bluuebunny/crossref_metadata_2025 huggingface dataset:
Fas‐mediated lysis of chronic lymphocytic leukaemia cells: role of type I versus type II cytokines and autologous FasL‐expressing T cells
Authors: Williams Jonathan F. , Petrus Matthew J. , Wright Jacob A. , Husebekk Anne , Fellowes Vicki , Read Elizabeth J. , Gress Ronald E. , Fowler Daniel H.
Published: March 2003

Given the known role of the fas cytolytic pathway in B‐cell regulation, we evaluated whether fas–fasL interactions might induce chronic lymphocytic leukaemia (CLL) cell death. De novo CLL cells expressed a low level of surface fas, and were not lysed by fasL‐bearing cells. CLL cells cultured in media containing the type I cytokines interleukin (IL)‐12 or interferon (IFN)‐α had increased fas expression, and were readily lysed by fasL‐bearing cells. In contrast, the type II cytokine IL‐4 did not increase CLL cell fas, and abrogated type I cytokine‐induced fas up‐regulation. With prolonged culture, IL‐4 exposed CLL cells expressed an intermediate level of fas; however, such CLL cells were resistant to fas‐mediated lysis. These results indicate that IL‐4 inhibits fas‐mediated killing of CLL cells at the level of both fas receptor expression and post‐receptor events. Additionally, we have defined in vitro culture conditions which generate fasL‐bearing T cells from CLL patients; such T cells efficiently mediated fas‐based lysis of autologous fas‐positive CLL cells. We therefore conclude that type I and type II cytokines differentially regulate the fas pathway in CLL cells, and that a combination of type I cytokines and fasL‐expressing T cells may represent a new approach to the immunotherapy of CLL.
Comparative serial quantitative measurements of chimaerism following unmanipulated allogeneic transplantation of peripheral blood stem cells and bone marrow
Authors: Miflin G. , Stainer C. J. , Carter G. I. , Byrne J. L. , Haynes A. P. , Russell N. H.
Published: March 2003

Serial samples were collected from 38 patients following allogeneic transplantation using either unmanipulated peripheral blood stem cells (PBSC) (n = 18) or bone marrow (BM) (n = 20) to assess the incidence of mixed chimaerism using PCR amplification of five VNTR regions. After amplification, products were analysed using the Applied Biosystems ABI PRISM 377 Automated DNA Sequencer and GeneScan software GenoTyper program to determine a quantitative measure of chimaerism. The sensitivity of detection using this method was 0.1%.

In the immediate post‐transplant period (up to day 30) a significantly lower incidence of mixed chimaerism (MC) occurred in recipients of PBSC compared to BM (P < 0.0005). Between 1 and 6 months there was a significantly lower incidence of low‐level MC in patients receiving PBSCT compared to BMT (4/14 v 8/11 respectively, P = 0.04) in patients who had not rejected their grafts or relapsed. Similarly, beyond 6 months 0/9 PBSCT patients compared to 4/9 BMT patients showed MC (P = 0.02). Beyond day 30 13/33 (39%) patients showed intermittent low‐level MC, but this was not predictive for subsequent relapse. A rapidly increasing proportion of recipient haemopoiesis was predictive of graft rejection or relapse. Stable continuous MC without relapse was seen in one patient transplanted with PBSC for severe aplastic anaemia. These results suggest that the incidence of intermittent low‐level MC is relatively high in the first 6 months following unmanipulated haemopoietic stem cell transplantation but reduces with time and is significantly lower in recipients of PBSC.
A bone marrow biopsy technique suitable for use in neonates
Authors: Sola Martha C. , Rimsza Lisa M. , Christensen Robert D.
Published: March 2003

Thrombocytopenia and neutropenia are common among neonates in intensive care units. Bone marrow aspirations are sometimes performed as part of their evaluation. However, marrow biopsies have not been reported from living neonates. Since architecture and cellularity cannot generally be accurately assessed from marrow aspirates, we devised a biopsy technique which we successfully applied to five cytopenic neonates (three with severe persistent thrombocytopenia and two with idiopathic neutropenia). This technique used a 19 gauge, half‐inch Osgood needle to obtain bone marrow clots from the tibias of small preterm neonates which enabled the assessment of marrow cellularity and architecture. On the basis of our initial experience we have ceased using the traditional bone marrow aspiration technique in neonates and now use this technique exclusively.
Bcl10 in chronic lymphocytic leukaemia and T‐cell prolymphocytic leukaemia
Authors: Yuille M. R. , Stone J. G. , Bradshaw P. S. , Houlston R. S.
Published: March 2003

Bcl10 is a cancer gene recently identified in B‐cell lymphomas of mucosa‐associated lymphoid tissues. It has been suggested as a target for mutation in multiple types of tumour including follicular lymphoma, T‐cell acute lymphoblastic leukaemia and Sezary syndrome. To evaluate further the role of Bcl10 in human adult haematological cancers, we screened for mutations samples from 24 patients with B‐cell chronic lymphocytic leukaemia (CLL) and 18 samples from patients with T‐cell prolymphocytic leukaemia (T‐PLL). No pathogenic mutations were detected in any of the samples analysed, strongly suggesting that Bcl10 is not involved in the development of CLL or T‐PLL and that its involvement may be restricted to other haematological maligancies.
Phospholipid binding of factor VIII in different therapeutic concentrates
Authors: Raut S. , Weller L. , Barrowcliffe T. W.
Published: March 2003

