bluuebunny/crossref_metadata_2025 huggingface dataset:
Common resistance mechanisms to deoxynucleoside analogues in variants of the human erythroleukaemic line K562
Authors: Dumontet Charles , Fabianowska‐Majewska Krystyna , Mantincic Danko , Callet Bauchu Evelyne , Tigaud Isabelle , Gandhi Varsha , Lepoivre Michel , Peters Godefridus J. , Rolland Marie Odile , Wyczechowska Dorota , Fang Xiao , Gazzo Sophie , Voorn Daphne A. , Vanier‐Viornery Armelle , Mackey John
Published: March 2003

Resistant variants of the human leukaemic line K562 were developed using selection with the deoxynucleoside analogues cytosine arabinoside, 2‐chlorodeoxyadenosine, fludarabine and gemcitabine. The resistant lines displayed a high degree of cross resistance to all deoxynucleoside analogues, with little or no cross resistance to other agents. There was a profound accumulation defect of all nucleoside analogues in the resistant variants but no significant defect in nucleoside transport in any of the variants. 5′ nucleotidase activity was strongly increased and deoxycytidine kinase activity was moderately reduced in all of the resistant variants, resulting in reduced accumulation of triphosphate analogues. In addition a deletion in one of the alleles of the deoxycytidine kinase was detected in the fludarabine‐resistant line. Ribonucleotide reductase activity was found to be strongly increased in the gemcitabine‐selected line and purine nucleoside phosphorylase was increased in the 2‐chlorodeoxyadenosine‐selected line. Free nucleotide pools were increased in the 2‐chlorodeoxyadenosine‐selected line. There was no expression of the mdr1 gene by the resistant lines. Karyotypic analysis and FISH experiments using a 6q21 specific probe showed alterations in the 6(q16‐q22) region which contains the 5′‐nucleotidase gene. Early events in the activation and degradation of deoxynucleoside analogues appear to constitute common mechanisms of resistance to these compounds.
Defective functional activity and accelerated apoptosis in neutrophils from children with cancer are differentially corrected by granulocyte and granulocyte‐macrophage colony stimulating factors in vitro
Authors: Lejeune Marylène , Cantinieaux Brigitte , Harag Saadia , Ferster Alina , Devalck Christine , Sariban Eric
Published: March 2003

We have previously shown that polymorphonuclear leucocytes (PMN) harvested from children with cancer and exposed to chemotherapy exhibit defective bactericidal activities against both Gram‐positive and Gram‐negative microorganisms as well as accelerated apoptosis. In this study, PMN from children with cancer were evaluated to compare in vitro the corrective effects of the two myeloid colony stimulating factors G‐CSF and GM‐CSF on these defective pathways. Both G‐CSF and GM‐CSF were able to increase the defective bactericidal activities against S. aureus and E. coli. However, GM‐CSF was consistently superior to G‐CSF in correcting PMN microbicidal activity; this correction was incomplete since it did not reach the level observed in normal PMN exposed to GM‐CSF.

The accelerated apoptosis of PMN was not affected by G‐CSF. In contrast, GM‐CSF significantly prolonged the survival of the PMN although it did not reach the level of survival observed with normal PMN exposed to GM‐CSF.

These observations were consistent with other studies indicating that in PMN, microbicidal activities and apoptosis are differentially sensitive to the myeloid growth factors G‐CSF and GM‐CSF.
All‐trans retinoic acid regulates adhesion mechanism and transmigration of the acute promyelocytic leukaemia cell line NB‐4 under physiologic flow
Authors: Brown David C. , Tsuji Hisashi , Larson Richard S.
Published: March 2003

The success of all‐trans retinoic acid (ATRA) in the therapy of acute promyelocytic leukaemia (APL) has received increased attention. Unfortunately, life‐threatening multiorgan failure commonly occurs, i.e. retinoic acid syndrome, and is thought to be the result of organ infiltration by leukaemic cells. We hypothesized that ATRA‐induced differentiation of APL cells leads to adhesion receptor alterations responsible for leucocyte extravasation from the blood into tissue. Changes in adhesive properties of the APL cell line NB‐4 in response to ATRA were investigated using a parallel plate flow chamber under conditions that recapitulate physiologic flow conditions. Untreated NB‐4 cells initially tether and roll on activated human umbilical vein endothelial cell monolayers using a combination of E‐selectin, P‐selectin and α4 integrin. After ATRA treatment, > 80% of initial NB‐4 cell attachment to endothelial cells was E‐selectin dependent. Stable arrest (firm adherence) of NB‐4 cells on activated endothelium was also altered by ATRA treatment. Untreated NB‐4 cells used α4 integrin to arrest on endothelium, but β2 integrin dependent arrest was induced by ATRA. With the acquisition of β2 integrin function, ATRA‐treated cells acquired the ability to transmigrate through activated endothelium. Thus, ATRA dramatically altered the adhesion phenotype on NB‐4 cells: ATRA induced rolling largely attributable to E‐selectin, abrogated α4 integrin dependent rolling, and promoted acquisition of β2 integrin dependent firm adherence and transmigration. These findings represent novel cellular and differentiation effects of ATRA, and, to our knowledge, are the first demonstration that a therapeutic agent differentially regulates α4 and β2 integrin on the same leucocyte.
Selective depletion of major and minor histocompatibility antigen reactive T cells: towards prevention of acute graft‐versus‐host disease
Authors: Van Dijk Astrid M. C. , Kessler Floortje L. , Stadhouders‐Keet Simone A. E. , Verdonck Leo F. , De Gast Gijsbert C. , Otten Henny G.
Published: March 2003

