Showing papers in "British Journal of Haematology in 1999"

Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate.

Abstract: Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colony-forming assay in which samples from early passages were plated at low densities of about 10 cells per cm2. Samples with high colony-forming efficiency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.

A simple, robust, validated and highly predictive index for the determination of risk-directed therapy in acute myeloid leukaemia derived from the MRC AML 10 trial. United Kingdom Medical Research Council's Adult and Childhood Leukaemia Working Parties.

Abstract: Data on 1711 patients, aged up to 55 years, in the MRC AML 10 trial were used to create a prognostic index for use in risk-directed therapy decision making for younger patients with acute myeloid leukaemia (AML). Two parameters, response after course 1 and cytogenetics, were strongly predictive of outcome. For patients with complete remission, partial remission and resistant disease, 5-year survival from the start of course 2 was 53%, 44% and 22% and relapse rates were 46%, 48% and 69% respectively, and for patients with favourable, intermediate and adverse karyotypic abnormalities, survival was 72%, 43% and 17% and relapse rates were 34%, 51% and 75% respectively (all P < 0.0001). Patients with FAB type M3 but no cytogenetic t(15;17) also had a low relapse rate (29%). These three factors were combined to give three risk groups: good (favourable karyotype or M3, irrespective of response status or presence of additional abnormalities), standard (neither good nor poor), poor (adverse karyotype or resistant disease, and no good-risk features). Survival for these three groups was 70%, 48% and 15% respectively and relapse rates were 33%. 50% and 78% (both P < 0.0001). The index is simple (based on just three parameters), robust (derived from 1711 patients), highly discriminatory (55% survival difference between good and poor risk) and validated, so can be applied in the clinical setting to assist with therapeutic decisions as in the current AML 12 trial.

Phosphatidylserine-related adhesion of human erythrocytes to vascular endothelium

Abstract: In pathological conditions such as sickle cell disease, falciparum malaria and diabetes, an abnormal adherence of erythrocytes to endothelium is concomitant with loss of phospholipid asymmetry resulting in phosphatidylserine (PS) exposure We have investigated the involvement of PS in this interaction by studying adhesion of human erythrocytes, treated with Ca2+-ionophore A23187 in combination with N-ethylmaleimide, to human umbilical vein endothelial cells in a flow-based assay Results showed that erythrocytes which exposed PS, massively adhered to HUVEC in a Ca2+-dependent manner This adhesion was inhibited by PS liposomes and by annexin V, giving clear evidence of the PS dependence of these interactions

Red blood cell membrane disorders

Abstract: The recent discovery of the specific molecular defects in many patients with hereditary spherocytosis and hereditary elliptocytosis/pyropoikilocytosis partially clarifies the molecular pathology of these diseases. HE and HPP are caused by defects in the horizontal interactions that hold the membrane skeleton together, particularly the critical spectrin self-association reaction. Single gene defects cause red cells to elongate as they circulate, by a unknown mechanism, and are clinically harmless. The combination of two defective genes or one severe alpha spectrin defect and a thalassaemia-like defect in the opposite allele (alphaLELY) results in fragile cells that fragment into bizarre shapes in the circulation, with haemolysis and sometimes life-threatening anaemia. A few of the alpha spectrin defects are common, suggesting they provide an advantage against malaria or some other threat. HS, in contrast, is nearly always caused by family-specific private mutations. These involve the five proteins that link the membrane skeleton to the overlying lipid bilayer: alpha and beta spectrin, ankyrin, band 3 and protein 4.2. Somehow, perhaps through loss of the anchorage band 3 provides its lipid neighbours (Peters et al, 1996), microvesiculation of the membrane surface ensues, leading to spherocytosis, splenic sequestration and haemolysis. Future research will need to focus on how each type of defect causes its associated disease, how the spleen aggravates membrane skeleton defects (a process termed 'conditioning'), how defective red, cells are recognized and removed in the spleen, and why patients with similar or even identical defects can have different clinical severity. Emphasis also needs to be given to improving diagnostic tests, particularly for HS, and exploring new options for therapy, like partial splenectomy, which can ameliorate symptoms while better protecting patients from bacterial sepsis and red cell parasites, and perhaps from atherosclerosis (Robinette & Franmeni, 1977) and venous thrombosis (Stewart et al, 1996).

c‐kit proto‐oncogene exon 8 in‐frame deletion plus insertion mutations in acute myeloid leukaemia

Abstract: Genomic DNA from 60 cases of acute myeloid leukaemia (AML) was screened for mutations in the c-kit gene. DNA from all 21 exons was subjected to polymerase chain reaction (PCR) amplification and analysis by conformation sensitive gel electrophoresis (CSGE); exons showing altered CSGE patterns were then sequenced. Mutations were identified only in those patients with inv(16) (3/7 cases) or t(8;21) (1/2 cases) and comprised three in-frame deletion plus insertion mutations (exon 8) and one point mutation (exon 10, GTA --> ATA, Val530Ile). Exons 8 and 10 were then analysed in 31 further cases of inv(16) (n = 14) and t(8;21) (n = 17), revealing four additional exon 8 in-frame deletion plus insertion mutations, all of which were in cases of inv(16). All exon 8 in-frame deletion plus insertion mutations (n = 7) involved the loss or replacement of the codon for Asp419 which is highly conserved cross species and is located in the receptor's extracellular domain. The high frequency of the c-kit proto-oncogene exon 8 deletion plus insertion mutations in AML suggests an essential role for this region of the receptor's extracellular domain. The association with inv(16) invites speculation as to the link between these two changes in the pathogenesis of AML.