Binding to anionic phospholipid (PL) is essential for the biological function of factor VIII (FVIII). We have developed a method to study the level of PL binding of FVIII in a variety of therapeutic concentrates, using the BIACORETM system which utilizes the Surface Plasmon Resonance (SPR) phenomenon. A HPA sensor chip was employed on to which synthetic phospholipid unilamellar vesicles were adsorbed to form a 3:1 phosphatidylcholine: phosphatidylserine lipid monolayer. Using this surface the interaction of unlabelled FVIII in concentrates was observed from which direct kinetic data (kon, koff and KD values) were obtained in real‐time. Marked differences in the binding to PL, as measured by KD values, between different products were observed. These fell into three categories: two recombinant FVIII products showed high affinities for PL with KD values around 0.05–0.14 nm; four high‐purity plasma derived products, two prepared by monoclonal antibody and two prepared by ion‐exchange chromatography, had 6–8‐fold lower affinities, and two intermediate‐purity products had 34–60‐fold lower affinities with KD values in the nm region. Measurements of kon and koff values for each product showed that the differences in the KD values expressed were primarily due to the differences in their respective kon values, although the recombinant products showed changes in the koff values. The study showed that the assessment of binding to PL by FVIII in concentrates was possible without prior purification and gave KD values in the range reported previously for other methods. The difference between the products requires further investigation but may be partly due to other proteins present, in particular the content and quality of von Willebrand factor which is known to affect PL binding of FVIII.
The methylenetetrahydrofolate reductase gene C677T polymorphism in patients with homozygous sickle cell disease and stroke
Authors: Cumming  , Olujohungbe  , Keeney  , Singh  , Hay  , Serjeant
Published: March 2003

Homozygosity for the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism may cause hyperhomocysteinaemia, a recognized risk factor for stroke, in individuals with folate deficiency. Homozygous sickle cell (SS) disease is associated both with increased demands for folic acid and a tendency to develop stroke. We therefore investigated a possible role of the MTHFR C677T polymorphism in SS disease patients with stroke. Investigation of the frequency of the polymorphism in 48 patients with stroke and in 48 age‐, sex‐ and racially‐matched SS controls without stroke failed to reveal a difference between the groups (Fisher exact test, P = 0.99). Homozygosity for the MTHFR C677T polymorphism is unlikely to be a risk factor for stroke in this population with SS disease.
Accessibility of abciximab to megakaryocytes and endothelial cells in the bone marrow compartment: studies on a patient receiving antithrombotic therapy
Authors: Poujol Christel , Durrieu‐Jais Catherine , Larrue Benoit , Nurden Alan T. , Nurden Paquita
Published: March 2003

Abciximab, chimaeric Fab fragments of the monoclonal antibody 7E3 (c7E3 Fab), has achieved widespread use as an anti‐platelet agent for blocking GP IIb–IIIa (αIIbβ3) function and preventing ischaemic complications after coronary artery angioplasty. However, its accessibility to the bone marrow compartment during therapy is unknown, as is its ability to bind αvβ3in vivo. Using electron microscopy and immunogold labelling, we have looked for abciximab in the bone marrow of a patient who became thrombocytopenic during treatment. The presence of abciximab was assessed on ultrathin frozen sections of a marrow aspirate, the drug being revealed by a rabbit antibody to c7E3 Fab. Labelling was maximal on fragmenting megakaryocytes (MK) and proplatelets in the vascular sinus and in direct access to the blood compartment. Not only the plasma membrane but also the demarcation membrane system (DMS) and the membranes of α‐granules were labelled. Abciximab was also revealed on the luminal surface of endothelial cells lining the marrow sinuses, thereby confirming for the first time its ability to bind to αvβ3in vivo. The study revealed no signs that abciximab had accumulated in the marrow.
Increased bleeding associated with protease inhibitor therapy in HIV‐positive patients with bleeding disorders
Authors: Wilde Jonathan T. , Lee Christine A. , Collins Peter , Giangrande Paul L. F. , Winter Mark , Shiach Caroline R.
Published: March 2003

The use of protease inhibitor (PI) drugs in treatment regimens for HIV‐infected patients with hereditary bleeding disorders has been associated with an increased bleeding tendency. To characterize the nature of this bleeding tendency, a retrospective case record analysis was performed on 67 HIV‐positive patients with hereditary bleeding disorders who had been treated with PI therapy. 34 patients (51%) developed an increased bleeding tendency on PI therapy, usually within the first few weeks of treatment. As well as an increase in usual joint bleeds, patients developed spontaneous atypical small joint, soft tissue and muscle bleeds. Haematuria was also common. Bleeding episodes tended to respond suboptimally to factor concentrate replacement. Ritonavir was most likely to be associated with bleeding. Nine patients switched first‐line PI therapy as a direct consequence of bleeding and seven had no further bleeding problem on their second PI. Factor concentrate usage was significantly increased during the first 6 months of PI therapy compared to the 6 months preceeding treatment. PI therapy is frequently associated with increased bleeding in patients with hereditary bleeding disorders. The mechanism of the bleeding tendency remains to be elucidated.
Identification and characterization of zebrafish thrombocytes
Authors: Jagadeeswaran P. , Sheehan J. P. , Craig F. E. , Troyer D.
Published: March 2003

To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface‐connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa‐like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin‐induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.
Intracellular trafficking of the αIIbβ3 receptor antagonist, abciximab, in normal and Glanzmann's disease megakaryocytes
Authors: Massé Jean‐Marc , Perlemuter Katy , Debili Najet , Letestu Rémi , Castaigne Alain , Cramer Elisabeth M.
Published: March 2003