Development of acute graft‐versus‐host disease (aGVHD) following HLA‐identical sibling bone marrow transplantation (BMT) remains a serious complication. Aselective depletion of T cells has proved to be effective in preventing aGVHD but is associated with relapse and increased incidence of infection. As aGVHD is directed mainly against epithelial tissues we examined whether it would be feasible to selectively deplete T cells reactive with epithelial cells whilst preserving other specificities. Donor T cells which express HLA‐DR, CD25, CD69 and CD71 activation markers after cocultivation with patient keratinocytes were depleted using magnetic cell separation techniques. Depletion of major as well as minor histocompatibility antigen activated T cells revealed a significant (P = 0.004 and P = 0.031, respectively) 10‐fold decrease in the frequency of donor T lymphocyte precursors reactive with patient keratinocytes. The frequency reactive with third‐party and patient peripheral blood mononuclear cells, including leukaemia cells, remained unchanged, supporting the notion that aGVHD and graft‐versus‐leukaemia (GVL) may be separable. This alloantigen‐specific depletion may be used in matched unrelated as well as HLA‐identical sibling BMT for reducing aGVHD whilst conserving GVL.
Unexplained aplastic anaemia, immunodeficiency, and cerebellar hypoplasia (Hoyeraal‐Hreidarsson syndrome) due to mutations in the dyskeratosis congenita gene, DKC1
Authors: Knight S. W. , Heiss N. S. , Vulliamy T. J. , Aalfs C. M. , McMahon C. , Richmond P. , Jones A. , Hennekam R. C. M. , Poustka A. , Mason P. J. , Dokal I.
Published: March 2003

Hoyeraal‐Hreidarsson (HH) syndrome is a multisystem disorder affecting boys characterized by aplastic anaemia (AA), immunodeficiency, microcephaly, cerebellar‐hypoplasia and growth retardation. Its pathogenesis is unknown. X‐linked dyskeratosis congenita (DC) is an inherited bone‐marrow‐failure syndrome characterized by skin pigmentation, nail dystrophy and leucoplakia which usually develop towards the end of the first decade of life. AA occurs in >90% of cases of DC. We speculated that mutations in the gene responsible for X‐linked DC (DKC1) may account for the HH syndrome, due to the phenotypic similarities between the disease in respect of AA and gender bias. We therefore analysed the DKC1 gene in two HH families. In one family a nucleotide change at position 361(A → G) in exon 5 was found in both affected brothers; in the other family a nucleotide change at position 146(C → T) in exon 3 was found in the affected boys. The finding of these two novel missense DKC1 mutations demonstrates that HH is a severe variant of DC. They also show that mutations in DKC1 can give rise to a very wide clinical spectrum of manifestations. Boys with unexplained AA or immunodeficiency should be tested for mutations in DKC1 even though they may lack diagnostic features of DC.
Cytotoxicity against lymphoblastoid cells mediated by a T‐cell clone from an aplastic anaemia patient: role of CD59 on target cells
Authors: Takami Akiyoshi , Zeng Weihua , Wang Hongbo , Matsuda Tamotsu , Nakao Shinji
Published: March 2003

In an attempt to elucidate the pathogenic role of a CD4+ cytotoxic T‐cell clone NT4.2 isolated from the bone marrow of a patient with cyclosporine‐dependent aplastic anaemia, we characterized the T‐cell clone as well as its cytotoxicity against an autologous Epstein‐Barr (EB) virus‐transformed B‐lymphoblastoid cell line (LCL). NT4.2 expressed BV21+ BJ2.7+ with a complementarity‐determining region (CDR) 3 motif of SQGQGEVEQY which was homologous to that of a T‐cell clone isolated from a patient with connective tissue disease. NT4.2 started to lyse LCL cells within 2 h and exerted maximal cytotoxicity within 3 h of incubation. The cytotoxicity required the presence of divalent cations and was not associated with DNA fragmentation of the target cells. Anti‐CD59 monoclonal antibodies (MoAb) blocked the cytotoxicity to the same degree as anti‐CD3, HLA‐DR or CD2 mAb. Flow cytometric analysis of the peripheral blood of this patient during remission after cyclosporine therapy revealed 1.7% of granulocytes to be deficient in CD59. These findings indicate that NT4.2 exerts its cytotoxicity through a perforin‐mediated pathway, not a Fas/Fas ligand‐dependent pathway, and that haemopoietic stem cells lacking CD59 may evade cytotoxic T lymphocytes, leading to the in vivo expansion of a paroxysmal nocturnal haemoglobinuria clone.
Clinical and laboratory evidence for a trilineage haematopoietic defect in patients with refractory Diamond–Blackfan anaemia
Authors: Giri  , Kang  , Tisdale  , Follman  , Rivera  , Schwartz  , Kim  , Young  , Rick  , Dunbar
Published: March 2003

Diamond–Blackfan anaemia (DBA) is a constitutional pure red cell aplasia presenting in early childhood. In some patients, neutropenia and/or thrombocytopenia have also been observed during the course of the disease. We have followed 28 patients with steroid‐refractory DBA for up to 13 years with serial peripheral blood counts and bone marrow (BM) aspirates and biopsies. In 21/28 (75%) patients, moderate to severe generalized BM hypoplasia developed, with overall cellularities ranging from 0% to 30%. Marrow hypoplasia correlated with the development of neutropenia (9/21; 43%) and/or thrombocytopenia (6/21; 29%) in many patients. No patient had either cytogenetic abnormalities or progressed to acute leukaemia, although one 13‐year‐old developed marked marrow fibrosis and trilineage dysplasia. We used the in vitro long‐term culture‐initiating cell (LTC‐IC) assay to quantify multilineage, primitive haematopoietic progenitors in a representative subset of these patients. LTC‐IC assays showed equivalent frequencies of cobblestone area‐forming cells (CAFCs) with a mean of 5·42/105 cells ± 1·9 SD and 6·13/105 cells ± 2·6 SD in nine patients and six normal controls respectively. The average clonogenic cell output per LTC‐IC, however, was significantly lower in DBA patients (mean 2·16 ± 1·2 SD vs. 7·36 ± 2·7 SD in normal controls, P = 0·0008). Our results suggest that the underlying defect in patients with severe refractory DBA may not be limited to the erythroid lineage, as was evidenced by the development of pancytopenia, bone marrow hypoplasia and reduced clonogenic cell output in LTC‐IC assays.
Quantitative expression of erythropoietin receptor (EPO‐R) on acute leukaemia cells: relationships between the amount of EPO‐R and CD phenotypes, in vitro proliferative response, the amount of other cytokine receptors and clinical prognosis
Authors: Takeshita Akihiro , Shinjo Kaori , Higuchi Masato , Miyawaki Shuichi , Takemoto Yoshinobu , Kishimoto Yuji , Saito Kenji , Takuchi Hirokuni , Kuriyama Kazutaka , Kimura Yukihiko , Asou Norio , Takahashi Masatomo , Hotta Tomomitu , Kanamaru Akihisa , Ueda Ryuzo , Ohno Ryuzo
Published: March 2003