Primary prophylaxis in severe haemophilia should be started at an early age but can be individualized.

Abstract: The frequency of joint bleeds and orthopaedic joint scores were evaluated in 121 patients with severe haemophilia who had started prophylactic treatment with clotting factor concentrates at least once weekly before the age of 10. 75 of the patients started before the age of 3, 31 at the age of 3-5 and 15 at the age of 6-9. Each subgroup was evaluated separately. In addition, a regimen of one infusion weekly was compared with that of two (haemophilia B) or three (haemophilia A) infusions weekly in each patient. A significant decrease in the overall number of joint bleeds per year was found after shortening the infusion interval (P<0.005), but the individual bleeding pattern varied. In survival analysis of the first pathologic joint score event, those who started prophylaxis before the age of 3 had a better outcome overall than those starting at later ages (P=0.001). However, in subgroup analysis, no significant difference was seen in the annual number of joint bleeds and the development of arthropathy between those starting with, or shifting to, the more intensive regimen before the age of 3 and those that were put on this regimen at the age of 3-5. Age at start of prophylaxis was found to be an independent predictor for the development of arthropathy (P=0.0002), whereas dose and infusion interval at start were not. Our data emphasize the importance of starting replacement therapy during the first years of life. However, it seems that when beginning the regimen it can be individualized and adjusted according to the bleeding pattern. In this way, the need for a venous access system may be assessed on an individual basis.

MLL rearrangements in haematological malignancies: lessons from clinical and biological studies

Abstract: Since the identi®cation of the Philadelphia chromosome almost 40 years ago, the study of speci®c chromosomal alterations in human cancer has had a major impact on the diagnosis of, and choice of treatment for, patients with haematological and other malignancies (Heim & Mitelman, 1995; Look, 1997). Karyotype analysis to identify chromosomal rearrangements is now a routine part of the diagnostic work-up for newly diagnosed and relapsed leukaemia patients. Due to years of correlative studies, many leukaemia-speci®c rearrangements can be used quite accurately for risk strati®cation and, in turn, to choose appropriate therapy. This paradigm has been demonstrated in paediatric acute lymphoblastic leukaemia (ALL) (reviewed by Rubnitz & Crist, 1997). For example, ALL patients with the t(1;19) translocation were initially found to be at higher risk of treatment failure. Treatment of these patients with more aggressive combinations of chemotherapy has resulted in long-term cure rates comparable to those of children with standard-risk ALL. In addition to serving as diagnostic and prognostic markers, recurring rearrangements are providing intriguing clues about the molecular events underlying leukaemogenesis. The cloning and functional analysis of genes whose structure or expression is altered as a result of chromosomal rearrangements is yielding unprecedented insights into the mechanism(s) of neoplastic transformation (Look, 1997). Among cancer-related chromosomal aberrations that are shedding new light on the origins of haematological malignancies, a family of rearrangements involving the MLL (Myeloid/Lymphoid Leukaemia or Mixed Lineage Leukaemia) gene on chromosome 11, band q23 is proving to be a fertile area of investigation. MLL rearrangements include non-constitutional or acquired deletions, duplications, inversions and reciprocal translocations at 11q23. As a group, these rearrangements account for 5±10% of acquired chromosomal rearrangements in children and adults with ALL, acute myeloblastic leukaemia (AML), poorly differentiated or biphenotypic leukaemias and myelodysplastic syndromes (MDS) (Heim & Mitelman, 1995). Strikingly, the majority of acute leukaemias in neonates and infants (children <1 year of age), as well as secondary or treatment-related leukaemias, have clonal rearrangements involving 11q23 (Chen et al, 1993; Reaman et al, 1985; Spier et al, 1984; Rubnitz et al, 1994a). Independent of their association with other high-risk features at presentation, 11q23 rearrangements are strongly predictive of poor clinical outcome. Despite the use of more aggressive induction regimens and stem cell transplant in ®rst remission, patients with 11q23 rearrangements continue to have poor long-term survival (Rubnitz et al, 1994a; Pui et al, 1991, 1994). Thus, in addition to their role in the neoplastic transformation of haemopoietic progenitors, 11q23 rearrangements may also affect the response of leukaemic blasts to chemotherapy. In this review we discuss some of the recent advances from clinical as well as biological studies of these rearrangements, with an emphasis on the possible mechanisms of leukaemic transformation by MLL fusion proteins.

Monitoring chronic myeloid leukaemia therapy by real-time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics

Abstract: We have developed a rapid real-time quantitative PCR method for measuring BCR-ABL mRNA levels in peripheral blood in chronic myeloid leukaemia (CML). The technique was used to monitor minimal residual disease for the early detection of relapse and as an assessment of treatment response. Normal BCR mRNA was quantitated to control for RNA degradation and the results reported as a percentage of BCR-ABL/BCR. Every patient measured at diagnosis (n = 21) had increased expression of BCR-ABL of up to 5-fold above the normal BCR levels. With effective treatment the BCR-ABL levels decreased. The molecular data was correlated with Philadelphia chromosome levels in bone marrow and a good correlation was found when treatment induced a cytogenetic response (Spearman correlation = 0.94, P < 0.0001, n = 67 samples). In patients receiving interferon-alpha therapy we found a significant difference in the BCR-ABL levels between cytogenetic response groups. The method was sensitive, reproducible, and readily detected a change in BCR-ABL transcript levels in serial blood samples. Sample throughput was high because post PCR processing was unnecessary. We conclude that real-time quantitative PCR monitoring of peripheral blood can be used to reliably monitor disease response in CML.