The αIIbβ3 platelet receptor antagonist abciximab (c7E3Fab, ReoPro®) has proved to be effective in preventing arterial thrombosis. However, its binding capacity to the platelet precursors, megakaryocytes (MKs), which also express αIIbβ3, is not known. The purpose of this study was to establish whether abciximab is able to react with αIIbβ3 located on human MKs, and to follow its subsequent intracellular trafficking. MKs were grown from CD34+ progenitors from normal subjects and from a patient with type I Glanzmann's thrombasthenia, and abciximab was added at day 10 of culture (4 μg/ml). Cells were fixed at day 12, cryosectioned, and immunolabelled for abciximab. Labelling was prominent on the MK plasma membrane; it also lined the demarcation membration system. Interestingly, α‐granule membranes were labelled showing that the antibody was internalized and further stored into MK secretory granules. Abciximab was also strongly detected on and in newly‐formed platelets. Glanzmann's disease MKs (which completely lacked αIIbβ3) were consistently negative, confirming that the antibody fragment was specifically interacting with αIIbβ3. In conclusion, this study demonstrated that abciximab: (i) binds MK plasma membrane and demarcation membranes, (ii) trafficks into α‐granules, and (iii) is expressed on and in nascent platelets. These findings could be taken in account when monitoring anti‐αIIbβ3 receptor therapy.
Role of P‐glycoprotein in all‐trans retinoic acid (ATRA) resistance in acute promyelocytic leukaemia cells: analysis of intracellular concentration of ATRA
Authors: Takeshita Akihiro , Shinjo Kaori , Naito Kensuke , Ohnishi Kazunori , Sugimoto Yoshikazu , Yamakawa Yoshinori , Tanimoto Mitsune , Kitamura Kunio , Naoe Tomoki , Ohno Ryuzo
Published: March 2003

We analysed the relationship between all‐trans retinoic acid (ATRA) resistance and P‐glycoprotein (P‐gp)‐associated multidrug resistance (MDR) in acute promyelocytic leukaemia (APL). There was no difference in the intracellular ATRA accumulation between NB4 cells and an MDR1 cDNA‐transduced NB4 subline and between ATRA‐resistant NB4 cells (NB4/RA) and an MDR1 cDNA‐transduced NB4/RA subline. PSC833, a MDR modifier, did not increase the intracellular accumulation of ATRA or affect the expression of CD11b, the nitroblue tetrazolium (NBT) reduction activity, the proportion of apoptotic cells or the morphology of these four ATRA‐treated cell lines. Similar results were obtained in the analysis of APL cells from five patients relapsed after ATRA‐induced complete remission.
Cladribine with or without prednisone in the treatment of previously treated and untreated B‐cell chronic lymphocytic leukaemia — updated results of the multicentre study of 378 patients
Authors: Robak  , Błoński  , Kasznicki  , Konopka  , Ceglarek  , Dmoszyńska  , Soroka‐Wojtaszko  , Skotnicki  , Nowak  , Dwilewicz‐Trojaczek  , Tomaszewska  , Hellmann  , Lewandowski  , Kuliczkowski  , Potoczek  , Zdziarska  , Hansz  , Kroll  , Komarnicki  , Hołowiecki  , Grieb
Published: March 2003

Between January 1992 and January 1999, we treated 378 B‐chronic lymphocytic leukaemia (CLL) patients with cladribine (2‐CdA), and 255 of the patients were also treated with prednisone. A total of 194 patients were previously untreated, and 184 had relapsed or refractory disease after previous other therapy. Complete response (CR) was obtained in 111 (29.4%) and partial response (PR) in 138 (36.5%) patients, giving an overall response (OR) rate of 65.9%. CR and OR were achieved more frequently in patients in whom 2‐CdA was a first‐line treatment (45.4% and 82.5% respectively) than in the pretreated group (12.5% and 48.4% respectively) (P < 0.0001). The median duration of OR for previously untreated patients was 14.7 months and for pretreated patients 13.5 months (P = 0.09). The median survival evaluated from the beginning of 2‐CdA treatment was shorter in the pretreated group (16.3 months) than in the untreated group (19.4 months) (P < 0.0001). A total of 117 (63.9%) patients died in the pretreated group and 63 (32.6%) in the untreated group. In pretreated patients, 2‐CdA + prednisone (P) and 2‐CdA alone resulted in similar OR (51.0% and 45.0% respectively; P = 0.4). In contrast, in untreated patients, 2‐CdA + P produced a higher OR (85.4%) than 2‐CdA alone (72.1%) (P = 0.04). Infections and fever of unknown origin, observed in 91 (49.4%) pretreated and 74 (38.1%) untreated patients (P = 0.03), were the most frequent toxic effects. Our results indicate that 2‐CdA is an effective, relatively well‐tolerated drug, especially in previously untreated CLL.
CD34+/CD105+ cells are enriched in primitive circulating progenitors residing in the G0 phase of the cell cycle and contain all bone marrow and cord blood CD34+/CD38low/− precursors
Authors: Pierelli Luca , Scambia Giovanni , Bonanno Giuseppina , Rutella Sergio , Puggioni Pierluigi , Battaglia Alessandra , Mozzetti Simona , Marone Maria , Menichella Giacomo , Rumi Carlo , Mancuso Salvatore , Leone Giuseppe
Published: March 2003

