bluuebunny/crossref_metadata_2025 huggingface dataset:
Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man
Authors: Jagadeeswaran Pudur , Gregory Michael , Johnson Stephen , Thankavel Bharath
Published: March 2003

Zebrafish were used as a model to study haemostasis, a vertebrate function of paramount importance. A limitation of the zebrafish model is the difficulty in assaying small amounts of blood to detect coagulation mutants. We report the use of a rapid total coagulation activity (TCA) assay to screen for coagulation defects in individual adult zebrafish. We screened the TCA in 1000 gynogenetic half‐tetrad diploids derived from 86 clutches. Each clutch was from a single F1 female offspring of males mutagenized with ethylnitrosourea (ENU). We found 30–50% defective zebrafish among six clutches, consistent with a heritable defect. The assay developed here provided a rapid screen to detect overall coagulation defects. However, because of the limited amounts of plasma, we could not detect defects in specific pathways. Therefore, a novel, ultra‐sensitive kinetic method was developed to identify specific pathway defects. To test whether the kinetic assay could be used as a screening tool, 1500 Florida wild‐type zebrafish pairs were analysed for naturally occurring coagulation defects. We detected 30 fish with extrinsic pathway defects, but with intact common and intrinsic pathways. We conclude that it is now possible to identify specific coagulation pathway defects in zebrafish.
Flow cytometry and the study of central nervous disease in patients with acute leukaemia
Authors: Subirá D. , Castañón S. , Román A. , Aceituno E. , Jiménez‐Garófano C. , Jiménez A. , García R. , Bernácer M.
Published: March 2003

Central nervous system (CNS) leukaemia is still a matter of debate and new technologies are required to improve the classic morphological definition. One hundred and sixty‐eight cerebrospinal fluid (CSF) samples from 31 patients with acute leukaemia were analysed by flow cytometry and conventional cytology. Concordant positive and negative findings were found in 158 samples but 10 produced discrepant results. Cytology seemed to offer more precise information in one CSF sample and flow cytometric accuracy could be demonstrated in five samples. We conclude that flow cytometry is of great help in confirming CNS leukaemia and eliminating other conditions. Therefore, leukaemic patients can benefit from double cytological and flow cytometric CSF studies.
Calcein assay for multidrug resistance reliably predicts therapy response and survival rate in acute myeloid leukaemia
Authors: Karászi Éva , Jakab Katalin , Homolya László , Szakács Gergely , Holló Zsolt , Telek Béla , Kiss Attila , Rejtô László , Nahajevszky Sarolta , Sarkadi Balázs , Kappelmayer János
Published: March 2003

In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory method for the identification of multidrug‐resistant phenotype in acute leukaemia. This study presents the results of the calcein tests obtained in two large haematological centres in Hungary. Assays were performed with blast cells of 93 de novo acute leukaemia patients, including 65 patients with acute myeloid leukaemia (AML). Results were expressed as multidrug resistance activity factor (MAF) values. AML patients were divided into responders and non‐responders and MAF values were calculated for each group. In both centres, responder patients displayed significantly lower MAF values than non‐responders (P = 0·0045 and P = 0·0454). Cut‐off values were established between the MAFR + SEM and MAFNR − SEM values. On the basis of these cut‐off levels, multidrug resistance (MDR) negativity showed a 72% predictive value for the response to chemotherapy, whereas MDR positivity was found to have an average predictive value of 69% for therapy failure. MDR activity was a prognostic factor for survival rate and the test was suitable for detecting patients at relapse. The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting. The assay detects both P‐glycoprotein and multidrug resistance‐associated protein activities, and identifies AML patients with unfavourable therapy responses.
Decreased prion protein expression in human peripheral blood leucocytes from patients with paroxysmal nocturnal haemoglobinuria
Authors: Dürig Jan , Giese Armin , Schmücker Ute , Kretzschmar Hans A. , Dührsen Ulrich
Published: March 2003

The cellular isoform of the prion protein (PrPC) is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosylphosphatidyl‐inositol (GPI) anchor. PrPC is involved in the pathogenesis of prion diseases and has recently been shown to play a role in haemopoietic cell activation and proliferation. We have used the PrPC‐specific monoclonal antibody (mAb) 3F4 in a flow cytometry approach to analyse the constitutive expression of PrPC on human peripheral blood (PB) cell populations from patients with paroxysmal nocturnal haemoglobinuria (PNH), which are characterized by a deficiency of GPI‐linked cell surface proteins. Comparable PrPC expression levels (P > 0·05), quantified as mean fluorescent intensity, were measured on lymphocytes isolated from normal donors (n = 10) and patients with PNH (n = 5), whereas PNH PB monocytes and granulocytes exhibited substantially lower PrPC surface immunoreactivity than their normal counterparts (P < 0·05). More detailed histogram analyses of the PNH PB leucocytes revealed that PrPC was absent in PNH granulocytes, but was normally expressed in lymphocytes from four out of five patients. However, in one patient a bimodal distribution of 3F4 mAb staining was observed, indicating the presence of a PrPC‐deficient lymphocyte subpopulation. In conclusion, our results show that PNH haemopoietic cells are deficient in cell surface‐bound PrPC.
Detection of diagnostically critical, often hidden, anomalies in complex karyotypes of haematological disorders using multicolour fluorescence in situ hybridization
Authors: Jalal S. M. , Law M. E. , Stamberg J. , Fonseca R. , Seely J. R. , Myers W. H. , Hanson C. A.
Published: March 2003