Bleeding and thrombosis in 55 patients with inherited afibrinogenaemia

Abstract: Knowledge of the spectrum of symptoms in patients with inherited afibrinogenaemia is limited by the rarity of this coagulation defect. We compared a large series of 55 afibrinogenaemic patients from Iran with 100 patients with severe factor VIII deficiency. In afibrinogenaemia there was a higher frequency of mucosal-type bleeding symptoms but joint and muscle bleeding was less frequent and severe than in haemophilia. Umbilical cord bleeding was relatively frequent only in afibrinogenaemic patients. Two young patients developed spontaneous thrombotic episodes and three women had recurrent abortions. Overall, in afibrinogenaemia bleeding symptoms are qualitatively different and less severe than in haemophilia. Afibrinogenaemia can also be accompanied by thrombotic manifestations.

Factor xiii deficiency

Abstract: Factor XIII (FXIII) is the last enzyme in the clotting cascade. Its main function is to convert the loose ®brin polymer into a ®rm, highly organized, cross-linked structure with increased tensile strength, ®rmly anchored to the site of the wound and possessing an in-built resistance to ®brinolysis. In FXIII de®ciency, standard clotting tests are normal, as the clotting end point is not affected by the absence of FXIII. It is the quality of the clot which is abnormal. Unless this is assessed, the diagnosis may be missed. Soon after its discovery by Robbins (1944), FXIII was aptly named: the `®brin stabilizing factor' or `FSF'. Fibrin formed in the absence of FSF was `unstable': it dissolved in weak acids, weak bases and 5 M urea. Addition of a small amount of plasma to the system `stabilized' the ®brin, it was no longer soluble in these reagents and it was also more resistant to ®brinolysis. Solubility of clots in 1% monochloroacetic acid or in 5 M urea still forms the basis of the standard laboratory screening test for inherited FXIII de®ciency. Initially it was believed that FSF combined with ®brin stoichiometrically, acting as a kind of glue, sticking molecules of ®brin together (Laki & Lorand, 1948; Lorand, 1950). Later it became obvious that FSF was an enzyme (Buluk et al, 1961; Loewy et al, 1961; reviewed in detail by Board et al, 1993) and the cross-link introduced into ®brin by FSF was found to be the «(g-glutamyl)lysine link (reviewed by Miloszewski & Losowsky, 1988), identifying FXIII as a member of the transglutaminase family of enzymes (see reviews by Greenberg et al, 1991; Aeschlimann & Paulsson, 1994). FXIII is the only transglutaminase found both intraand extracellularly and the only one requiring thrombin as well as calcium for activation. Thus, in common with other clotting factors, it exists as a pro-enzyme (Lorand, 1977). In plasma, FXIII circulates in the form of a tetramer composed of two catalytic A subunits bound to two carrier protein B subunits (A2B2) (Schwartz et al, 1973). FXIII is found in plasma, platelets and monocytes (Muszbek et al, 1985, 1996). Intracellular FXIII is a dimer of two catalytic A subunits (A2). In inherited FXIII de®ciency the A subunit is absent from plasma, platelets and monocytes. The plasma level of the B subunit is usually reduced, and very rarely both A and B subunits are absent (Girolami et al, 1985). The clinical relevance of FXIII became apparent 16 years after its discovery, when Duckert et al (1960) described the case of a boy with a severe bleeding diathesis in whom the only abnormality in the clotting tests was the solubility of his clots in 5 M urea. His clots became insoluble when some normal plasma was mixed with his plasma in vitro. He formed insoluble clots, after blood or plasma transfusion which also controlled his bleeding temporarily. Since that original description, over 200 cases have been reported from all parts of the world. Most patients with inherited FXIII de®ciency suffer from a severe, lifelong, crippling bleeding diathesis with a very high risk of death in early life from intracranial haemorrhage. In a minority of patients defective wound healing has been reported. Women with the de®ciency are unable to carry a pregnancy to term and habitual abortion is usual (Fisher et al, 1966). Inherited FXIII de®ciency affects all races and both sexes equally. Inheritance is autosomal recessive. The genes for both the A and B subunits were cloned about 10 years ago (detailed in Board et al, 1993). Five years ago only ®ve mutations were tentatively identi®ed as probably being responsible for the de®ciency (Board et al, 1993). In recent years developments in DNA technology have facilitated detailed studies of the FXIII gene in families with inherited de®ciency as well as in normal individuals. Thirty-six different mutations resulting in FXIII de®ciency have now been identi®ed. Studies on the structural and functional consequences of the various mutations and the normal polymorphisms in the gene have resulted in a much better understanding of FXIII function. Discussion of these exciting developments in the ®eld will form the major part of this review.

Human osteoclasts derive from CD14-positive monocytes.

Abstract: Osteoclasts have been defined as calcitonin (CT) and vitronectin (VN) receptor (R) positive, and CD14-, CD11b- and CD11c-negative cells which resorb bone. The aim of this study was to identify the phenotype of the osteoclast precursor. Osteoclasts were generated by co-culturing peripheral blood mononuclear cells (PBMNCS) with the rat osteoblastic UMR 106 cell line. On days 2-4 at least 80% of CTR-positive cells co-expressed CD14, CD11b and CD11c (monocyte markers), but by day 14 < 3.3% expressed these markers. Selection of CD14-positive monocytes from PBMNCS enhanced osteoclastic bone resorption 2-4-fold compared to unfractionated PBMNCS. This study demonstrates that osteoclasts derive largely from CD14-positive monocytes.