A subset of circulating CD34+ cells was found to express CD105 antigen. Sorting experiments showed that most granulocyte–macrophage colony‐forming units (GM‐CFU) and burst‐forming units — erythroid (BFU‐E) were retained in the CD34+/CD105− fraction, whereas rare GM‐CFU/BFU‐E were generated from CD34+/CD105+ cells. Megakaryocytic aggregates were entirely retained in the CD34+/CD105+ fraction. Neutralizing doses of an anti‐TGF‐β1 antibody demonstrated CD34+/CD105+ cells capable of colony‐forming activity without any significant effect on CD34+/CD105− cells. Cloning of secondary colonies revealed that CD34+/CD105+ cells had a significantly higher secondary cloning efficiency than CD34+/CD105− cells. CD34+/CD105+ cells had a significantly higher long‐term culture‐initiating cell (LTC‐IC) frequency than CD34+/CD105− cells. Kinetic analysis showed that 75% of CD34+/CD105+ cells consisted of DNA 2n G0Ki‐67− cells whereas 82% of CD34+/CD105− were DNA 2n G1Ki‐67+ cells, and this latter subset showed a RNA content consistently higher than CD34+/CD105+ cells. CD34+/CD105+ progenitors were CD25+, whereas CD34+/CD105− contained a small CD25+ subset. Three‐colour analysis of bone marrow and cord blood CD34+ cells demonstrated that all the CD34+/CD38low/− primitive precursors were contained in CD34+/CD105+ cells. Extensive characterization of these CD105+ precursors indicated that they have biological properties associated with primitive haematopoietic precursors.
Preliminary experience of allogeneic stem cell transplantation for lymphoproliferative disorders using BEAM–CAMPATH conditioning: an effective regimen with low procedure‐related toxicity
Authors: Cull G. M. , Haynes A. P. , Byrne J. L. , Carter G. I. , Miflin G. , Rebello P. , Hale G. , Waldmann H. , Russell N. H.
Published: March 2003

Autologous transplantation has an established role in the treatment of lymphoproliferative disorders, but allogeneic transplantation remains controversial. In an attempt to reduce the high procedure‐related mortality reported with allografting in lymphoma, we have used BEAM (BCNU, etoposide, cytarabine and melphalan), a standard conditioning regimen for autologous transplantation. As BEAM may be insufficiently immunosuppressive to permit durable engraftment in the allogeneic setting, patients received additional pretransplant immunosuppression with the anti‐CD52 antibody CAMPATH‐1G from day −5 to day −1.

Twelve patients (median age 46 years) underwent allogeneic transplantation for lymphoma (n = 11) or chronic lymphocytic leukaemia (n = 1) from HLA‐identical (n = 9) or mismatched (n = 3) sibling donors. Cyclosporin A and methotrexate were used as graft‐versus‐host disease (GVHD) prophylaxis. One patient died of progressive lymphoma at day +12, the remaining 11 patients engrafted rapidly, with eight demonstrating full donor chimerism. One patient had an episode of rejection and received a further stem cell infusion with sustained recovery. Only one patient developed GVHD (grade I). The low incidence of acute GVHD may be in part related to persisting levels of in vivo CAMPATH‐IG at the time of transplantation. Of 11 evaluable patients, nine achieved complete remission (CR), and a further patient achieved CR after donor lymphocyte infusion at 5 months.

Our preliminary experience is that this regimen was well tolerated with a low risk of GVHD and appears no more toxic than a BEAM autograft. Further follow‐up is required to see whether the low incidence of GVHD impacts upon relapse risk.
Octreotide in the treatment of gastrointestinal bleeding caused by angiodysplasia in two patients with von Willebrand's disease
Authors: Bowers Margaret , McNulty Orla , Mayne Elizabeth
Published: March 2003

Two cases of von Willebrand's disease and angiodysplasia with intractable gastrointestinal bleeding are presented. Replacement therapy with cryoprecipitate and variable purity von Willebrand factor (VWF) was ineffective, as were other treatments including steroids, immunoglobulin and hormonal replacement. Both patients required massive blood transfusion and product support. The efficacy of somatostatin and an analogue is described. In one patient, we observed a rise in von Willebrand factor activity after octreotide infusion.
A new mutation (G51C) in the iron‐responsive element (IRE) of l‐ferritin associated with hyperferritinaemia–cataract syndrome decreases the binding affinity of the mutated IRE for iron‐regulatory proteins
Authors: Camaschella  , Zecchina  , Lockitch  , Roetto  , Campanella  , Arosio  , Levi
Published: March 2003

Hereditary hyperferritinaemia–cataract syndrome is an autosomal dominant disorder characterized by a constitutively increased synthesis of l‐ferritin in the absence of iron overload. The disorder is associated with point mutations in the iron‐responsive element (IRE) of
l‐ferritin mRNA. We report a new mutation, G51C, identified in two members of a Canadian family, presenting a moderate increase in serum ferritin and a clinically silent bilateral cataract. Gel retardation assays showed that the binding of the mutated IRE to iron‐regulatory proteins (IRPs) was reduced compared with the wild type. Structural modelling predicted that the G51C induces a rearrangement of base pairing at the lateral bulge of the IRE structure which is likely to modify IRE conformation.
Mastocytosis cells bearing a c‐kit activating point mutation are characterized by hypersensitivity to stem cell factor and increased apoptosis
Authors: Dror Yigal , Leaker Michael , Caruana Georgina , Bernstein Alan , Freedman Melvin H.
Published: March 2003