Multicolour fluorescence in situ hybridization (M‐FISH) simultaneously detects all 24 human chromosomes in unique fluorescent colours. The identification of diagnostically critical gene rearrangement(s) in complex karyotypes of haematological disorders continues to be a challenge. We present five cases in which t(9;11), complex t(8;22), t(12;21) and t(11;14) were detected primarily using M‐FISH and were confirmed using locus‐specific probes. We conclude that M‐FISH can be effective in complete characterization of critical gene rearrangements in haematological disorders.
Recombinant human factor VIIa in the management of amyloid‐associated factor X deficiency
Authors: Boggio Lisa , Green David
Published: March 2003

Factor X deficiency is an important complication of amyloidosis. It is associated with severe bleeding that is difficult to control with plasma or prothrombin complex concentrates. Splenectomy ameliorates the factor X deficiency, but achieving satisfactory haemostasis for this operation is problematic. We report that a new clotting concentrate, recombinant factor VIIa, readily controls bleeding and makes splenectomy feasible.
Telomere length changes in patients with aplastic anaemia
Authors: Lee Je‐Jung , Kook Hoon , Chung Ik‐Joo , Na Jeung‐A , Park Moo‐Rim , Hwang Tai‐Ju , Kwak Jae‐Yong , Sohn Sang‐Kyun , Kim Hyeoung‐Joon
Published: March 2003

To investigate telomere changes in patients with aplastic anaemia (AA) and clinical factors influencing the telomere dynamics, telomere length (TL) was measured in peripheral blood mononuclear cells using Southern blot analysis of 42 patients with AA and 39 healthy normal controls. Nineteen patients received supportive treatment only, while the remaining 23 patients received immunosuppressive therapy with anti‐thymocyte globulin or anti‐lymphocyte globulin ± cyclosporin A. In AA patients, TL was on average 1·41 kb shorter than that of age‐matched normal controls (P < 0·001). In patients treated with immunosuppression, the mean TL of non‐responders was significantly shorter than that of age‐matched normal controls (P < 0·001), while no difference in TL was detected in responders compared with controls. Positive correlation was observed between the extent of telomere shortening, the severity of neutropenia (P = 0·05) and the degree of mean corpuscular volume elevation (P = 0·005) at the time of the study. However, there was no correlation with time elapsed since diagnosis (P = 0·214). These findings suggest that haematopoietic stem cells in patients with AA rapidly lose TL at the onset of the disease. The TL shortening may reflect the severity of impairment of haematopoiesis.
Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia
Authors: Matsushita Maiko , Ikeda Hideyuki , Kizaki Masahiro , Okamoto Shinichiro , Ogasawara Masahiro , Ikeda Yasuo , Kawakami Yutaka
Published: March 2003

PRAME (Preferentially expressed antigen of melanoma) has been previously identified as a melanoma antigen recognized by cytotoxic T cells (CTLs) and found to be expressed in a variety of cancer cells including leukaemic cells. We have screened 98 Japanese patients with leukaemia and lymphoma for expression of the PRAME gene using semiquantitative reverse transcription polymerase chain reaction (RT‐PCR). Forty‐one patients (42%) showed high levels of PRAME expression. Eight of these patients were then monitored using real‐time PCR for a period of 10–37 months. Significant reductions in the PRAME expression were observed in all patients after chemotherapy. An increased expression was detected in the two patients who relapsed, one of which was before cytological diagnosis. These changes were correlated with those of other known genetic markers, such as the bcr‐abl gene. Therefore, quantitative monitoring of the PRAME gene using real‐time PCR method may be useful for detecting minimal residual disease and to predict subsequent relapse, especially in patients without known genetic markers. In addition, a PRAME‐positive leukaemia cell line and fresh leukaemic cells were found to be susceptible to lysis by PRAME‐specific CTLs established from a patient with melanoma, suggesting that the PRAME peptide can also be a target leukaemia antigen for T cells.
Microangiopathic haemolytic anaemia in metastasizing malignant tumours is not associated with a severe deficiency of the von Willebrand factor‐cleaving protease
Authors: Fontana S. , Gerritsen H. E. , Hovinga J. Kremer , Furlan M. , Lämmle B.
Published: March 2003

Complete deficiency of von Willebrand factor‐cleaving protease (VWF‐cp) has recently been identified as a pathogenetically important factor for thrombotic thrombocytopenic purpura (TTP). Microangiopathic haemolytic anaemia (MAHA) with thrombocytopenia in patients with metastasizing neoplasms is clinically similar to TTP, however, the pathogenesis of the condition is unclear. Partial deficiency of VWF‐cp in metastasizing malignancy has recently been reported in patients without MAHA. Our study shows normal or subnormal VWF‐cp activity in four patients with metastasizing neoplasia‐associated MAHA but, in contrast to classical TTP, no complete deficiency of VWF‐cp despite the full clinical picture of MAHA.
Molecular characterization of the PK‐LR gene in sixteen pyruvate kinase‐deficient patients
Authors: Zanella Alberto , Bianchi Paola , Fermo Elisa , Iurlo Alessandra , Zappa Manuela , Vercellati Cristina , Boschetti Carla , Baronciani Luciano , Cotton Frederic
Published: March 2003

We studied the PK‐LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042–1044); one four nucleotide duplication (nt 1515–1518, GGTC); one splice site [IVS6(−2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042–1044, dupl 1515–1518, IVS6(−2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042–1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515–1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(−2)t leads to an alteration of the 5′ and 3′ splice site consensus sequence; the cDNA analysis shows a 67‐bp deletion in the first part of exon 11 (del 1437–1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.
Comparison of ‘sequential’ versus ‘standard’ chemotherapy as re‐induction treatment, with or without cyclosporine, in refractory/relapsed acute myeloid leukaemia (AML): results of the UK Medical Research Council AML‐R trial
Authors: Liu Yin John A. , Wheatley Keith , Rees John K. H. , Burnett Alan K. ,   the UK MRC Adult Leukaemia Working Party
Published: March 2003