Expression of erythropoietin (EPO) receptor (EPO‐R) was analysed in leukaemia cells from 150 patients with acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL). EPO‐R was expressed in 81 (60%) out of 136 AML, and in vitro treatment with EPO led to proliferation of leukaemia cells in 13 (16%) out of 81 AML examined. EPO‐R expression and in vitro response to EPO were observed in all subtypes of AML according to the French–American–British (FAB) classification. All eight patients with FAB‐M6 expressed EPO‐R, and one out of four showed an in vitro response to EPO. Although there was no significant correlation (r = 0.2522) between the amount of EPO‐R and the in vitro response to EPO, all of the AML patients who showed in vitro response expressed EPO‐R. Stem cell factor significantly enhanced both EPO‐R expression and in vitro response to EPO. Interleukin‐3 tended to increase in vitro response to EPO. CD phenotypes, the amount of granulocyte colony‐stimulating factor (G‐CSF) receptors and the amount of TPO receptors had no significant relationship with the amount of EPO‐R. Patients with both EPO‐R expression and in vitro response to EPO had shorter duration of complete remission than those without EPO‐R (P = 0.0053). EPO‐R was expressed in four (29%) out of 14 ALL, and none out of five ALL showed in vitro response to EPO.
Jamaican Sβ+‐thalassaemia: mutations and haematology
Authors: Donaldson  , Old  , Fisher  , Serjeant  , Serjeant
Published: March 2003

The sickling disorders are a common cause of morbidity and mortality in Jamaica. Sickle cell β+‐thalassaemia is the fourth commonest form, occurring in one in every 3000 births. This is a heterogeneous condition, producing HbS, HbF and HbA2 with variable amounts of HbA, depending on the mutation and, within a defined population, only a few β‐thalassaemia mutations occur at high frequency. This study establishes the frequency of β‐thalassaemia mutations in Sβ+‐thalassaemia patients in Jamaica. In addition, comparison of the haematological phenotypes is possible by looking at the ‘average steady‐state haematology’ of the different mutational groups. Blood samples from 132 unrelated Sβ+‐thalassaemia patients attending the MRC Sickle Cell Unit at the University of the West Indies were analysed by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) or sequencing to determine the nature and frequencies of the underlying β‐thalassaemia mutations. Ten mutations were identified, four of which accounted for 93% of patients studied. These were −29(A → G) in 71 (54%), −88(C → T) in 27 (20%), polyA(T → C) in 17 (13%) and IVS1‐5(G → C) in nine (7%). The six remaining mutations found at low frequency were C24(T → A) in two patients and one each of IVS2‐848(C → A), −90(C → T), IVS1‐5(G → T), IVS1‐5(G → A) and IVS1‐6 (T → C). In one individual, no mutation was found. The three commonest mutations were all associated with haemoglobin levels of greater than 10 g/dl, whereas IVS1‐5 (G → C) had a more severe haematological phenotype. The predominance of −29(A → G) and −88(C → T) is in keeping with other studies on populations of African origin. IVS1‐5(G → C) is found chiefly in Indian populations, and all affected families acknowledged Indian ancestry, reflecting the prominent Indian community in Jamaica.
Induction of mitochondrial manganese superoxide dismutase confers resistance to apoptosis in acute myeloblastic leukaemia cells exposed to etoposide
Authors: Mäntymaa Pentti , Siitonen Timo , Guttorm Tanja , Säily Marjaana , Kinnula Vuokko , Savolainen Eeva‐Riitta , Koistinen Pirjo
Published: March 2003

We investigated the possible roles of mitochondrial manganese superoxide dismutase (MnSOD) and bcl‐2 in etoposide‐induced cell death in acute myeloblastic leukaemia (AML) using two subclones of the OCI/AML‐2 cell line, the etoposide‐sensitive (ES) and the etoposide‐resistant (ER), as models. Cell death after 24 h exposure to 10 μmol/l etoposide was about 60% and 70% in the ES subclone and about 20% and 25% in the ER subclone, when analysed by trypan blue and annexin V respectively. Cytochrome c efflux from mitochondria to cytosol was observed after 4 h of exposure in both subclones, whereas the activation of caspase‐3 was not detectable until after 12 h of exposure in the ES subclone and 24 h of exposure in the ER subclone, using Western blotting. The decrease in mitochondrial membrane potential, when analysed by the JC‐1 probe fluorocytometrically, also appeared to take place later in the ER than in the ES subclone. Both subclones showed evident basal expression of MnSOD and bcl‐2 by Western blotting. Etoposide caused a potent induction of MnSOD, more than 400% at 12 h, in the ER but not in the ES subclone. No significant change in bcl‐2 expression could be observed in either of the subclones during exposure to etoposide when analysed by Western blotting or flow cytometry. In conclusion, we suggest that MnSOD might have a special role in the protection of AML cells against etoposide‐induced cell death. Although unable to influence the cytochrome c efflux to cytosol, MnSOD might prevent the disruption of mitochondrial membrane potential, which evidently leads to cell death by releasing various activators of apoptosis.
Prevalence of the post‐thrombotic syndrome in young women with previous venous thromboembolism
Authors: McColl  , Ellison  , Greer  , Tait  , Walker
Published: March 2003