Mastocytosis is characterized by abnormal infiltration of mast cells into various organs. An activating mutation in c‐kit, involving an A → T substitution at nucleotide 2648 has recently been described in some patients with mastocytosis. We describe a 12‐year‐old girl with this mutation in her bone marrow cells at diagnosis with a myelodysplastic syndrome (MDS) without evidence of mastocytosis, and then in peripheral blood mononuclear cells 1 year later after the emergence of mastocytosis. The role of the c‐Kit receptor and its ligand stem cell factor (SCF) in the pathogenesis of the disease was analysed in marrow cell clonogenic assays. We show that the genetic abnormalities in the patient resulted in factor‐independent growth and hypersensitivity of primitive progenitors to SCF, with increased production of mast cells. Increased apoptosis and cluster formation, consistent with the myelodysplastic nature of the disorder, accompanied accumulation of abnormal cells with increasing concentrations of SCF.
Fas‐mediated apoptosis of peripheral blood mononuclear cells in patients with hepatitis C
Authors: Taya Noriyasu , Torimoto Yoshihiro , Shindo Motohiro , Hirai Katsuyuki , Hasebe Chitomi , Kohgo Yutaka
Published: March 2003

In the present study, we demonstrated that a close relationship exists between hepatitis C virus (HCV) infection of peripheral blood mononuclear cells (PBMCs) and cell‐surface Fas expression in patients with hepatitis C, and showed the possibility of PBMCs apoptosis via a Fas‐mediated system. The expression of Fas on PBMCs was found by flowcytometric analysis to be significantly increased in these patients. In addition, the treatment of patients' PBMCs with anti‐Fas antibody induced cell death, with nuclear condensation and fragmentation and cellular DNA fragmentation. These data indicate that the patients' PBMCs expressed a large amount of functional Fas on the cell surface and were susceptible to stimulation against Fas, causing apoptotic cell death. We then quantified the serum‐soluble Fas ligand (sFasL), which was known to bind to Fas and induce the apoptotic signals into the sensitized cells. The patients' serum sFasL levels were significantly higher than those of normal subjects and showed a good negative correlation with their PBMC number. To demonstrate the correlation between Fas expression and HCV infection, nested reverse transcriptase polymerase chain reaction (RT‐PCR) was performed to detect HCV RNA. Interestingly, HCV RNA was preferentially detected from Fas‐positive cells but not from Fas‐negative cells, which had been isolated from PBMCs by magnetic beads. These results suggest that HCV infection of PBMCs might induce Fas expression and additional stimulation such as sFasL might induce apoptosis in these Fas‐expressing cells. These mechanisms, in addition to hypersplenism, may explain the decrease in the number of PBMCs observed in patients with chronic hepatitis C.
Exclusive expression of G‐CSF receptor on myeloid progenitors in bone marrow CD34+ cells
Authors: Ebihara Yasuhiro , Xu Ming‐jiang , Manabe Atsushi , Kikuchi Akira , Tanaka Ryuhei , Kato Shunichi , Nakahata Tatsutoshi , Tsuji Kohichiro
Published: March 2003

Although granulocyte colony‐stimulating factor (G‐CSF) has been reported to act on cells of neutrophilic lineage, the administration of G‐CSF to induce the mobilization of various haematopoietic progenitors into the circulation. We analysed the expression of receptors for G‐CSF (G‐CSFR) on human bone marrow and G‐CSF‐mobilized peripheral blood CD34+ cells, and examined the proliferation and differentiation capabilities of sorted CD34+G‐CSFR+ and CD34+G‐CSFR− cells using methylcellulose clonal culture. Flow cytometric analysis showed that G‐CSFR was expressed on 14.9 ± 4.9% of bone marrow CD34+ cells, most of which were included in CD34+CD33+ and CD34+CD38+ cell fractions. In clonal cultures, CD34+G‐CSFR+ cells produced only myeloid colonies, whereas CD34+G‐CSFR− cells produced erythroid bursts, megakaryocyte and multilineage colonies. When incubated with the cytokine cocktail for 5 d, CD34+G‐CSFR− cells generated CD34+G‐CSFR+ myeloid progenitors. In G‐CSF‐mobilized peripheral blood, CD34+ cells contained 10.8 ± 5.8% of G‐CSFR+ cells, most of which were also myeloid progenitors, although CD34+G‐CSFR− cells contained a substantial number of myeloid progenitors. These results indicated that the expression of G‐CSFR on CD34+ cells is restricted to myeloid progenitors, suggesting that the specific activity of G‐CSF on myelopoiesis depends on the exclusive expression of its receptor on myeloid progenitors, and that the mobilization of various haematopoietic progenitors is not a direct effect of G‐CSF in humans.
Determination of serotonin release from platelets by enzyme immunoassay in the diagnosis of heparin‐induced thrombocytopenia
Authors: Harenberg J. , Huhle G. , Giese CH. , Wang L. C. , Feuring M. , Song X. H. , Hoffmann U.
Published: March 2003