This aim of the acute myeloid leukaemia (AML)‐R trial was to compare sequential (Seq) ADE (cytarabine, daunorubicin, etoposide) with standard (Std) ADE as remission re‐induction treatment and to assess any benefit of cyclosporine (CSA) as a multidrug resistance modulator in refractory/relapsed AML patients. Seq ADE, based on the concept of Timed Sequential Therapy, comprised the same drugs as Std ADE but given at higher doses and in a different sequence. Between 1992 and 1997, 235 patients with relapsed (175) and refractory (60) AML were entered: 170 were randomized between Std versus Seq ADE and 213 between CSA versus no CSA. CSA was initially given at a dose of 5 mg/kg/d and increased to 10 mg/kg/d in the latter part of the trial. Overall, the complete remission (CR) rate was 43%, with Std ADE being significantly better than Seq ADE (54% versus 34%, P = 0·01). CR rates did not differ between the CSA and no CSA arms (41% versus 45%, P = 0·6). Overall, 3 year disease‐free survival (DFS) of remitters was 16%, with a relapse risk of 70%. DFS was not significantly different between the chemotherapy or the CSA arms. Overall, 3 year survival was 8%. Survival with Std ADE was significantly better than with Seq ADE (12% versus 6%, P = 0·03). CSA did not affect overall survival, except in patients ≥ 60 years, who fared worse on CSA (P = 0·0003). No difference in haematological toxicity between the chemotherapy or CSA arms was seen. Survival was better with longer first CR duration (P < 0·0001). We conclude that Std ADE was superior to Seq ADE for CR achievement and survival, with no benefit with CSA, at the doses used in this study.
Two further British families with the ‘cryohydrocytosis’ form of hereditary stomatocytosis
Authors: Haines Philip G. , Jarvis Helen G. , King Simon , Noormohamed Faruq H. , Chetty Margaret C. , Fisher Julie , Hill P. , Nicolaou Anna , Stewart Gordon W.
Published: March 2003

We describe two families with the ‘cryohydrocytosis’ form of stomatocytosis. Both show a mild stomatocytic anaemia with Hb levels of 12–16 g/dl and reticulocyte counts of 4·3–24%, with very marked autohaemolysis at refrigerator temperatures and pseudohyperkalaemia as a result of loss of K from red cells on storage at room temperature. The ouabain + bumetanide‐insensitive ‘passive leak’ K influx showed a ‘U’‐shaped temperature dependence, with a minimum at 23°C. In one family, there was consistent variation in haematological severity within the pedigree. In the other, the parents of the proposita were normal, but all three of her children were affected, consistent with a new mutation of a dominant condition. Cold storage of the red cells led to a very marked increase in osmotic fragility and macrospherocytosis, explaining why a diagnosis of ‘hereditary spherocytosis’ can easily be reached in these pedigrees.
Class switch recombination was specifically targeted to immunoglobulin (Ig)G4 or IgA in Hodgkin's disease‐derived cell lines
Authors: Irsch Johannes , Wolf Jürgen , Tesch Hans , Diehl Volker , Radbruch Andreas , Staratschek‐Jox Andrea
Published: March 2003

In T cell‐dependent immune responses, class switch recombination occurs in germinal centres. There is now evidence that Hodgkin/Reed–Sternberg cells are derived from germinal centre B cells. Cytokines specifically determine the direction of class switching, i.e the isotype of the new antibodies. We performed restriction analyses and polymerase chain reaction on the immunoglobulin heavy chain loci for five Hodgkin's disease‐derived B‐cell lines and one Hodgkin's disease‐derived T‐cell line in order to analyse class switch recombination. In all the B‐cell lines, class switch recombination had been targeted to Cα4 or Cα1/2. This showed that cell‐line precursors had undergone class switching, probably under the influence of TH2 or TH3 cell‐derived cytokines. Deletions comprising several constant region genes were observed in cell lines L428, L1236, L591 and KMH2. Karyotype analyses of two of these revealed translocational breakpoints within the immunoglobulin heavy chain gene locus. Our data support the view that a chromosomal instability may occur during class switch recombination in Hodgkin/Reed–Sternberg cells causing chromosomal breaks. Thus, as in other germinal centre B cell‐derived lymphomas, the immunoglobulin gene locus may be frequently involved in structural chromosomal aberrations in Hodgkin's disease.
Identification of both myeloid CD11c+ and lymphoid CD11c− dendritic cell subsets in cord blood
Authors: Borràs Francesc E. , Matthews Nick C. , Lowdell Mark W. , Navarrete Cristina V.
Published: March 2003

Dendritic cells (DCs) are the most potent antigen‐presenting cells described to date. In human peripheral blood, both myeloid and lymphoid subsets of DCs have been identified. In contrast, cord blood (CB) DCs have recently been described as being exclusively of the immature CD11c− lymphoid DC subset. Using an alternative method of enrichment, based on a negative selection system, both lymphoid (HLA‐DR+ CD123+++ CD11c− CD33−) and myeloid (HLA‐DR++ CD123+ CD11c+ CD33+) DCs were identified in CB. Although the majority of CB DCs showed a lymphoid phenotype, a significant number of CD11c+ myeloid DCs (25·6% ± 14·5%, n = 13) were also present. Other markers, such as CD80 and CD83, were negative in both subsets. Analyses of the allostimulatory capacity of both subsets showed that freshly isolated CB lymphoid DCs failed to induce a potent allostimulation of naive CB T cells. These features are therefore consistent with previous work reporting an immature phenotype for lymphoid DCs in adult blood. The significance of the inverted CD11c+/CD11c− ratio observed in CB DCs (1:3) with respect to adult blood DCs (3:1) remains to be explained.
A modified high‐dose dexamethasone regimen for primary systemic (AL) amyloidosis
Authors: Palladini Giovanni , Anesi Ernesto , Perfetti Vittorio , Obici Laura , Invernizzi Rosangela , Balduini Carlo , Ascari Edoardo , Merlini Giampaolo
Published: March 2003