The prevalence of mild, moderate and severe post‐thrombotic syndrome (PTS) among 43 young women with a previous single episode of deep vein thrombosis (DVT) was 67%, 7% and 0% respectively. Subjects were assessed at a mean 51 months after the event. Moderate PTS was more common in women with recurrent (n = 9) DVT (44%, P < 0.001). Chronic venous insufficiency, assessed by light reflection rheography (LRR), was significantly (P < 0.05) more prevalent in women with single previous DVT (n = 40), recurrent DVT (n = 9) and isolated pulmonary embolism (PE) (n = 19) compared with healthy age‐matched controls (odds ratios 10.9, 52.4 and 3.8 respectively). LRR findings correlated with moderate, but not mild, PTS. There was no correlation between development of PTS and body mass index.
Recurrent B‐cell non‐Hodgkin's lymphoma in two brothers with X‐linked lymphoproliferative disease without evidence for Epstein–Barr virus infection
Authors: Strahm  , Rittweiler  , Duffner  , Brandau  , Orlowska‐Volk  , Karajannis  , Stadt  , Tiemann  , Reiter  , Brandis  , Meindl  , Niemeyer
Published: March 2003

We present two male siblings suffering from recurrent manifestations of B‐cell non‐Hodgkin's lymphoma (NHL) and recurrent infections of the lower respiratory tract associated with bronchiectasis. Immunodeficiency could not be demonstrated by any laboratory investigation. In both patients, lymphomas developed without evidence for Epstein–Barr virus (EBV) infection, i.e. no antibody response to EBV‐specific antigens, negative EBV‐PCR (polymerase chain reaction) in peripheral blood cells, and absence of latent membrane protein (LMP) and EBV‐encoded RNA (EBER) in lymphoma cells. Molecular analysis of the SH2D1A, the gene for X‐linked lymphoproliferative disease (XLP) led to the identification of a deletion in the first exon in both patients. Therefore, we postulate that the genetic defect and the following dysregulation of the B‐/T‐cell interaction rendered these patients susceptible to the early onset of B‐cell NHL and that EBV infection is not an obligate prerequisite.
The UK experience in treating relapsed childhood acute lymphoblastic leukaemia: a report on the Medical Research Council UKALLR1 study
Authors: Lawson  , Harrison  , Richards  , Oakhill  , Stevens  , Eden  , Darbyshire  ,   on behalf of the mrc childhood leukaemia working party
Published: March 2003

We have examined the toxicity and overall outcome of the Medical Research Council UKALL R1 protocol for 256 patients with relapsed childhood acute lymphoblastic leukaemia (ALL). Second remission was achieved in over 95% of patients. Two patients died during induction and seven patients died of resistant disease. The overall actuarial event‐free survival (EFS) at 5 years for all patients experiencing a first relapse was 46% (95% CI 40–52). Duration of first remission, site of relapse, age at diagnosis and sex emerged as factors of prognostic significance. Five‐year EFS was only 7% for children relapsing in the bone marrow within 2 years of diagnosis, but was 77% for those relapsing without bone marrow involvement > 2.5 years from diagnosis. All analyses in this report are by treatment received. For those receiving chemotherapy alone, the 5‐year EFS was 48%; for autologous bone marrow transplantation (BMT), the 5‐year EFS was 47%; for unrelated donor BMT, it was 52%; and for related donor BMT, the 5‐year EFS was 45%. The groups, however, were not comparable with respect to risk factor profile, and therefore direct comparison of EFS is misleading. Adjustment for time to transplant and prognostic factors was used to reduce the effects of biases between treatment groups, but did not suggest benefit for any particular treatment. There was failure of our planned randomization scheme in this trial with only 9% of those eligible being randomized, which highlights the difficulties in running randomized trials especially in patients who have relapsed from a previous trial. The optimal treatment for relapsed ALL therefore remains uncertain. Alternative approaches are clearly needed for those with early bone marrow relapse if outcome is to improve.
Spontaneous monoclonal immunoglobulin‐secreting peripheral blood mononuclear cells as a marker of disease severity in multiple myeloma
Authors: Cordoba Franck , Lavabre‐Bertrand Thierry , Salhi Sharon Lynn , Huguet Marie France , Gerfaux Jacqueline , Rossi Jean Francois , Vendrell Jean Pierre
Published: March 2003

Peripheral blood from patients with multiple myeloma (MM) contains a small number of plasma cells related to the bone marrow tumour cells by their cytoplasmic immunoglobulin (Ig), their cell membrane antigen expression and/or their gene rearrangements, but hitherto the monoclonal Ig (M‐Ig) production by circulating cells has not been reported. Using a two‐colour ELISPOT assay, Ig‐secreting cells (Ig‐SCs) were detected in the blood of 28 MM and five Waldenstrom's macroglobulinaemia (WM) patients. The number of cells that spontaneously produced an Ig isotype similar to that of the M‐Ig in serum was greater than that of the other Ig‐SCs. MM patients presented an excess of circulating heavy‐chain (α or γ) Ig‐SCs (0.38% of the PBMC) with κ or λ light chains (0.48%) compared with the number of cells secreting the other heavy‐ (0.02%) and light‐chain isotypes (0.03%). WM patients also presented high numbers of cells secreting the μ‐heavy‐chain isotype (0.66%). The Ig synthesized in vitro was characterized as monoclonal, and the M‐Ig secretory capacity of the peripheral blood cells was similar to that observed for Ig‐SCs from polyclonal activated B cells in vivo. The number of these monoclonal cells was significantly increased in patients in an advanced stage of MM (I/II vs. III, P < 0.001) and correlated with the serum beta‐2 microglobulin concentration (r = 0.69; P < 0.0003). The number of M‐Ig‐SCs in MM patients could be a useful marker for evaluating the progression of multiple myeloma.
Histone deacetylase inhibitor but not arsenic trioxide differentiates acute promyelocytic leukaemia cells with t(11;17) in combination with all‐trans retinoic acid
Authors: Kitamura Kunio , Hoshi Shinetsu , Koike Mitsuru , Kiyoi Hitoshi , Saito Hidehiko , Naoe Tomoki
Published: March 2003

