vWFAg and vWF activity were not detected in plasma and in platelet and megakaryocyte a‐granules. After transplantation with normal marrow, platelet vWFAg and platelet vWF activity were normal and high molecular weight multimers and numerous tubular structures were present in a‐granules.

Before transplantation, the normal pig had normal plasma and platelet vWFAg‐vWF activity, normal multimeric pattern, and the platelet and megakaryocyte a‐granules displayed many tubular structures, eccentrically located in one of their poles, coinciding with immunogold staining vWFAg. After transplantation with homozygous vWD marrow, platelet and megakaryocyte a‐granules lacked tubular structures. Alpha‐granule immunogold staining for vWF was consistently negative, although plasma vWF was at a normal level. In conclusion, this study shows that, unlike other plasma proteins such as fibrinogen, vWF endocytosis does not occur from plasma to the platelet a‐granules. Platelet and megakaryocyte vWF solely originates from megakaryocyte endogenous synthesis and is independent of plasma vWF.
A point mutation (Arg271→Cys) of a homozygote for dysfunctional prothrombin, prothrombin Obihiro, which has a region of high sequence variability
Authors: MIYATA TOSHIYDKI , ZHENG YAN‐ZHEN , KATO ATSUSHI , KATO HISAO
Published: March 2008

Summary. The molecular defect of a congenitally dysfunctional form of prothrombin, prothrombin Obihiro, was identified in a patient with a severe bleeding tendency. He showed reduced fibrinogen clotting activity, despite a normal prothrombin antigen level. Nucleotide sequencing of amplified DNA revealed a C→T change at nucleotide 7311 of exon VIII of the prothrombin gene. This resulted in the substitution of Arg271 by Cys at the factor Xa cleavage site which precludes normal activation of prothrombin Obihiro by factor Xa and the generation of thrombin. The proband was homozygous for this mutation. In addition, seven new nucleotide changes were identified in the prothrombin gene of this patient by comparison with the published gene sequence. Three polymorphisms at nucleotides 4291, 4298 and 7223 were exclusively identified in the prothrombin gene from Japanese. Particularly in the region containing exon VI and introns E and F (nucleotides 4048‐4303), the prothrombin gene proved to be of high‐sequence variability.
Low‐intensity oral anticoagulation in sickle‐cell disease reverses the prethrombotic state: promises for treatment?
Authors: WOLTERS HUGO J. , CATE HUGO TEN , THOMAS LAMBERTUS L. M. , BRANDIES DESIDERIUS P. M. , VAN DER ENDE ABRAHAM , VAN DER HEIDEN YVONNE , STATIUS VAN EPS LODEWIJK W.
Published: March 2008

Summary. Increased plasma levels of prothrombin fragment 1 + 2 (Fl + 2) found in patients with sickle‐cell disease reflect enhanced endogenous thrombin generation. We postulate that hypercoagulability contributes to vaso‐ occlusion. The intensity of acenocoumarol treatment required to reduce the Fl + 2 level to 50% of pretreatment level was investigated in seven patients with symptomatic sickle‐cell anaemia during steady‐state disease for a period of 2 months. All patients had increased levels of Fl + 2 compared with an age‐matched control group. Normalization of the Fl + 2 was achieved at a median INR of 1.64 (range 1.18‐2.2). It is concluded that low‐intensity oral anticoagulation normalizes the hypercoagulability in sickle‐cell disease.