The AML1-ETO chimaeric transcription factor in acute myeloid leukaemia: biology and clinical significance.

Abstract: Acute myeloid leukaemia (AML) is a heterogenous disease, with individual cases showing variability in clinical presentation, blast cell morphology, therapeutic response and long-term prognosis. This heterogeneity extends to the molecular genetic lesions underlying the pathogenesis of AML. Although nonrandom clonal chromosomal aberrations are present in the majority of cases, each abnormality affects only a limited subset of cases. Nonetheless, recent studies have demonstrated that several chromosomal rearrangements, and the molecular abnormalities they produce, identify distinct patient subgroups with predictable clinical features and therapeutic responses. One of the most frequent cytogenetic abnormalities in AML is t(8;21)(q22;q22). The cloning of the genes targeted by this translocation led to the identi®cation of AML1 as the DNA-binding subunit of the AML1/CBFb core binding factor transcription complex, a critical regulator of normal haemopoiesis. Subsequent studies have demonstrated that the genes encoding this transcription factor complex are the most common targets of chromosomal rearrangements in human leukaemia. In this review I will summarize our understanding of the normal function of AML1/CBFb in haemopoiesis, and will then describe the mechanism through which t(8;21) induces leukaemia. Throughout I will emphasize the clinical signi®cance of this genetic lesion and the general principles of haemopoietic transformation that have emerged from study of the activity of the encoded chimaeric oncoprotein.

Fetal cells in the maternal circulation: feasibility for prenatal diagnosis

Abstract: Over the past 25 years the utilization of prenatal diagnosis by expectant couples and their physicians has expanded due primarily to two trends: smaller family size, with an increased emphasis on assurance of the ‘normalcy’ of each child, and advancing parental age. In the United States it is currently considered ‘standard of care’ to offer prenatal cytogenetic diagnosis to pregnant women who will be 35 years or older at the time of delivery (American College of Obstetricians and Gynecologists, 1996). Such cytogenetic diagnoses are facilitated by obtaining fetal nucleated cells via an invasive technique such as chorionic villus sampling (CVS) or amniocentesis. 35 years of age was established as the threshold for these procedures because the liveborn incidence of an infant with the most common autosomal aneuploidy, trisomy 21, is roughly equal to the chance of a miscarriage secondary to these procedures (approximately 1 in 250). Despite the safety and accuracy of these techniques, to date there has been little impact upon the incidence of trisomy 21, which is still close to 1 per 1000 live births. A major reason for this is that prenatal diagnostic techniques are directed towards a minority of pregnant women. Although, individually, older pregnant women are at increased risk for having a baby with trisomy 21, as a group they are having a relatively small fraction of the total births. 80% of the newborn infants with Down syndrome are born to women under age 35, who are not offered the invasive prenatal diagnostic techniques because the risk of a procedural complication is greater than the incidence of Down syndrome in a given fetus. Therefore, over the past decade, increased attention has been paid to noninvasive techniques of screening for fetal trisomy 21 that can be offered to all pregnant women. Trisomy 21 is used as a benchmark because it is the most common liveborn aneuploidy associated with mental retardation and serious congenital anomalies. At present, screening for trisomy 21 consists initially of assessing the maternal age, because the risk of fetal chromosomal abnormalities due to nondisjunction increases as maternal age advances. In addition, second-trimester maternal serum screening for proteins such as human chorionic gonadotropin (hCG), oestriol, and alphafetoprotein (AFP) has been incorporated into routine obstetric care. Results are measured in absolute values, expressed in terms of multiples of the median, and interpreted as a mathematical risk for fetal trisomy 21. Women who have a test result that indicates a fetal Down syndrome risk of greater than 1 in 270 are offered amniocentesis. Because of the large-scale success of the serum-screening programmes which have been employed in the United States, Europe and Asia, research has been directed towards improving both their sensitivity and specificity. Current tests detect 60–70% of the cases of trisomy 21 with a calculated false positive rate of 5% (Wald et al, 1988). Addition of another marker such as dimeric inhibin A raises the sensitivity to 75% (Wald et al, 1996). More recently, a study measuring serum markers expressed during the first trimester, b-hCG and pregnancy-associated plasma protein A (PAPP-A), concluded that the sensitivity of screening at 10– 13 weeks of gestation is nearly as good as the secondtrimester test (Haddow et al, 1998). An independent and newer method to screen for fetal trisomy 21 is the sonographic assessment of a fluid-filled space at the back of the fetal neck known as the nuchal translucency (NT) measurement. An increased NT measurement over that expected for gestational age is associated with an increased risk of fetal chromosome and cardiac abnormalities (Nicolaides et al, 1992; Hyett et al, 1997). In a study of 96 127 singleton pregnancies, the combination of maternal age and NT measurement enabled detection of 77% of the fetuses with trisomy 21 in the 5% of patients with the largest NTs (Snijders et al, 1998). However, by recommending amniocentesis or CVS following a positive screening test, 30 invasive tests are required to find one affected fetus. According to an editorial that accompanied the above study, antenatal screening research for Down syndrome is likely to place more emphasis on lowering the false-positive rate to 1% or less by discovering new markers and reconfiguring existing screening techniques (Haddow, 1998). It is into the existing context of noninvasive screening for Down syndrome that fetal cells in maternal blood must be placed. Successful isolation of fetal cells from maternal blood represents a source of fetal chromosomes or DNA obtained non-invasively by maternal venepuncture. This test could be used as a primary screen, a secondary screen designed to be used in concert with the aforementioned screening tests (to reduce the 5% false-positive rate) (Farina & Bianchi, 1998), or, ultimately, as a diagnostic test. This review will summarize the key areas related to this field: the clinical implications of this test for the clinical practice of obstetrics and gynaecology, the surprising insights into the biology of pregnancy that have come from the study of fetal cells in maternal blood, and the technical challenges associated with rare event cell separation. British Journal of Haematology, 1999, 105, 574–583

Unexplained aplastic anaemia, immunodeficiency, and cerebellar hypoplasia (Hoyeraal‐Hreidarsson syndrome) due to mutations in the dyskeratosis congenita gene, DKC1

Abstract: 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.