Acute promyelocytic leukaemia (APL) with t(11;17)/PLZF‐RARα responds poorly to all‐trans retinoic acid (ATRA) and arsenic trioxide (As2O3), in contrast to APL with t(15;17)/PML‐RARα. Molecular studies have shown that histone deacetylase (HDAC) recruited by PLZF‐RARα is associated with the ATRA resistance. Here, we analysed in vitro the differentiation of APL cells with t(11;17) using ATRA, As203, granulocyte colony‐stimulating factor (G‐CSF), HDAC inhibitor trichostatin A (TSA), or combinations of these. Although 1 μm ATRA, which stimulated the differentiation of APL cells with t(15;17), was insufficient to induce differentiation, 3 μm ATRA induced terminal differentiation into granulocytes. As203 alone or in combination with ATRA induced neither differentiation nor apoptosis. However, the combination of TSA and 1 μm ATRA had a potent differentiating effect, although TSA alone had little effect. The combination of 1 μm ATRA and G‐CSF did not induce differentiation. These results indicate that APL cells with t(11;17) need a higher concentration of ATRA than those with t(15;17) to differentiate and suggest that HDAC inhibitor is a promising differentiation enhancer in APL with t(11;17).
Antibodies to tissue‐type plasminogen activator in plasma from patients with primary antiphospholipid syndrome
Authors: Cugno Massimo , Dominguez Miguel , Cabibbe Mara , Bisiani Giuliana , Galli Monica , Angles‐Cano Edouard , Agostoni Angelo
Published: March 2003

A reduction in fibrinolysis has been described in association with thrombosis in the primary antiphospholipid syndrome (PAPS). In this study, we measured anti‐tissue‐type plasminogen activator (t‐PA) antibodies and anti‐fibrin‐bound t‐PA antibodies as possible causes of hypofibrinolysis in 39 patients with PAPS. We also evaluated the differences in anti t‐PA antibodies between patients without previous thrombosis (20 patients) and patients with previous episodes of thrombosis (19 patients: deep vein thrombosis in nine, ischaemic stroke in six, arterial leg thrombosis in one, hepatic vein thrombosis in one, thrombophlebitis in one and cerebral venous thrombosis in one). Anti‐t‐PA antibodies were measured by an enzyme‐linked immunosorbent assay (ELISA), and anti‐t‐PA fibrin‐bound antibodies were measured by a solid‐phase fibrin immunoassay (SOFIA) in 39 patients with PAPS and in 39 controls matched for gender and age. High levels of IgG anti‐t‐PA were found in three out of 39 patients with PAPS, and all three patients had a history of thrombosis; four other patients, one of whom had a history of thrombotic events, had high titres of antibodies directed against fibrin‐bound t‐PA. In addition, patients with ischaemic stroke had significantly higher levels of IgG anti‐t‐PA than patients without thrombosis (P = 0·029). In conclusion, our data showed that, in patients with PAPS, the highest levels of anti‐t‐PA antibodies were present in subjects with previous thrombotic events. The discrepancy in the results obtained with two methods of detection of anti‐t‐PA antibodies, ELISA and SOFIA, indicates a different interaction of the antibodies with the t‐PA molecules, which are directly bound to polystyrene plates in ELISA and bound to fibrin as a bridging molecule in SOFIA.
Relapse after bone marrow transplantation: evidence for distinct immunological mechanisms between adult and paediatric populations
Authors: Guimond Martin , Busque Lambert , Baron Chantal , Bonny Yvette , Bélanger Robert , Mattioli Johanne , Perreault Claude , Roy Denis Claude
Published: March 2003

Donor lymphocyte infusions are particularly effective for remission induction in malignant cells in patients who relapse after allogeneic progenitor cell transplantation (PCT) and who remain sensitive to the administration of unprimed donor T and/or natural killer (NK) cells present in donor lymphocyte infusions. To determine whether relapse after unmanipulated PCT could be ascribed to donor T and/or NK cell loss or tolerization, we evaluated the chimeric status of 81 patients with haematological malignancies who were receiving allogeneic unmanipulated PCT. The incidence of mixed chimaerism (MC) in unfractionated mononuclear leucocyte samples decreased rapidly after transplant, and was not detectable 4 months after PCT, even in patients who subsequently relapsed. The chimeric status of immune effector cell subsets was then evaluated in 15 patients at the time of relapse. All adults demonstrated complete donor haematopoiesis (CDH) for all cell lineages, whereas T‐ and NK‐cell MC was only found in patients younger than age 13 years (P = 0·004). MC was not found in T nor NK cells of a control group consisting of age‐matched paediatric patients in remission after allogeneic PCT. Thus, in adults, T and NK cell MC disappears early after unmanipulated allogeneic PCT and is absent at the time of relapse. However, the identification of donor T and NK cell loss in the paediatric relapsed but not remission patients suggests a distinct mechanism of relapse.
Simultaneous detection and quantification of minimal residual disease in childhood acute lymphoblastic leukaemia using real‐time polymerase chain reaction
Authors: Kwan Edward , Norris Murray D. , Zhu Ling , Ferrara Daniella , Marshall Glenn M. , Haber Michelle
Published: March 2003