High‐dose dexamethasone (HD‐Dex) has been reported to benefit AL amyloidosis patients with varying response rates. Our preliminary experience with the usual HD‐Dex schedule indicated that the induction phase was rather toxic in AL patients. We therefore adopted a milder schedule consisting of dexamethasone 40 mg on d 1–4 q21 d for up to eight cycles. Overall 8 out of 23 (35%) treated patients responded to treatment in a median time of 4 months (range 2–6 months) without significant toxicity. This regimen may be considered front‐line therapy when autologous stem cell transplantation is not feasible and when a rapid response is particularly important.
Successful treatment of severe intra‐abdominal bleeding associated with disseminated intravascular coagulation using recombinant activated factor VII
Authors: Moscardó Federico , Pérez Francisca , De La Rubia Javier , Balerdi Begoña , Lorenzo José I. , Senent Maria L. , Aznar Ismael , Carceller Soledad , Sanz Miguel A.
Published: March 2003

Recombinant activated factor VII (rFVIIa) is indicated mainly for the treatment of patients with haemophilia and inhibitors. However, little information is available on the use of rFVIIa in the treatment of the severe bleeding associated with disseminated intravascular coagulation (DIC). We report a pregnant woman with DIC, who developed severe intra‐abdominal bleeding after caesarean section. Despite treatment with fresh‐frozen plasma, fibrinogen, platelet transfusions and surgery, the abdominal bleeding persisted and intravenous treatment with rFVIIa was initiated. The response to treatment was rapid, with control of the bleeding and resolution of the coagulopathy. No side‐effects related to rFVIIa were noted. This case suggests a potential role for rFVIIa in the treatment of severe and refractory bleeding associated with DIC.
HLA‐A3 increases and HLA‐DR1 decreases the risk of acute graft‐versus‐host disease after HLA‐matched sibling bone marrow transplantation for chronic myelogenous leukaemia
Authors: Clark Richard E. , Hermans Jo , Madrigal Alejandro , Nachbaur David , Kropshofer Gabriele , Gratwohl Alois , Apperley Jane , Niederwieser Dietger ,   for the Chronic Leukaemia Working Party of the European Group of Blood and Bone Marrow Transplantation
Published: March 2003

Frequencies of human leucocyte antigens (HLA)‐A, ‐B and ‐DR were determined in 751 patients with chronic myelogenous leukaemia (CML) reported to the European Group for Blood and Marrow Transplantation after bone marrow transplantation from HLA‐identical family donors and related to the occurrence of graft‐versus‐host disease (GVHD). HLA‐A3 and DR1 were significantly associated with acute GVHD, the first with a higher risk (44% in HLA‐A3+ versus 34% in HLA‐A3− patients) and the latter with a lower risk (28% in HLA‐DR1+ versus 38% in HLA‐DR1− patients) for developing acute GVHD grade II–IV. Both factors were independent of known variables for GVHD as shown in a multivariate analysis. The results show that MHC alleles independently influence the incidence of GVHD in bone marrow transplantation from an HLA‐identical donor for first chronic‐phase CML. Possible mechanisms might include an HLA antigen‐specific allele‐associated effect, and/or non‐specific allele‐associated immune hypo‐ or hyper‐responsiveness.
Reduced serum ceruloplasmin levels in hereditary haemochromatosis
Authors: Cairo Gaetano , Conte Dario , Bianchi Laura , Fraquelli Mirella , Recalcati Stefania
Published: March 2003

As ceruloplasmin (Cp) seems to be involved in iron mobilization, serum Cp levels were measured in 35 patients with hereditary haemochromatosis (HH), 12 with acquired iron overload (AIO) and 36 healthy subjects. Cp was lower in HH patients than in controls (P < 0·001); no difference was found between untreated HH patients and those on a phlebotomy programme (P = 0·07) and between the HH patients carrying the normal and the mutated alleles of the HFE gene (P = 0·8). Cp levels in AIO subjects were significantly higher than in HH patients (P < 0·004) and similar to those of controls (P = 0·2). No differences in albumin, α1 acid glycoprotein and copper serum levels were observed in the three groups.
Microsatellite instability occurs in defined subsets of patients with acute myeloblastic leukaemia
Authors: Das‐Gupta Emma P. , Seedhouse Claire H. , Russell Nigel H.
Published: March 2003

Using a sensitive fluorescent‐polymerase chain reaction technique we looked for microsatellite instability (MSI) as functional evidence of mismatch repair defects in 71 cases of acute myeloblastic leukaemia (AML). MSI was assessed at 11 loci in matched leukaemic and constitutional DNA. Nine out of 71 patients (13%) were found to have MSI. Four of these patients had therapy‐related leukaemia and the remaining five were all over the age of 60 years. There was a high incidence of adverse‐risk cytogenetics in the patients with MSI, including abnormalities of chromosomes 5 and/or 7. Of the nine cases of t‐AML included in this study, four (44%) had MSI. MSI was also seen in five of 51 cases (10%) over the age of 60 years but not in any cases under the age of 60 years with de novo AML. Using a sensitive assay, our results suggest that MSI occurs in two subgroups of patients with AML: those with t‐AML and the elderly (> 60 years), but is rare in younger patients.
Persistent polyclonal B‐cell lymphocytosis is an expansion of functional IgD+CD27+ memory B cells
Authors: Himmelmann Andreas , Gautschi Oliver , Nawrath Michael , Bolliger Ursula , Fehr Jörg , Stahel Rolf A.
Published: March 2003