Bryostatin and CD40‐ligand enhance apoptosis resistance and induce expression of cell survival genes in B‐cell chronic lymphocytic leukaemia

Abstract: Modulating signal transduction pathways represents a promising approach for altering the biological behaviour of haemopoietic malignancies. B-cell chronic lymphocytic leukaemia (B-CLL) cells were treated in vitro with CD40-ligand (CD40L) (CD154) or the protein kinase C modulator Bryostatin-1, exploring the effects on: (a) sensitivity to apoptosis induction by chemotherapeutic drugs (fludarabine, dexamethasone) or anti-Fas antibody; (b) expression of apoptosis-regulatory proteins (Bcl-2, Bcl-X, Mcl-1, Bax, Bak, BAG-1, Flip, XIAP); (c) expression of cell surface co-stimulatory antigens (CD80 [B7.1]; CD54 [ICAM-1]; CD70); and (d) expression of immune modulatory receptors (CD27, CD40, CD95 [Fas]). CD40L and Bryostatin decreased both spontaneous and drug-induced apoptosis in most B-CLL specimens tested. Apoptosis resistance was associated with CD40L- and Bryostatin-induced elevations in the anti-apoptotic Bcl-2 family protein Mcl-1. CD40L also induced striking increases in the levels of the anti-apoptotic protein Bcl-XL in B-CLLs. CD40L stimulated increases in the surface expression of CD40, CD54, CD69, CD70, CD80 and CD95, whereas Bryostatin induced expression of CD40, CD54, CD69 and CD95 but not the co-stimulatory molecules CD70 and CD80. Despite elevations in the expression of CD95 (Fas), anti-Fas antibodies failed to induce apoptosis of CD40L- and Bryostatin-treated B-CLL cells. This Fas-resistance was associated with increased expression of the Fas-antagonist Flip in CD40L-treated, and with elevations in the caspase inhibitor XIAP in Bryostatin-treated B-CLLs. The potential anti-apoptotic properties of CD40L and Bryostatin should be taken into consideration when employing these agents in clinical trials involving patients with B-CLL.

A randomized controlled trial of itraconazole versus fluconazole for the prevention of fungal infections in patients with haematological malignancies. U.K. Multicentre Antifungal Prophylaxis Study Group.

Abstract: Fluconazole is widely used as antifungal prophylaxis but it is ineffective against Aspergillus. Itraconazole has a broader spectrum of activity but the capsules give erratic bioavailability in neutropenic patients. We compared itraconazole oral solution (which has an improved pharmacokinetic profile) with fluconazole for antifungal prophylaxis. Adults with haematological malignancies receiving chemotherapy or bone marrow transplants were randomly allocated 5 mg/kg/d itraconazole (itra) solution (288 episodes) or 100 mg fluconazole suspension (flu) (293 episodes) from before the onset of neutropenia until neutrophil recovery or suspected fungal infection. Outcomes were assessed by independent reviewers unaware of the prophylaxis allocation. More proven systemic fungal infections occurred in flu (Aspergillus four, Candida tropicalis one, C. krusei one) than itra (C. albicans one) and more of these were fatal (four versus nil). This difference reached statistical significance when first study episodes were considered separately (six flu versus nil itra, P = 0.03). Significantly more deaths of presumed fungal origin occurred in flu than itra (seven versus nil, P = 0.024). There were significantly more cases of proven aspergillosis in flu than itra (six versus nil, P = 0.038, 5/6 cases were fatal) if those occurring outside the study period are included. Significantly more patients receiving flu required amphotericin B (58 v 39, P = 0.043) but this may have been affected by the fact that the study was not blinded. There were 11 proven mucosal candidal infections in flu and four in itra. Itraconazole solution and fluconazole provide effective prophylaxis against Candida but itraconazole affords greater protection against fatal aspergillosis.

The Italian Consensus Conference on Diagnostic Criteria for Myelofibrosis with Myeloid Metaplasia.

Abstract: The purpose of this work was to develop a definition of myelofibrosis with myeloid metaplasia (MMM) using diagnostic criteria that would remain valid within the set of patients with chronic myeloproliferative disorders or myelodysplastic syndromes A list of 12 names for the disease and 37 diagnostic criteria were proposed to a Consensus Panel of 12 Italian experts who ranked them in order so as to identify a core set of criteria The Panel was then asked to score the diagnosis of 46 patient profiles as appropriate or not appropriate for MMM Using the experts' consensus as the gold standard, the performance of 90 possible definitions of the disease obtained through the core set was evaluated ‘Myelofibrosis with myeloid metaplasia’ ranked as the preferred name of the disease Necessary criteria consisted of ‘diffuse bone marrow fibrosis’ and ‘absence of Philadelphia chromosome or BCR-ABL rearrangement in peripheral blood cells’ The six optional criteria in the core set consisted of: splenomegaly of any grade; anisopoikilocytosis with tear-drop erythrocytes; the presence of circulating immature myeloid cells; the presence of circulating erythroblasts; the presence of clusters of megakaryoblasts and anomalous megakaryocytes in bone marrow sections; myeloid metaplasia The definition of the disease with the highest final score was as follows: necessary criteria plus any other two criteria when splenomegaly is present or any four when splenomegaly is absent The use of this definition will help to standardize the conduct and reporting of clinical studies and should help practitioners in clinical practice

Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children.