[ 14C]‐Serotonin release assay (14C‐SRA) from platelets is considered to be the most sensitive test for laboratory confirmation of heparin‐induced thrombocytopenia (HIT). This study compared 14C‐SRA with an enzyme immunoassay (EIA) to determine the release of serotonin from platelets in the presence of heparin and serum from HIT patients. The normal range (median, 2·5 and 97·5 percentiles) of serotonin release from platelets in healthy subjects (n = 149) is 38 ng/ml (19 and 62) measured by EIA‐SRA. Serum from HIT patients (n = 42) released 2548 ng/ml (244 and 7987) serotonin in the presence of 0·1 IU/ml heparin and 29 ng/ml (13 and 76) in the presence of 100 IU/ml heparin. One hundred per cent and 15% of HIT samples exhibited an elevated serotonin release from platelets in the presence of 0·1 IU/ml low molecular weight (LMW) heparin, 2100 ng/ml (869 and 5968), or danaparoid, 272 ng/ml (143 and 403), respectively. The sensitivity and specificity of the EIA‐SRA was 100% and 97·4% and of the 14C‐SRA 100% and 92·9% in HIT patients. No false‐positive results were found in patients receiving heparin (n = 28), in patients with elevated levels of bilirubin (n = 5), in patients with antiphospholipid antibody syndrome (n = 10) or in non‐HIT patients (n = 78) with both assays. The EIA technique to quantify serotonin release from platelets provides a reliable non‐radioactive method to diagnose heparin‐induced thrombocytopenia and to assess in vitro crossreactivity of low molecular weight heparins and heparinoid.
Effect of factor VIII concentrate on antigen‐presenting cell (APC)/T‐cell interactions in vitro: relevance to inhibitor formation and tolerance induction
Authors: Hodge G. , Han P.
Published: March 2003

Inhibitor formation in patients with haemophilia receiving factor VIII (FVIII) concentrate is a common problem requiring tolerance induction therapy. Immune tolerance is dependent on defective T cell/antigen‐presenting cell (APC) interactions and inhibitor antibody formation is associated with effective T‐cell/B‐cell interaction. We studied the expression of the cell‐surface molecules involved with these interactions using multiparameter flow cytometry and a whole blood stimulation assay–phytohaemaglutinin (PHA) to activate T cells and Escherichia coli lipopolysaccharide (LPS) to activate monocytes and B cells. Up‐regulation of T‐cell co‐stimulatory receptors CD11a, CD40 ligand (CD40L) and CTLA4 were inhibited in a dose‐dependent manner by plasma‐derived (pd)FVIII, but CD28 was unchanged. Up‐regulation of monocyte and B‐cell co‐stimulatory ligands CD4O, B7‐1 (CD80) and B7‐2 (CD86) were also inhibited in a dose‐dependent manner by pdFVIII, but LFA‐3 (CD58) was unchanged. The combined inhibitory effect of prednisolone, an immunosuppressive agent used in several tolerance induction protocols, with pdFVIII on co‐stimulatory molecules, was additive. There was no significant alteration in T‐cell/APC adhesion or co‐stimulatory molecules noted in the presence of recombinant (rh)FVIII concentrate. The inhibitory effect of pdFVIII on molecules involved in interaction between T cells and APCs may result in immune tolerance in recipients of pdFVIII concentrate. The inhibitory effect of pdFVIII on CD40/CD40L up‐regulation may result in defective antibody formation. We now provide evidence that the use of pdFVIII, through interfering with APC/T‐cell interactions, may be more appropriate than rhFVIII for tolerance induction.
Remission status defined by immunofixation vs. electrophoresis after autologous transplantation has a major impact on the outcome of multiple myeloma patients
Authors: Lahuerta Juan José , Martinez‐Lopez Joaquín , Serna Javier de la , Bladé Joan , Grande Carlos , Alegre Adrián , Vazquez Lourdes , García‐Laraña José , Sureda Ana , Rubia Javier de la , Conde Eulogio , Martinez Rafael , Perez‐Equiza Katy , Moraleda José M. , León Angel , Besalduch Juan , Cabrera Rafael , Miguel José D. Gonzalez‐San , Morales Alfonso , García‐Ruíz Juan C. , Diaz‐Mediavilla Joaquín , San‐Miguel Jesús ,   for the G ethand P ETHEMA G roups
Published: March 2003

We have retrospectively analysed 344 multiple myeloma (MM) patients (202 de novo patients) treated in a non‐uniform way in whom high‐dose therapy and autologous stem cell transplantation (ASCT) response was simultaneously measured by both electrophoresis (EP) and immunofixation (IF). Patients in complete remission (CR) by EP were further subclassified as CR1 when IF was negative and CR2 when it remained positive. Partial responders (PR) were also subclassified as PR1 (very good PR, > 90% reduction in M‐component) or PR2 (50–90% reduction). CR1 patients showed a significantly better event‐free survival (EFS) [35% at 5 years, 95% confidence interval (CI) 17–53, median 46 months] and overall survival (OS) (72% at 5 years, CI 57–86, median not reached) compared with any other response group (univariate comparison P < 0·00000 to P = 0·004). In contrast, comparison of CR2 with PR1 and with PR2 did not define different prognostic subgroups (median EFS 30, 30 and 26 months respectively, P = 0·6; median survival 56, 44 and 42 months respectively, P = 0·5). The non‐responding patients had the worst outcome (5‐year OS 8%, median 7 months). Multivariate analysis confirmed both the absence of differences among CR2, PR1 and PR2 and the highly discriminatory prognostic capacity of a three‐category classification: (i) CR1 (ii) CR2 + PR1 + PR2, and (iii) non‐response (EFS P < 0·00000; OS P < 0·00000; both Cox models P < 0·00000). In the logistic regression analysis, the factors significantly associated with failure to achieve CR1 were the use of two or more up‐front chemotherapy lines, status of non‐response pre‐ASCT and inclusion of total body irradiation (TBI) in the preparative regimen. Tandem transplants or the use of multiple agents (busulphan and melphalan) in the preparative regimen resulted in a higher CR1 level; none of the biological factors explored influenced the possibility of achieving CR1. These results confirm that, in MM patients undergoing ASCT, achieving a negative IF identifies the patient subset with the best prognosis; accordingly, therapeutic strategies should be specifically designed to achieve negative IF.
Increased expression of Fas (APO‐1, CD95) on CD34+ haematopoietic progenitor cells after allogeneic bone marrow transplantation
Authors: Saheki Kaname , Fujimori Yoshihiro , Takemoto Yoshinobu , Kakishita Eizo
Published: March 2003