Persistent polyclonal B‐cell lymphocytosis (PPBL) is a rare disorder of unknown aetiology affecting predominantly young to middle‐aged women. It is characterized by a polyclonal expansion of B cells, including typical binucleated lymphocytes, and is associated with the presence of the translocation t(14;18), involving the bcl‐2 oncogene. The stage of differentiation of the B cells expanded in PPBL is not known. We analysed the immunophenotype of the expanded B‐cell subset in five new patients with PPBL and found a large uniform expansion of a recently defined human memory B‐cell population, IgD+CD27+ memory B cells. After in vitro stimulation with interleukin 2 (IL‐2) and Staphylococcus aureus Cowan strain I, B cells from PPBL patients produced high levels of IgM immunoglobulins, which is a characteristic feature of IgD+CD27+ memory B cells. Using a quantitative real‐time polymerase chain reaction method, we found a high frequency of the translocation t(14;18) in the range of 1000–3000 per 106 B cells in PPBL patients. In contrast, a much smaller number of cells with a t(14;18) was found in B cells from healthy individuals. Our finding that PPBL is an accumulation of memory B cells further suggests that chronic antigeneic stimulation plays an important part in the pathogenesis of this disorder. This IgD+CD27+ memory B‐cell population might harbour a certain number of ‘physiological’ t(14;18) translocations that increases as this population expands in PPBL patients and constitutes the majority of peripheral blood lymphocytes.
Favourable effect of the combination of acute and chronic graft‐versus‐host disease on the outcome of allogeneic peripheral blood stem cell transplantation for advanced haematological malignancies
Authors: Brunet Salut , Urbano‐Ispizua Alvaro , Ojeda Emilio , Ruiz Dolores , Moraleda José Ma. , Díaz Miguel Angel , Caballero Dolores , Bargay Joan , De La Rubia Javier , Solano Carlos , Zuazu Javier , Diez José Luis , De La Serna Javier , Espigado Idelfonso , Alegre Adrián , Torres J. Pio , Jurado Manuel , Fernández Manuel , Vivancos Pilar , Carreras Enric , Hernández Fernando , Maldonado Juan , Sierra Jorge , Rozman Ciril , Of Allogeneic–PBT For The Spanish Group ,   Spain
Published: March 2003

To assess the influence of graft‐versus‐host disease (GVHD) on the outcome of patients with advanced haematological malignancies (AHM) who received a primary, unmodified allogeneic peripheral blood progenitor cells transplant (allo‐PBT) from a human leucocyte antigen (HLA) identical sibling donor, we analysed 136 patients with myeloid neoplasms (n = 70) or lymphoproliferative disorders (n = 66), transplanted at 19 Spanish institutions. Median age was 35 years (range 1–61). The cumulative incidence of relapse for all patients was 34% (95% CI, 26–42%), 41% (95% CI, 33–49) for patients without GVHD and 14% (95% CI, 3–25) (P = 0·001) for patients with acute and chronic GVHD. After a median follow‐up of 11 months (range 2–49), 60 (44%) patients remained alive with an actuarial probability of overall survival and disease‐free survival (DFS) at 30 months of 31% (95% CI, 21–41%) and 28% (95% CI, 17–39%) respectively. In patients surviving > 100 d, the low incidence of relapse in those with acute and chronic GVHD led to a DFS of 57% (95% CI, 38–76%) compared with a DFS of 34% (95% CI, 17–51%) in the remaining patients (P = 0·03). Our results indicate a reduced incidence of relapse for patients with AHM receiving an unmodified allo‐PBT and developing acute and chronic GVHD, which results in an improved DFS.
Disparity for the minor histocompatibility antigen HA‐1 is associated with an increased risk of acute graft‐versus‐host disease (GvHD) but it does not affect chronic GvHD incidence, disease‐free survival or overall survival after allogeneic human leucocyte antigen‐identical sibling donor transplantation
Authors: Gallardo David , Aróstegui Juan Ignacio , Balas Antonio , Torres Antonio , Caballero Dolores , Carreras Enric , Brunet Salut , Jiménez Antonio , Mataix Rodolfo , Serrano David , Vallejo Carlos , Sanz Guillermo , Solano Carlos , Rodríguez‐Luaces Marta , Marín Julián , Baro Julio , Sanz César , Román Jose , González Marcos , Martorell Jaume , Sierra Jorge , Martín Carmen , De La Cámara Rafael , Grañena Albert ,   on behalf of the GvHD Subcommittee of the GETH (Grupo Español de trasplante hemapoyético
Published: March 2003

Disparity for the minor histocompatibility antigen HA‐1 between patient and donor has been associated with an increased risk of acute graft‐versus‐host disease (GvHD) after allogeneic human leucocyte antigen (HLA)‐identical sibling donor stem cell transplantation (SCT). However, no data concerning the impact of such disparity on chronic GvHD, relapse or overall survival are available. A retrospective multicentre study was performed on 215 HLA‐A2‐positive patients who received an HLA‐identical sibling SCT, in order to determine the differences in acute and chronic GvHD incidence on the basis of the presence or absence of the HA‐1 antigen mismatch. Disease‐free survival and overall survival were also analysed. We detected 34 patient–donor pairs mismatched for HA‐1 antigen (15·8%). Grades II–IV acute GvHD occurred in 51·6% of the HA‐1‐mismatched pairs compared with 37·1% of the non‐mismatched. The multivariate logistic regression model showed statistical significance (P: 0·035, OR: 2·96, 95% CI: 1·07–8·14). No differences were observed between the two groups for grades III–IV acute GvHD, chronic GvHD, disease‐free survival or overall survival. These results confirmed the association between HA‐1 mismatch and risk of mild acute GvHD, but HA‐1 mismatch was not associated with an increased incidence of chronic GvHD and did not affect relapse or overall survival.
Synergism between fludarabine and rituximab revealed in a follicular lymphoma cell line resistant to the cytotoxic activity of either drug alone
Authors: Di Gaetano N. , Xiao Y. , Erba E. , Bassan R. , Rambaldi A. , Golay J. , Introna M.
Published: March 2003