Abstract: We examined mRNA expression and internal tandem duplication of the Fms-like tyrosine kinase 3 (FLT3) gene in haematological malignancies by reverse transcriptase-polymerase chain reaction (RT-PCR) and genomic PCR followed by sequencing. By RT-PCR, expression of FLT3 was detected in 45/74 (61%) leukaemia cell lines and the frequency of expression of FLT3 was significantly higher in undifferentiated type (B-precursor acute lymphoblastic leukaemia; ALL) than in differentiated type cell lines (B-ALL) (P = 0.0076). Using the genomic PCR method, 194 fresh samples including 87 acute myeloid leukaemias, 60 ALLs, 32 myelodysplastic syndromes (MDSs) and 15 juvenile chronic myelogenous leukaemias (JCMLs) were examined. Tandem duplication was found in 12 (13.8%) AMLs and two (3.3%) ALLs. Sequence analyses of the 14 samples with the duplication revealed that eight showed a simple tandem duplication and six a tandem duplication with insertion. Most of these tandem duplications occurred within exon 11, and two duplications occurred from exon 11 to intron 11 and exon 12. No tandem duplications of FLT3 gene were detected in MDS or JCML. The frequency of tandem duplication of FLT3 gene in childhood AML was lower than that in adult AML so far reported. All of the 12 AML patients with the duplication died within 47 months after diagnosis, whereas two ALL patients with the duplication have survived 44 and 72 months, respectively. These two ALL patients expressed both lymphoid and myeloid antigens and were considered to have biphenotypic leukaemia. These results suggest that tandem duplication is involved in ALL in addition to AML, but not in childhood MDS or JCML, and that childhood AML patients with the tandem duplication have a poor prognosis.

Identification and characterization of zebrafish thrombocytes

Abstract: To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.

Factor VIII, von Willebrand factor and the risk of major ischaemic heart disease in the Caerphilly Heart Study

Abstract: The relationships of three measurements of the factor VIII/von Willebrand factor (VWF) complex (factor VIII activity, FVIIIc (one-stage assay); VWF antigen, VWF Ag (ELISA); and VWF activity, VWF act, measured by a recently-developed ELISA) to major ischaemic heart disease (IHD) events were studied in 1997 men aged 49-65 years, in the second phase of the Caerphilly Heart Study. These variables were related using logistic regression analysis to myocardial infarction or IHD death, which occurred in 129 men during an average follow-up period of 61 months. All three measurements were highly correlated (r = 0.63-0.77), and each was significantly associated with incident major IHD on univariate analyses (relative odds in highest fifth compared to lowest fifth, 1.68-1.90; P = 0.028-0.006) and on multivariate analyses adjusting for major IHD risk factors and for baseline IHD. Neither FVIIIc nor VWF act was significantly related to incident IHD following adjustment for VWF Ag. We therefore suggest that the associations between these three measurements of the factor VIII/VWF complex and incident IHD might have at least three explanations: VWF Ag is a marker of arterial endothelial disturbance; VWF act promotes platelet adhesion/aggregation and hence the platelet component of arterial thrombosis; and FVIIIc promotes fibrin formation and hence the fibrin component of arterial thrombosis.

Circulating platelet–neutrophil complexes represent a subpopulation of activated neutrophils primed for adhesion, phagocytosis and intracellular killing

Abstract: Platelets play a prominent role in linking the processes of inflammation, haemostasis and thrombosis. Recent studies have shown that platelets form heterotypic aggregates with leucocytes via platelet CD62P and leucocyte beta2 integrins. These interactions have been observed in vitro in blood taken from healthy volunteers and in clinical conditions in which thrombosis and inflammation are prominent. This study investigated the properties of platelet-neutrophil complexes (PNCs) in anticoagulated whole blood. At rest, neutrophils in PNCs exhibit a significantly more activated adhesion molecule profile than free neutrophils with increased CD11b expression and activation (increased binding of the CD11b/CD18 'activation reporter' monoclonal antibody 24) and decreased CD62L expression. In addition, neutrophils in PNCs phagocytosed significantly more Neisseria meningitidis and produced more toxic oxygen metabolites than free neutrophils. Stimulation with the platelet agonist adenosine diphosphate (ADP) led to further increases in CD11b expression and activation, loss of CD62L as well as increased phagocytosis and toxic oxygen metabolite production throughout the whole neutrophil population. When these experiments were repeated with the CD62P blocking antibody G1 the effects were inhibited to a variable extent, dependent upon the parameter under investigation. These results indicate that both soluble and contact-dependent factors contribute to platelet-mediated neutrophil activation. Platelet neutrophil complexes represent a large subpopulation of neutrophils with a more activated adhesion molecule profile, and a greater capacity for phagocytosis and toxic oxygen metabolite production. This study provides further support for a role for PNCs in both health and disease.