A number of prospective studies have indicated the clinical utility of measuring minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia (ALL) and have highlighted the need for improved methodology for quantification of residual disease. We describe a novel real‐time polymerase chain reaction (PCR) strategy for MRD analysis based on the exonuclease activity of Taq polymerase to cleave a fluorescently labelled probe. Using a consensus probe designed to the framework 2 region of the IgH gene, together with leukaemia‐specific primers, the utility of this technique for simultaneous detection and quantification of MRD was demonstrated in samples from six ALL patients. This technique provides a rapid quantitative assay for determining MRD levels which lends itself to the routine monitoring of minimal residual leukaemia.
Second chronic phase before transplantation is crucial for improving survival of blastic phase chronic myeloid leukaemia
Authors: Visani Giuseppe , Rosti Gianantonio , Bandini Giuseppe , Tosi Patrizia , Isidori Alessandro , Malagola Michele , Stanzani Marta , Martinelli Giovanni , Piccaluga Pierpaolo , Testoni Nicoletta , Ricci Paolo , Tura Sante
Published: March 2003

Because successful outcome after transplantation seems to depend in acute myeloid leukaemia (AML) and in chronic phase chronic myeloid leukaemia (CML) on disease status at the time of transplantation, we investigated whether FLAN (fludarabine, cytosine arabinoside, mitoxantrone) induction before allogeneic stem cell transplantation (allo‐SCT) may be useful in blastic phase (BP)‐CML. Twenty patients with BP‐CML were studied: 10 patients received FLAN induction chemotherapy before proceeding to early allo‐SCT, whereas 10 patients were submitted to bone marrow transplantation (BMT) without remission induction. Eight out of 10 (80%) patients achieved second chronic phase after one course of therapy with FLAN and seven patients (six in second chronic phase and one with partial response) were then submitted to allo‐SCT. Of the six patients transplanted in the second chronic phase, all achieved molecular remission, four are still in second chronic phase, with intervals ranging from 10 to 54 months, whereas one patient died from infection having relapsed 14 months after SCT and one died of transplant‐related complications in the second chronic phase. Mean durations of second chronic phase and survival after allo‐SCT were both significantly longer than in the group of 10 BP‐CML patients submitted to allo‐SCT without FLAN remission induction treatment [22·4 (range 1–61) vs. 3·5 months (range 1–10) with FLAN and 22·7 (range 2–61) vs. 6·4 (range 1–16) months without FLAN]. We conclude that FLAN induction therapy followed by early allo‐SCT appears to be effective in the treatment of BP‐CML and could provide a curative possibility for BP‐CML patients.
Essential role for T cells in human B‐cell lymphoproliferative disease development in severe combined immunodeficient mice
Authors: Johannessen I. , Asghar M. , Crawford D. H.
Published: March 2003

Epstein–Barr virus (EBV)‐positive B‐cell lymphoproliferative disease (BLPD)‐like lesions develop in severe combined immunodeficient (SCID) mice inoculated with peripheral blood mononuclear cells (PBMCs) from EBV‐seropositive donors. We used this model to investigate the pathogenesis of EBV‐associated BLPD. Tumour incidence fell from 81% to 11% when only B cells were inoculated, suggesting a key role for T cells in tumour formation. This was further underlined by the reduction in tumour incidence from 76% to 7% when PBMCs were depleted of CD4 positive (+ve) helper T cells. Tumour outgrowth was also reduced when PBMC were depleted of CD8 +ve, CD45RA +ve or CD45RO +ve T cells. The majority of PBMC‐derived tumours analysed by reverse transcriptase–polymerase chain reaction (RT–PCR) expressed mRNA for interleukin (IL) 2, 4, 6, 10 and interferon (IFN) γ. This is the cytokine pattern seen in activated T cells and includes B‐cell growth factors. In situ hybridization studies confirmed that the tumour cells themselves express the growth factors, which is consistent with autocrine‐stimulated tumour growth.

Our results suggest the following sequence of events: (1) T cells are essential for the initial outgrowth of tumorigenic EBV +ve B cells in vivo; (2) the neoplasm sustains its growth in an autocrine, cytokine‐stimulated manner; and (3) established tumours become independent of T‐cell help.
The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors
Authors: Drayer A. Lyndsay , Sibinga Cees T. Smit , Blom Nel R. , De Wolf Joost T. M. , Vellenga Edo
Published: March 2003

Increasing the number of megakaryocyte progenitors in stem cell transplants by ex vivo expansion culture may be an approach to accelerate platelet recovery in patients undergoing high‐dose chemotherapy. We evaluated the effect of three different cytokine combinations on expansion, with special emphasis on the type of colony formation and migration of megakaryocytic cells. The number of clonogenic megakaryocyte progenitors (colony‐forming units–megakaryocyte; CFU‐Mk) with high‐ (> 20 cells/colony) and low‐proliferative capacity (5–20 cells/colony) and the number of megakaryocytic (CD61+) cells were significantly increased by including interleukin 3 (IL‐3) or IL‐3 + IL‐6 + IL‐11 + Flt3‐ligand to cultures containing megakaryocyte growth and development factor (MGDF) plus stem cell factor (SCF). No difference in the maturation of megakaryocytes from all three cytokine combinations to platelets were observed, as demonstrated by electron microscopy. In chemotaxis experiments, the migration towards stromal cell‐derived factor 1 (SDF‐1) was shown to be reduced for CD61+ cells and megakaryocyte progenitors cultured in other cytokines besides MGDF + SCF. The reduced migration was related to a lower expression of CXCR4, the receptor for SDF‐1, on megakaryocytes from the proliferating cultures. These in vitro results demonstrate that expansion in IL‐3 and other cytokines besides MGDF + SCF significantly impair the capacity of megakaryocytic cells to migrate.
Expression of urokinase plasminogen activator and the urokinase plasminogen activator receptor in myeloma cells
Authors: Hjertner Öyvind , Qvigstad Gunnar , Hjorth‐Hansen Henrik , Seidel Carina , Woodliff Jeffery , Epstein Joshua , Waage Anders , Sundan Anders , Börset Magne
Published: March 2003