We have shown previously that the anti‐CD20 chimaeric monoclonal antibody rituximab exerts its effects on neoplastic B‐lymphoma cell lines in part via complement‐dependent cytotoxicity. In addition, membrane expression levels of complement inhibitory proteins CD55 and CD59 play a role in determining susceptibility to lysis. We have identified one t(14;18)‐positive human B‐cell non Hodgkin's lymphoma cell line (Karpas 422) that is resistant to rituximab and complement and used it for subsequent studies on the possible interaction between this novel therapeutic agent and established antineoplastic drugs. We have exposed Karpas to several chemotherapeutic agents (doxorubicin, idarubicin, cisplatin, taxol) for different time periods and subsequently exposed the cells to rituximab and human complement. The combination of these drugs with rituximab induced an additive cytotoxic effect. In contrast, exposure to fludarabine (1 µg/ml for 48–72 h) showed a synergistic effect, with cell lysis increasing from 10% to 20% using fludarabine or rituximab and complement alone to about 70% with both cytotoxic agents. Analysis of the mechanism for this synergistic effect showed that fludarabine downmodulates the membrane expression of CD55 (from 96% to 55% positive cells) without significantly altering CD20 levels. Northern analysis demonstrated that fludarabine induced a general downmodulation of steady state mRNA levels with no change in transcription rate detected in run‐off assays. The study of the effect of fludarabine and rituximab in six freshly isolated B‐cell chronic lymphocytic leukaemia (B‐CLL) samples showed that, in most cases, fludarabine has an additive cytotoxic activity with rituximab and complement. This report gives a rational support for clinical studies with combinations of drugs, including monoclonal antibodies and fludarabine.
Loss of CD20 expression following treatment with rituximab (chimaeric monoclonal anti‐CD20): a retrospective cohort analysis
Authors: Foran James M. , Norton Andrew J. , Micallef Ivana N. M. , Taussig David C. , Amess John A. L. , Rohatiner Ama Z. S. , Lister T. Andrew
Published: March 2003

A retrospective analysis of CD20 expression following rituximab for B‐cell non‐Hodgkin's lymphoma demonstrated a significant change in immunophenotype in 6/25 (24%) patients with persistent bone marrow (BM) infiltration. In three out of six patients, the B cells were uniformly CD20−/CD79α+, consistent with frank loss of CD20 expression. In the remaining three cases, the BM infiltrate was predominantly (> 80%) CD20−/CD79α+. Two of the former but none of the latter three cases achieved a clinical response. In three further cases, the post‐treatment BM infiltrate was composed entirely of benign or reactive CD3+ T cells. Frank loss of CD20 was not seen in 25 post‐treatment lymph node biopsies. Immunophenotyping is therefore an important adjunct in the diagnosis of BM infiltration following rituximab.
Dendritic cells in acute promyelocytic leukaemia
Authors: Rigolin Gian Matteo , Porta Matteo Della , Bigoni Renato , Tieghi Alessia , Cuneo Antonio , Castoldi Gianluigi
Published: March 2003

Dendritic cell (DC) differentiation was investigated in samples from two acute promyelocytic leukaemia (APL) patients with classic translocation t(15;17)(q22;q21). After 18 d of culture in the presence of granulocyte–macrophage colony‐stimulating factor, interleukin 4 and tumour necrosis factor alpha, 10–15% of pathological promyelocytes had differentiated into DC‐like cells, as demonstrated by immunological and functional characteristics and by analysis of CD1a+ cells. In one patient, analysed at relapse and after developing a picture of secondary myelodysplastic syndrome (MDS), three different populations of DCs were demonstrated, two of which derived from pathological myeloid precursors (the APL and the MDS clones). This patient's DCs also presented abnormal dextran uptake. Our results demonstrated that pathological myeloid precursors in APL can differentiate into DC‐like elements and that different populations of pathological DCs may coexist in the same patient.
Increased lipid peroxidation correlates with platelet activation but not with markers of endothelial cell and blood coagulation activation in patients with antiphospholipid antibodies
Authors: Martinuzzo Marta E. , Forastiero Ricardo R. , Kordich Lucía , Carreras Luis O.
Published: March 2003