Interaction of thrombopoietin with the platelet c‐mpl receptor in plasma: binding, internalization, stability and pharmacokinetics

Abstract: Thrombopoietin (TPO) is the primary regulator of platelet production and acts through binding its receptor, c-mpl, found on megakaryocyte progenitor cells, megakaryocytes and platelets. Circulating levels of TPO are regulated primarily by the clearance of TPO after it binds to c-mpl receptors on circulating platelets. In this study the interaction of TPO with the platelet c-mpl receptor has been analysed under physiological conditions using radiochemical and pharmacokinetic approaches. 125I-rHuTPO was prepared using a novel method of gentle iodination that preserved its biological activity and used to demonstrate that platelets, but not endothelial cells, have a single class of binding sites (56 +/- 17 binding sites/platelet) with high affinity (Kd = 163 +/- 31 pM). Cross-linking experiments confirmed that TPO, but not erythropoietin (EPO), specifically associated with the 95 kD platelet c-mpl receptor. Upon addition of TPO to platelets, 80% of the TPO binding sites were internalized within an hour and were not recycled. TPO that was not bound by platelets was stable for up to 6 d in both platelet-poor and platelet-rich plasma. Using unlabelled recombinant human TPO (rHuTPO), standard pharmacokinetic analysis demonstrated that platelets have an average TPO clearance of 1.24 +/- 0.38 ml/h/109 platelets and that TPO clearance was reduced by low temperature but not by a number of drugs or metabolic inhibitors. The maximal amount of TPO removed by platelets in vitro was identical to that predicted by the total number of TPO binding sites. These results provide a biochemical and pharmacokinetic basis for the clinical use of TPO and for understanding possible disorders of platelet production.

Angiogenic growth factors and endostatin in non‐Hodgkin's lymphoma

Abstract: A number of clinical studies have demonstrated the prognostic significance of angiogenesis and angiogenic growth factors in solid tumours; however, very little is known about the relevance of these parameters in haematological malignancies. We evaluated circulating levels of angiogenic growth factors and endostatin in 36 non-Hodgkin's lymphoma (NHL) patients. Baseline vascular endothelial growth factor (VEGF) levels of patients in complete remission (CR) after a median follow-up of 21 months were significantly lower than those of patients with progressive disease (P = 0.016). Event-free survival (EFS) rate was significantly higher in patients who had baseline VEGF and basic-fibroblast growth factor (b.FGF) levels below the median values of 147 and 19.5 pg/ml (P = 0.018 and 0.039 by log-rank test, respectively). Conversely, the levels of endostatin, angiogenin and leptin were not different in CR patients compared to relapsed patients and did not correlate with EFS. Our data suggest that b-FGF and, particularly, VEGF might be considered prognostic factors in NHL staging and management.

Secondary leukaemia and myelodysplasia after autografting for lymphoma: results from the EBMT

Abstract: Between 1978 and 1996 more than 7500 lymphoma transplants have been reported to the European Bone Marrow Transplantation (EBMT) Lymphoma Registry. This has been examined to establish the incidence of secondary leukaemia and myelodysplasia and to relate this to possible prognostic factors. 131 centres representing 4998 patients responded to a questionnaire. This identified 66 patients with post transplant myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML). The actuarial risk for MDS/AML at 5 years post-transplant (+/-95% CI) was 4.6% (3.1-6.8) for Hodgkin's disease and 3.0% (2.0-4. 3) for non-Hodgkin's lymphoma. Multivariate analysis for all patients demonstrated an effect of age at transplant, radiotherapy at conditioning, number of transplants and interval between diagnosis and transplant as risk factors. For patients with NHL, grade of histology was important (low grade > intermediate or high-grade); for Hodgkin's disease, female sex was identified as a risk factor. These findings suggest that the incidence of MDS/AML may not be greater following an autograft than after conventional chemotherapy.

Associations of blood rheology and interleukin-6 with cardiovascular risk factors and prevalent cardiovascular disease.

Abstract: Haemorheological variables (whole-blood, plasma and relative blood viscosity, haematocrit, red cell aggregation, white cell count and fibrinogen) were measured in 753 men and 821 women aged 25–74 years, and related to cardiovascular risk factors and prevalent cardiovascular disease (CVD). Men had higher levels than women of blood viscosity, haematocrit, corrected viscosity and relative viscosity. Post-menopausal women had higher levels than pre-menopausal women of blood viscosity, haematocrit, corrected blood viscosity, plasma viscosity and fibrinogen: each of these differences was completely or partly abolished by use of hormone replacement therapy. Serum total cholesterol, triglycerides, diastolic blood pressure, body mass index and smoking markers showed positive associations with most rheological variables, whereas HDL-cholesterol, plasma vitamin C and social class showed inverse associations. Rheological variables were associated with prevalent CVD after age-adjustment. However, after multiple risk factor adjustment only plasma viscosity and red cell aggregation showed significant (P < 0.04) associations in both men and women (comparing top to bottom quarters). Plasma interleukin-6 (measured in a 25% subsample of 196 men and 221 women) correlated significantly with age, fibrinogen, white cell count, plasma and blood viscosity, current smoking, and (in men) with low serum vitamin C levels; but not with other major risk factors or with prevalent cardiovascular disease.

Mcl-1 and Bcl-xL are co-regulated by IL-6 in human myeloma cells.

Abstract: Multiple myeloma (MM) is a slowly proliferative malignancy in which malignant plasma cells accumulate within the bone marrow. The expression of several anti-apoptotic proteins was evaluated by immunoblotting in human myeloma cell lines and in highly purified native myeloma cells. Expression of Bcl-xL, Mcl-1 and Bcl-2 was found in most of the samples; expression of Bcl-xL and Mcl-1 seemed to be related on myeloma cells. In a system of apoptosis by growth factor deprivation on myeloma cells, we showed that the effect of Bcl-2 seemed minimal whereas Mcl-1 and Bcl-xL were tightly regulated by interleukin (IL)-6. These findings underline the important role of Mcl-1 and Bcl-xL instead of Bcl-2 in IL-6-induced survival of myeloma cells.