Up‐regulation of Fas/APO‐1 (CD95) on haematopoietic progenitors and Fas‐mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas‐mediated suppression of donor‐derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti‐Fas antibody (Ab) (CH‐11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte–macrophage colony‐forming units (GM‐CFU) and erythroid burst‐forming units (BFU‐E) after BMT. Pretreatment by blocking anti‐Fas Ab (ZB4) abrogated the Fas‐mediated GM‐CFU and BFU‐E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas‐mediated colony suppression. Thus, donor‐derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas‐mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.
Caspases influence the mode but not the extent of cell death induced by purine analogues in chronic lymphocytic leukaemia
Authors: Pettitt A.R. , Cawley J.C.
Published: March 2003

Caspases are known to be involved in the apoptotic killing of chronic lymphocytic leukaemia (CLL) cells by fludarabine. However, it is unclear whether these enzymes are required for the induction of such killing, or whether they simply determine the mode of cell death. To address this question, we examined the effect of the broad‐spectrum caspase inhibitor Z‐VAD.fmk on six different manifestations of nucleoside cytotoxicity. Our results indicate that while caspase activity is required for nucleoside‐induced poly(ADP‐ribose) polymerase (PARP) cleavage and DNA fragmentation, other manifestations of cell death (mitochondrial depolarization, exposure of phosphatidyl serine, cell membrane disruption and cell shrinkage) are caspase independent. By showing that caspases influence the mode, but not the extent, of nucleoside cytotoxicity, our results exclude defects in these enzymes as a mechanism of nucleoside resistance in CLL.
Methotrexate resistance in relapsed childhood acute lymphoblastic leukaemia
Authors: Rots Marianne G. , Pieters Rob , Peters Godefridus J. , Noordhuis Paul , Van Zantwijk Christina H. , Henze Günter , Janka‐Schaub Gritta E. , Veerman Anjo J. P. , Jansen Gerrit
Published: March 2003

Treatment failure in childhood acute lymphoblastic leukaemia (ALL) might be associated with methotrexate (MTX) resistance. Little is known about MTX resistance in relapsed ALL. In this study, we determined ex vivo MTX resistance in precursor‐B ALL at relapse (rALL) and determined possible defects in MTX membrane transport and polyglutamylation. Using the in situ thymidylate synthase inhibition assay, 21 rALL samples were threefold more MTX resistant than 63 initial precursor‐B ALL samples, both after short‐term and after continuous MTX exposure (P ≤ 0·01). [3H]‐MTX membrane transport did not differ between eight rALL and 25 precursor‐B ALL samples. Incubation for 24 h with 1 µm[3H]‐MTX resulted in a trend towards a lower accumulation of MTX in 20 relapsed than in 83 initial samples of precursor‐B ALL samples (906 vs. 1364 pmol/109 cells; P = 0·07). Accumulation of long‐chain MTX polyglutamates (MTX‐Glu4−6) did not differ between relapsed and newly diagnosed samples (746 and 889 pmol/109 cells; P = 0·1). Activities of the enzymes involved in polyglutamylation (folylpolyglutamate synthetase and folylpolyglutamate hydrolase) did not differ between rALL and untreated c/pre‐B‐ALL. This study demonstrates that leukaemic cells of children with relapsed precursor‐B ALL are relatively MTX resistant, but that this MTX resistance is not associated with major impairments in MTX uptake or polyglutamylation.
Restricted usage of T‐cell receptor α‐chain variable region (TCRAV) and T‐cell receptor β‐chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation
Authors: Matsutani Takaji , Yoshioka Takeshi , Tsuruta Yuji , Iwagami Shoji , Toyosaki‐Maeda Tomoko , Horiuchi Takahiro , Miura Akira B. , Watanabe Arata , Takada Goro , Suzuki Ryuji , Hirokawa Makoto
Published: March 2003

We analysed T‐cell receptor α‐chain variable region (TCRAV) and T‐cell receptor β‐chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo‐BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6–7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1‐4 (26%, 11 of 41 recipients), VA3‐1 (32%) and VB24‐1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity‐determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0·05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T‐cell regeneration via a thymic‐dependent pathway in children.
Genetic instability in myelodysplastic syndrome: detection of microsatellite instability and loss of heterozygosity in bone marrow samples with karyotype alterations
Authors: Maeck Lienhard , Haase Detlef , Schoch Claudia , Hiddemann Wolfgang , Alves Frauke
Published: March 2003