Recent studies have shown that patients with antiphospholipid antibodies (aPL) have increased lipid peroxidation. We evaluated the urinary excretion of 11‐dehydro thromboxane B2 (11‐DH‐TXB2) and isoprostane F2αIII (IPF2αIII), reflecting platelet activation and lipid peroxidation in vivo, and plasma soluble markers of endothelial cell, platelet and blood coagulation activation: soluble vascular cell adhesion molecule‐1 (sVCAM‐1), P‐ and E‐selectin (sPsel and sEsel), F1 + 2 fragment of prothrombin (F1 + 2), thrombin–antithrombin complexes (TAT) and D‐Dimer (DD). We studied 79 patients with aPL (47 with previous thrombosis), 45 healthy volunteers (normal controls, NC), 12 patients with systemic lupus erythematosus (SLE) without aPL and a thrombosis control group (TCG) without thrombophilia (n = 16). Urinary levels (mean, range) of eicosanoids and isoeicosanoids were significantly increased in 39 patients with aPL compared with 25 NC, 11‐DH‐TXB2 164·0 ng/mmol creatinine (9·5–1162·8) versus 43·4 ng/mmol creatinine (4·2–87·6), P < 0·001; IPF2αIII 56·9 pg/mg creatinine (5·5–388·7) versus 27·0 pg/mg creatinine (4·6–87·6), P = 0·03. Both metabolites were significantly correlated (ρ= 0·49, P = 0·014), but none correlated with any clinical manifestation or antibody profile. The aPL group presented increased levels of sPsel, sEsel, sVCAM‐1, TAT, F1 + 2 and DD, but any soluble marker correlated with IPF2αIII. Urinary 11‐DH‐TXB2 correlated with sPsel (ρ= 0·39, P = 0·04). Compared with SLE controls, the SLE group with aPL had higher levels of F1 + 2. Plasma levels of F1 + 2 and DD were significantly increased and a trend to higher sPsel was found in aPL patients with thrombosis compared with the TCG. Platelet activation, lipid peroxidation and blood coagulation activation seem to be important in the pathophysiology of antiphospholipid syndrome.
Chimaeric anti‐CD20 monoclonal antibody (rituximab) in post‐transplant B‐lymphoproliferative disorder following stem cell transplantation in children
Authors: Faye Albert , Quartier Pierre , Reguerre Yves , Lutz Patrick , Carret Anne‐Sophie , Dehée Axelle , Rohrlich Pierre , Peuchmaur Michel , Matthieu‐Boué Anne , Fischer Alain , Vilmer Etienne
Published: March 2003

Post‐transplant lymphoproliferative disorder (PTLD) after haemopoietic stem cell transplantation is a serious complication that occurs in 8–22% of patients with high‐risk factors. We retrospectively investigated tolerance and efficacy of humanized anti‐CD20 monoclonal antibody (rituximab) as first‐line treatment in 12 children with B‐cell PTLD. At diagnosis, eight patients had tumoral involvement. The other four patients had fever, associated with raised Epstein–Barr virus (EBV) viral load and monoclonal gammopathy. Rituximab was given at the dose of 375 mg/m2 once a week by intravenous infusion (1–9 infusions). Only 1/48 infusions was associated with a grade 2 clinical adverse event. Eight out of 12 (66%) patients responded to the treatment and were in complete remission. All patients without tumoral involvement responded to the treatment. A rapid decrease in fever within 1 week was observed in all responders. Non‐responders did not show any clinical response during the first week. Tumoral involvement and immunodepression seemed to be more marked in non‐responders. Rituximab was an effective and well‐tolerated treatment of B‐cell PTLD. Early treatment before tumoral involvement seemed to be the most effective approach. Lack of rapid response should lead to intensification of PTLD treatment. Pre‐emptive treatment should be considered and evaluated in further longitudinal multicentre studies.
Classification of the kinetics of factor VIII inhibitors in haemophilia A: plasma dilution studies are more discriminatory than time‐course studies
Authors: Ling M. , Duncan E. M. , Rodgers S. E. , Somogyi A. A. , Crabb G. A. , Street A. M. , Lloyd J. V.
Published: March 2003

Factor VIII inhibitors have previously been classified as type I or type II using complex experiments that study the time course of inactivation of factor VIII and the effect of varying the antibody concentration. Classification may be important to better understand inhibitor behaviour in vivo. To determine the most reliable method of classifying the kinetics of factor VIII inactivation, we studied 11 patients with haemophilia A, comprising five severe, three mild and three acquired cases, and compared the classification obtained from plasma dilution studies and time‐course studies. The plasma dilution studies showed two distinctly different patterns: a steep slope with complete FVIII:C inactivation at high antibody concentrations for type I inhibitors and a FVIII:C plateau with incomplete inactivation for type II inhibitors. Six type I (four severe, one mild and one acquired) and two type II (one mild and one acquired) inhibitors were classified using either plasma samples or purified and concentrated IgG, while the remaining were undetermined owing to insufficient available plasma. In contrast, the time‐course studies could not discriminate between these groups. We recommend that plasma dilution studies be used for the classification of in vitro kinetics of factor VIII inhibitors.
Dose‐reduced conditioning for allografting in 44 patients with chronic myeloid leukaemia: a retrospective analysis
Authors: Bornhäuser Martin , Kiehl Michael , Siegert Wolfgang , Schetelig Johannes , Hertenstein Bernd , Martin Hans , Schwerdtfeger Rainer , Sayer Herbert G. , Runde Volker , Kröger Nikolaus , Theuser Catrin , Ehninger Gerhard ,   the Cooperative German Transplant Study Group
Published: March 2003

This retrospective study describes the outcome of patients with chronic myeloid leukaemia after allografting using dose‐reduced conditioning with fludarabine and busulphan. Forty‐four Philadelphia chromosome (Ph)‐positive patients were transplanted in nine German centres; 26 patients were in chronic phase, 11 in accelerated phase and seven in blast crisis. Thirty‐four patients achieved complete remission, with 18 alive and disease‐free at a median follow‐up of 562 d (range 244–922 d). Grade II–IV acute graft‐versus‐host disease (GVHD) incidence was 43%. Twenty patients died, 15 of causes unrelated to relapse. Risk factors predisposing to graft failure by univariate analysis were an unrelated donor (8/23 compared with a related donor 2/21, P = 0·07) and interferon therapy within 90 d of transplant (4/6 versus 3/17, P = 0·025). At the last follow‐up, of 31 patients for whom molecular or cytogenetic data were available, 16 (52%) were polymerase chain reaction‐negative, and seven (23%) were Ph‐negative by fluorescent in situ hybridization. These findings demonstrate that dose‐reduced conditioning with fludarabine and busulphan provides durable engraftment and a low rate of relapse. However, in this population, many of whom were not eligible for high‐dose conditioning due to age, reduced performance status, previous complications or extensive pre‐treatment, these data highlight the need for effective anti‐infectious and GVHD prophylaxis. In addition, this study supports the discontinuation of interferon therapy at least 90 d before transplant
Tumour necrosis factor‐α‐induced apoptosis in cord blood T lymphocytes: involvement of both tumour necrosis factor receptor types 1 and 2
Authors: Yang Ya‐Chien , Hsu Tsuey‐Ying , Chen Jen‐Yang , Yang Czau‐Siung , Lin Rong‐Hwa
Published: March 2003