Binding of urokinase (uPA) to its receptor (uPAR; CD87) focuses proteolytic activity on the cell surface and this system is of importance in malignant matrix degradation and tumour invasion. By immunocytochemistry and flow cytometry, we found that primary myeloma cells and myeloma cell lines expressed uPA and uPAR. Soluble uPA was present in cell line supernatants and lysates in low concentrations. In cell lines, uPA and uPAR were located both on the cell surface and intracellularly, but the expression of both proteins was low. Higher levels of uPAR was detected on the cell surface of primary myeloma cells. When primary myeloma cells were gated by CD45 expression, stronger expression was found on immature CD45+ cells than on mature CD45−/dim cells. Finally, both myeloma cell lines and primary cells were able to cleave a uPA‐specific substrate showing that the uPA system is functionally active. We conclude that myeloma cells are able to produce uPA and uPAR. This opens up a possible role of the uPA system in myeloma cell invasion and in the proteolytic digestion of bone matrix.
Elevated interleukin (IL)‐18 levels during acute graft‐versus‐host disease after allogeneic bone marrow transplantation
Authors: Fujimori Yoshihiro , Takatsuka Hiroyuki , Takemoto Yoshinobu , Hara Hiroshi , Okamura Haruki , Nakanishi Kenji , Kakishita Eizo
Published: March 2003

Acute graft‐versus‐host disease (aGVHD) after allogeneic bone marrow transplantation (BMT) is mediated by grafted T lymphocytes after their polarization into type 1 T cells. Interleukin (IL)‐18, a novel immunoregulatory cytokine, strongly stimulates type 1 T cells, therefore we postulated that IL‐18 may be involved in the pathogenesis of aGVHD. Using an enzyme‐linked immunosorbent assay (ELISA), we serially measured serum levels of IL‐18 in 37 patients with haematological malignancy before and after allogeneic BMT. Patients with aGVHD had high levels of IL‐18 that strongly correlated with the severity of aGVHD. We also found that they showed reduced serum levels of IL‐18 after appropriate treatment or at a state of resolution. IL‐18 levels were not affected by the pretransplant regimen, engraftment or bacterial infection. Compared with circulating interferon (IFN)‐γ or IL‐12 levels, serum levels of IL‐18 showed a more sensitive and specific correlation with the disease status of aGVHD. These findings suggest that IL‐18 may play important roles in the pathogenesis of aGVHD and that measurement of serum IL‐18 levels can be useful indicator of aGVHD.
Functional analysis of the CD8+CD57+ cell population in normal healthy individuals and matched unrelated T‐cell‐depleted bone marrow transplant recipients
Authors: Rowbottom A. W. , Garland R. J. , Lepper M. W. , Kaneria S. S. , Goulden N. J. , Oakhill A. , Steward C. G.
Published: March 2003

The biological activities of CD8+ that co‐express CD57 remain poorly defined. It is unclear whether all CD8+ cells have the potential to become CD57+ or whether they represent a unique subset with distinct functions. Several studies have reported the association between elevated numbers of CD8+CD57+ and a wide range of clinical disorders such as viral reactivation of human cytomegalovirus (HCMV). In this study, we have investigated the relationship between viral reactivation and the effect of diminished interleukin (IL)‐2 production. Using CD8+ cells isolated from patients at various times after allogeneic transplants and in vitro models of HCMV infection, we determined their combined effect on CD8+CD57+. Our results show that high numbers of CD8+CD57+ correlated with diminished killing of HCMV‐infected targets. In addition, we showed a synergistic effect between IL‐2 and HCMV in the expansion of CD8+CD57+ cells. Furthermore, these cells after anti‐CD3 stimulation did not produce tumour necrosis factor (TNF)‐α or interferon (IFN)‐γ. Interestingly, IL‐10 production was elevated in several patients which appeared to be associated with the time from transplant.
Stem cell factor and erythropoietin inhibit apoptosis of human erythroid progenitor cells through different signalling pathways
Authors: Sui Xingwei , Krantz Sanford B. , Zhao Zhizhuang Joe
Published: March 2003

Erythropoietin (EPO) and stem cell factor (SCF) are two important factors in human erythropoiesis. We have recently demonstrated that SCF and EPO synergistically activate mitogen‐activated protein (MAP) kinase, thereby promoting growth of human erythroid colony‐forming cells (ECFCs). In the present study, we have examined the intracellular mechanisms by which SCF and EPO maintain survival of these cells. In the absence of SCF and EPO, human ECFCs underwent rapid apoptosis. The process was significantly inhibited by addition of a single factor and was totally prevented in the presence of both factors. Treatment of ECFCs with wortmannin, a specific inhibitor of phosphoinositide 3‐kinase (PI3K), inhibited the antiapoptotic effect of SCF but had no effect on that of EPO, indicating that SCF but not EPO inhibits apoptosis through the PI3K pathway. In contrast, treatment of ECFCs with PD98059, a specific inhibitor of MAP kinase/ERK kinase (MEK), inhibited cell growth but had no effect on the antiapoptotic activity of either SCF or EPO, suggesting that SCF and EPO prevent apoptosis of human ECFCs independent of the extracellular signal‐regulated kinase (ERK) pathway. Interestingly, both EPO and SCF induced activation of PI3K. However, through PI3K, SCF caused activation of protein kinase B (PKB), an anti‐apoptosis signal, whereas EPO led to activation of ERKs. Furthermore, the SCF‐ and EPO‐maintained expression of antiapoptotic protein Bcl‐XL was correlated with the activation of ERKs and was inhibited by PD98059, suggesting that Bcl‐XL may not have a major role in preventing apoptosis of human ECFCs. Phosphorylated BAD was not affected by SCF, EPO or wortmannin. Taken together with our previous results, the present study indicates that SCF and EPO support survival and growth of human ECFCs through different signalling pathways and that they transduce distinctly different signals through activation of PI3K.
Anti‐HPA‐5b‐induced neonatal alloimmune thrombocytopenia: antibody titre as a predictor
Authors: Ohto Hitoshi , Yamaguchi Tomiko , Takeuchi Chikako , Tohyama Yuriko , Sato Akira , Morita Shoji ,   Collaborative Study Group
Published: March 2003