Using a polymerase chain reaction (PCR)‐based approach, we examined the prevalence of loss of heterozygosity (LOH) and microsatellite instability (MSI) in relation to chromosomal imbalances in myelodysplastic syndrome (MDS). Two of 26 patients displayed MSI (8%), one of them at five loci. LOH was detected in six out of 26 cases (23%), predominantly involving markers IRF1 [5q31] and WT1 [11p]. Two patients displayed a corresponding chromosomal deletion by conventional cytogenetics. Supporting the mutator phenotype hypothesis, a significant coincidence of LOH, MSI and chromosome abnormalities was observed (P < 0·025). Moreover, our data suggest that LOH represents an initial rather than a secondary genetic event in MDS, promoting genetic instability in a subset of patients.
Regenerating normal B‐cell precursors during and after treatment of acute lymphoblastic leukaemia: implications for monitoring of minimal residual disease
Authors: Van Wering Elisabeth R. , Van der Linden‐Schrever Birgit E. M. , Szczepański Tomasz , Willemse Marja J. , Baars Ed A. , Van Wijngaarde‐Schmitz Hanne M. , Kamps Willem A. , Van Dongen Jacques J. M.
Published: March 2003

We studied 57 childhood acute lymphoblastic leukaemia (ALL) patients who remained in continuous complete remission after treatment according to the Dutch Childhood Leukaemia Study Group ALL‐8 protocols. The patients were monitored at 18 time points during and after treatment [640 bone marrow (BM) and 600 blood samples] by use of cytomorphology and immunophenotyping for the expression of TdT, CD34, CD10 and CD19. Additionally, 60 BM follow‐up samples from six patients were subjected to clonality assessment via heteroduplex polymerase chain reaction (PCR) analysis of immunoglobulin V h‐J
h gene rearrangements. We observed substantial expansions of normal precursor B cells in regenerating BM not only after maintenance therapy but also during treatment. At the end of the 2‐week intervals after consolidation and reinduction treatment, B‐cell‐lineage regeneration was observed in BM with a large fraction of immature CD34+/TdT+ B cells. In contrast, in regenerating BM after cessation of maintenance treatment, the more mature CD19+/CD10+ B cells were significantly increased, but the fraction of immature CD34+/TdT+ B cells was essentially smaller. Blood samples showed a profound B‐cell lymphopenia during treatment followed by a rapid normalization of blood B cells after treatment, with a substantial CD10+ fraction (10–30%). Heteroduplex PCR analysis confirmed the polyclonal origin of the expanded precursor B cells in regenerating BM. This information regarding the regeneration of BM is essential for the correct interpretation of minimal residual disease studies.
Chimaeric mice with disruption of the gene coding for phosphatidylinositol glycan class A (Pig‐a) were defective in embryogenesis and spermatogenesis
Authors: Lin Shu‐Rung , Yu I‐Shing , Huang Pei‐Hsin , Tsai Chan‐Wu , Lin Shu‐Wha
Published: March 2003

Mutations in the gene encoding PIG‐A (phosphatidylinositol glycan class A) are found in patients with paroxysmal nocturnal haemoglobinuria (PNH), an acquired haematopoietic stem cell disorder. Individuals with hereditary PIG‐A mutations have never been identified, which is also manifested by the difficulties in generating Pig‐a knockout (KO) mice. This study investigated the effect of Pig‐a mutations on the development of visceral and genital organs in addition to the haematopoietic system by the generation of Pig‐a KO chimaeric mice. Of a total of 54 live births out of 1684 blastocysts injected, chimaerism for Pig‐a knockout was detected in 29 mice, suggesting the importance of Pig‐a in embryogenesis and in live birth. Quantification of the degree of chimaerism in different organs of the surviving chimaeric mice revealed extremely low levels of Pig‐a KO cells in the liver and spleen. In contrast, high levels of KO signals were usually detected in the brain, heart, lung and kidney. Haematopoiesis proceeded normally in these chimaeric mice (as measured by ‘complete blood cell counting’) and the Pig‐a KO cells were present at low levels in red blood cells and B lymphocytes but at high levels in T lymphocytes, although these KO cells did not gain any growth advantage. The effect of Pig‐a knockout was also prominent in the reproductive system, another organ with high mitotic activity. Breeding the male chimaeras revealed a high rate of infertility and abnormality in the male genital organs, including abnormally shaped testes, epididymis and seminal vesicles. Even in the absence of gross abnormalities of the genital organs, low counts of motile sperm were also discernible. Pig‐a KO sperm was detected in these organs; however, no transmission of the KO allele was observed. The results suggest a possible mechanism underlying the non‐transmission of the Pig‐a KO gene in germlines.
A transient assay for regulatory gene function in haemopoietic progenitor cells
Authors: McIvor Zoe J. , Heyworth Clare M. , Johnson Barbra A. , Pearson Stella , Fiegler Heike , Hampson Lynn , Dexter T. Michael , Cross Michael A.
Published: March 2003

This work aimed to provide a means of assaying directly the effects of transient expression of introduced genes on the survival, proliferation, lineage commitment and differentiation of haemopoietic progenitor cells. For this purpose, we have developed a system that allows isolation of productively transfected, mulitipotent haemopoietic cells within a few hours of the introduction of test genes. We have shown that FDCP‐mix cells productively transfected with expression plasmids encoding green fluorescent protein (GFP) differentiate normally and retain colony‐forming potential. We constructed an expression vector consisting of a bicistronic cassette in which a GFP marker gene and a test gene are driven from the same promoter. The vector design has been optimized for co‐expression and the test gene was shown to be biologically active. The expression profile from a transiently transfected template under different growth conditions reveals that active expression continues for at least 2 d after transfection. The transient transfection of FDCP‐mix cells with the vectors described provides a powerful tool for analysis of the immediate early effects of test gene overexpression during haemopoietic differentiation.