BCR-ABL influences the antileukaemic efficacy of alkylphosphocholines.

Abstract: We have compared the antileukaemic efficacy of a series of new i.v. injectable alkylphosphocholines (APC) with their clinically used congeners miltefosine and perifosine. The test system consisted of four leukaemic cell lines carrying the bcr-abl rearrangement (K-562, LAMA-84, CML-T1 and BV-173) and two other leukaemic cell lines (HL-60 and SKW-3) without this genetic alteration. The prototype of i.v. injectable APC, erucylphosphocholine, was more active against BCR-ABL-positive cell lines than the two reference APC. It induced programmed cell death in HL-60 and SKW-3 cells after exposure for 24 h, and in bcr-abl expressing cells after a prolonged incubation period (48 h). LAMA-84 cells responded to i.v. injectable APC with increased conversion to an adherent, fibroblast-like phenotype. Experiments with a cell-free system showed that the target structures of APC are localized within the cytoplasmic compartment. Blockade of ceramide synthase by fumonisin B1 was insufficient to prevent oligonucleosomal DNA fragmentation. Using RT-PCR we confirmed that K-562 and LAMA-84 cells carry the b3a2 fusion type, and CML-T1 and BV-173 the b2a2 variant. BV-173 cells had the lowest level of bcr-abl mRNA which correlated with their increased sensitivity. Transfection of K-562 cells with antisense oligonucleotides directed against bcr-abl caused a specific suppression of K-562 clonogenicity. Our data indicated that i.v. injectable alkylphosphocholines are potent inducers of apoptosis and display increased antileukaemic efficacy against BCR-ABL-positive blasts as compared with miltefosine and perifosine. The expression of BCR-ABL cannot prevent apoptosis but delays erucylphosphocholine-induced programmed cell death. Transfection with bcr-abl directed antisense oligonucleotides reduces the clonogenicity of K-562 cells.

Common resistance mechanisms to deoxynucleoside analogues in variants of the human erythroleukaemic line K562.

Abstract: Variants of the human K562 were developed against the nucleoside analogues cytosine arabinoside, 2 chlorodeoxyadenosine, fludarabine and gemcitabine. The resistant lines displayed a high degree of cross-resistance to all nucleoside analogues, with little or no cross resistance to other agents. There was a profound accumulation defect of the different nucleoside analogues in all of the variants. There was a strong overexpression of 5’ nucleotidase, measured by rt-PCR and enzyme activity, in all resistant variants. There was a two fold increase of ribonucleotide reductase in the fludarabine resistant line and increased expression of purine nucleoside phosphorylase in the 2 chlorodeoxyadenosine selected line. Karyotypic analysis revealed the loss of a 6(q16;q22) deletion present in the parental line in all of the resistant lines. This portion of chromosome 6 has been shown to contain the gene for 5′nucleotidase. Early events in the transport and metabolism appear to be involved in the resistance mechanisms to nucleoside analogues and are responsible for broad cross resistance to this family of compounds.

Aberrant expression and reverse signalling of CD70 on malignant B cells.

Abstract: In normal lymphoid tissues the tumour necrosis factor-receptor family member CD27 and its ligand CD70 have a restricted expression pattern. Previously, we reported that expression of CD27 is deregulated in B-cell leukaemias and lymphomas. Here we show that, although infrequently expressed by normal human B cells in vivo, CD70 is found on 50% of B-CLLs, 33% of follicle centre lymphomas, 71% of large B-cell lymphomas, and 25% of mantle cell lymphomas. Interestingly, in the majority of leukaemias and lymphomas examined, CD70 was found to have a capped appearance, a feature that coincided with co-expression of CD27. Functional analysis showed that a subset of B-CLLs could proliferate vigorously in response to CD70 mAb but not to CD27 mAb. This response was synergistically enhanced by ligation of CD40 but inhibited by the presence of IL-4. Additional experiments indicated that the proliferative response was due to an agonistic signal delivered via CD70, rather than blocking of negative signalling by CD27. Thus, next to its role as ligand, in a subset of malignant B cells CD70 can operate as receptor and as such might contribute to progression of these B-cell malignancies.

A prospective study of protein‐specific assays used to investigate idiopathic thrombocytopenic purpura

Abstract: Idiopathic thrombocytopenic purpura (ITP) is a disorder in which platelets, sensitized by autoantibodies, are destroyed by the reticuloendothelial system. The diagnosis of ITP has been a clinical one because assays measuring platelet-associated IgG (PAIgG) have low specificity. The recently introduced assays that measure antibodies against specific platelet glycoproteins (GP) offer the possibility of improved specificity. In this report we describe two prospective studies. In the first study we compared two protein-specific assays (AC and MAIPA) looking for the presence of autoantibodies against GP IIb/IIIa in 81 patient samples. These results were compared with an immunoradiometric assay for PAIgG. The second study investigated the enhanced sensitivity of measuring anti-GP Ib/IX autoantibodies in 76 patient samples. The protein-specific assays were able to differentiate immune from non-immune thrombocytopenia (specificity 91%, sensitivity 39%), whereas the PAIgG assay could not (specificity 19%, sensitivity 78%). The addition of the Ib/IX AC assay maintained a specificity of 92% while increasing the diagnostic sensitivity to 66%. In contrast to the PAIgG assay, there was no correlation between the platelet count and the likelihood or degree of positivity within the control samples using the glycoprotein assays. These studies confirm that glycoprotein assays can be used as diagnostic tests for ITP.