Cord blood T cells are much more likely to be induced to apoptosis in vitro than adult T cells. Nevertheless, the expression of Fas is markedly lower on cord blood lymphocytes than on peripheral blood lymphocytes. In the current investigation, we determined the capacity of tumour necrosis factor‐α (TNF‐α) to induce apoptosis in human naïve T cells in cord blood, and assessed the roles of two distinct TNF receptors (TNFRs) in mediating death signals. After activation, cord blood T cells were sensitive to TNF‐α‐induced apoptosis, and interleukin 2 (IL‐2) could prevent this apoptotic response. Both TNFR1 (p55) and TNFR2 (p75) expressed on activated cord blood T cells were able to transmit apoptotic signals. Moreover, a synergistic effect was observed by a combination of TNFR1‐ and TNFR2‐signals. Additionally, CD4+ T cells showed higher sensitivity to TNFR‐mediated apoptosis than CD8+ T cells. These data suggest that TNF‐α probably is a mediator of apoptosis in cord blood T cells in vivo and may contribute to the low incidence of graft‐versus‐host disease in cord blood transplantation.
Waldenström macroglobulinaemia: presenting features and outcome in a series with 217 cases
Authors: García‐Sanz Ramón , Montoto Silvia , Torrequebrada Agustín , De Coca Alfonso García , Petit José , Sureda Anna , Rodríguez‐García José Antonio , Massó Pilar , Pérez‐Aliaga Ana , Monteagudo María Dolores , Navarro Isabel , Moreno Gemma , Toledo Carmen , Alonso Aránzazu , Besses Carles , Besalduch Joan , Jarque Isidro , Salama Perla , Rivas José Angel Hernández , Navarro Blanca , Bladé Joan , Miguel Jesús F. San
Published: March 2003

In this report we analyse the presenting features of a series of patients diagnosed with Waldenström macroglobulinaemia (WM) in Spain over the last 10 years. Criteria for diagnosis required a serum monoclonal IgM protein  30 g/l and > 20% bone marrow lymphocytes. Two hundred and seventeen patients were included in the study, with a median age of 69 years and male/female ratio of 2:1. The most common symptoms at diagnosis were anaemia (38%), hyperviscosity (31%), B symptoms (23%), bleeding (23%) and neurological symptoms (22%). Sixty‐one patients (27%) were asymptomatic at diagnosis and, to date, 32 of them have not received chemotherapy. Variables predicting a shorter survival free of therapy were haemoglobin < 12·5 g/dl and high β2microglobulin (β2M). The 83% of patients who did receive treatment were distributed as follows: chlorambucil/prednisone (43%), intermittent chlorambucil (11%), continuous chlorambucil (26%), cyclophosphamide/vincristine/prednisone (COP, 13·5%) and other (6·5%). Response to therapy was complete in 2%, partial in 48% and minor in 10%. Finally, 28% and 13% of patients presented stable and progressive disease, respectively, which was more common among patients treated with COP. Progression‐free survival was 43% at 5 years, with three independent predictors for shorter progression‐free survival (PFS): COP treatment, age > 65 and B symptoms at diagnosis. The 10‐year projected overall survival (OS) was 55%. The two most frequent causes of death were development of second malignancies (31%), or infections (19%). The two main variables predicting a poor OS were hyperviscosity and high β2M. In summary, this study favours the use of chlorambucil‐based therapy as the standard treatment for WM, and describes a subset of patients who should be considered as suffering a smouldering form and who therefore do not require treatment for a long period of time.
Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG ± Ida) and second allogeneic stem cell transplant
Authors: Pawson R. , Potter M. N. , Theocharous P. , Lawler M. , Garg M. , Yin J. A. Liu , Rezvani K. , Craddock C. , Rassam S. , Prentice H. G.
Published: March 2003

Acute leukaemias in relapse after allogeneic stem cell transplantation (SCT) respond poorly to donor leucocyte infusions (DLI) compared with chronic myeloid leukaemia (CML), at least in part because of faster disease kinetics. Fludarabine‐containing ‘non‐myeloablative’ chemotherapy followed by further allo SCT may offer more rapid and effective disease control. We report 14 patients with relapse after allo SCT for acute leukaemia [seven acute myeloid leukaemia (AML), five acute lymphoblastic leukaemia (ALL)] or refractory anaemia with excess blasts in transformation (RAEB‐t, n = 2) treated with fludarabine, high‐dose cytosine arabinoside (ara‐C) and granulocyte colony‐simulating factor (G‐CSF) with (n = 10) or without (n = 2) idarubicin (FLAG ± Ida) or DaunoXome (FLAG‐X) (n = 2) and second allo SCT from the original donor. Donors were fully human leucocyte antigen (HLA) ‐matched in 13 cases with a single class A mismatch in one. Actuarial overall survival was 60% and disease‐free survival was 26% at 58 months. Remissions after the second SCT were longer than those after the first bone marrow transplantation (BMT) in eight of the 13 assessable patients to date. Haematopoietic recovery was rapid. Transplants were well tolerated with no treatment‐related deaths. The major complication was graft‐versus‐host disease (GvHD, acute  grade II−2 cases, chronic – eight cases, two limited, six extensive) although there have been no deaths attributable to this. FLAG ± Ida and second allo SCT is a safe and useful approach and may be more effective than DLI in the treatment of acute leukaemias relapsing after conventional allo SCT.