Anti‐HPA‐5b is the most commonly found platelet‐specific antibody among pregnant women, but it does not cause severe fetal–neonatal alloimmune thrombocytopenia in the majority of affected infants. However, as the sequelae of the affected children may become severe, it is necessary to identify the risk factors for neonatal alloimmune thrombocytopenia. Of 21 354 consecutive pregnant women, 138 [0·65%; 95% confidence interval (CI) 0·54–0·75%], corresponding to 13·2% of the 1049 HPA‐5b− women caculated by the gene frequency, were positive for anti‐HPA‐5b at the first trimester. Anti‐HPA‐5b was titrated in specimens obtained at the third trimester and antibody‐positive women and their neonates were HPA‐5 genotyped. Platelet counts in cord blood and 3 d after birth were assessed in the infants born to these mothers. Chi‐square analysis showed no significant relationship between the titres of maternal antibody to HPA‐5b and the number of pregnancies. There was a significant difference in platelet counts at d 3 between neonates who were compatible (267 × 109/l) or incompatible (220 × 109/l, P < 0·05) with maternal anti‐HPA‐5b. HPA‐5b antibody titres ≥ 64 were related to the development of thrombocytopenia (< 150 × 109/l) in neonates 1 d and 3 d after birth. A high titre (≥ 64) had a positive predictive value of 50% for thrombocytopenia 3 d after birth when the infant was HPA‐5b+ and a negative predictive value of 100%. These results indicate that a high titre (≥ 64) of anti‐HPA‐5b is associated with a higher risk of neonatal thrombocytopenia, even if anti‐HPA‐5b‐induced severe thrombocytopenia rarely develops.
Characterization of apoptosis induced by protein kinase C inhibitors and its modulation by the caspase pathway in acute promyelocytic leukaemia
Authors: Amin Hesham M. , Ergin Melek , Denning Mitchell F. , Quevedo Maria E. , Alkan Serhan
Published: March 2003

Acute promyelocytic leukaemia (APL;M3) is a unique form of acute myelogenous leukaemia characterized by t(15;17) translocation. The induction of apoptosis via inhibiting protein kinase C (PKC) has been recently viewed as a promising tool for the eradication of several malignant disorders. In the present study, we investigated the effect of two different protein kinase C inhibitors, Gö6976 and safingol, on the induction of apoptosis in the APL cell line NB4 and its all trans retinoic acid (ATRA)‐resistant variant NB4.306. The effect of the PKC inhibitors on leukaemic cells obtained from three APL patients was also studied. We also evaluated the possible involvement of the caspases in apoptosis induced by PKC inhibitors. Significant time‐ and concentration‐dependent apoptotic changes were demonstrated using Gö6976 and safingol. In addition, our results demonstrated that the caspases were involved in the apoptosis induced by the PKC inhibitors. In conclusion, our study illustrates that the PKC inhibitors Gö6976 and safingol induce apoptosis in APL and hence could be potential therapeutic agents for the treatment of this disease.
In vitro drug resistance profiles of adult versus childhood acute lymphoblastic leukaemia
Authors: Styczynski Jan , Pieters Rob , Huismans Dieuwke R. , Schuurhuis Gerrit J. , Wysocki Mariusz , Veerman Anjo J. P.
Published: March 2003

The difference in the current cure rates between adult and childhood acute lymphoblastic leukaemia (ALL) may be caused by differences in drug resistance. Earlier studies showed that in vitro cellular drug resistance is a strong independent adverse risk factor in childhood ALL. Knowledge about cellular drug resistance in adult ALL is still limited. The present study compared the in vitro drug resistance profiles of 23 adult ALL patients with that of 395 childhood ALL patients. The lymphoblasts were tested by the MTT assay. The group of adult ALL samples was significantly more resistant to cytosine arabinoside,
l‐asparaginase, daunorubicin, dexamethasone and prednisolone. The resistance ratio (RR) was highest for prednisolone (31·7‐fold) followed by dexamethasone (6·9‐fold),
l‐asparaginase (6·1‐fold), cytosine arabinoside (2·9‐fold), daunorubicin (2·5‐fold) and vincristine (2·2‐fold). Lymphoblasts from adult patients were not more resistant to mercaptopurine, thioguanine, 4‐HOO‐ifosfamide, mitoxantrone and teniposide. There were no significant differences in drug resistance between adult T‐cell (T‐) ALL (n = 11) and adult common/pre‐B‐cell (B‐) ALL (n = 10). Additionally, adult T‐ALL did not differ from childhood T‐ALL (n = 69). There were significant differences between adult common/pre‐B‐ALL and childhood common/pre‐B‐ALL (n = 310) for prednisolone (RR = 302, P = 0·008), dexamethasone (RR = 20·9, P = 0·017) and daunorubicin (RR = 2·7, P = 0·009). Lymphoblasts from adults proved to be relatively resistant to drugs commonly used in therapy. This might contribute to the difference in outcome between children and adults with ALL.