bluuebunny/crossref_metadata_2025 huggingface dataset:
Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte‐mediated inhibition of CFU‐GM and alterations in T‐cell receptor Vβ profiles
Authors: Molldrem Jeffrey J. , Jiang Yin Zheng , Stetler‐Stevenson Maryalice , Mavroudis Dimitrios , Hensel Nancy , Barrett A. John
Published: March 2003

We have demonstrated that 44% of myelodysplastic syndrome (MDS) patients with cytopenia have a haematological response to antithymocyte globulin (ATG). Three ATG responders and two non‐responders with refractory anaemia were further studied for lymphocyte‐mediated inhibition of bone marrow using a standard CFU‐GM assay. In responders, peripheral blood lymphocytes (PBL) added at a 5:1 ratio suppressed CFU‐GM by 54 ± 9% (P = 0.04) and was reversed by ATG treatment. Pre‐treatment marrow depleted of CD3 lymphocytes, increased CFU‐GM by 32% (P = 0.02) in an ATG responder, but not in a non‐responder. CD3 lymphocytes from 6‐month post‐treatment marrow did not inhibit pre‐treatment CFU‐GM, indicating ATG had affected the T cells. Pre‐treatment marrow depleted of CD8 lymphocytes, increased CFU‐GM by 60% (P = 0.01) and 49% (P = 0.03) in two ATG responders, but not in a non‐responder. Inhibition required cell–cell interaction through MHC I. TCR Vβ families, analysed by SSCP, changed from clonal to polyclonal in one ATG responder after 6 months, but clones persisted in a non‐responder. These results indicate patients with refractory anaemia who respond to ATG have CD8 T‐cell clones that mediate MHC‐I‐restricted suppression of CFU‐GM which are replaced by polyclonal T cells that do not suppress CFU‐GM after ATG treatment.
Effect of the MTHFRC677T variant on risk of venous thromboembolism: interaction with factor V Leiden and prothrombin (F2G20210A) mutations
Authors: Brown Karen , Luddington Roger , Baglin Trevor
Published: March 2003

Odds ratios for the MTHFRC677T variant were determined in a large case–control study of 558 unselected patients with venous thromboembolism and 500 control subjects. The odds ratios for MTHFRC677T heterozygosity and homozygosity were 1.07 (95%CI 0.84–1.36) and 0.71 (95%CI 0.48–1.03). In patients with the factor V Leiden or the F2G20210A mutations there was no apparent increase in risk of venous thromboembolism due to the MTHFRC677T polymorphism.

Thrombophilia testing should not include genotyping for the MTHFRC677T polymorphism.
Mechanisms involved in the antiplatelet activity of Escherichia coli lipopolysaccharide in human platelets
Authors: Sheu  , Hung  , Kan  , Lee  , Yen
Published: March 2003

In this study, Escherichia coli lipopolysaccharide (LPS) dose‐dependently (100–300 μg/ml) and time‐dependently (10–60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LPS also dose‐dependently inhibited the phosphoinositide breakdown and the intracellular Ca+2 mobilization in human platelets stimulated by collagen. LPS (300 μg/ml) also significantly inhibited the thromboxane A2formation stimulated by collagen in human platelets. Moreover, LPS (100–300 μg/ml) dose‐dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. In addition, LPS (200 and 300 μg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 μg/ml) also significantly increased the production of nitrate within a 30 min incubation period. Rapid phosphorylation of a platelet protein of Mr 47 000, a marker of protein kinase C activation, was triggered by phorbol‐12‐13‐dibutyrate (PDBu, 50 n
M). This phosphorylation was markedly inhibited by LPS (200 μg/ml) within a 30 min incubation period.

These results indicate that the antiplatelet activity of LPS may be involved in two important pathways. (1) LPS may induce conformational changes in the platelet membrane, leading to change in the activity of phospholipase C. (2) LPS also activated the formation of nitric oxide (NO)/cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. Therefore, LPS‐mediated alteration of platelet function may contribute to bleeding diathesis in septicaemic and endotoxaemic patients.
Bioactive substances in buffy‐coat‐derived platelet pools stored in platelet‐additive solutions
Authors: Edvardsen Lisbeth , Taaning Ellen , Mynster Tommie , Hvolris Jesper , Drachman Ole , Nielsen Hans JØrgen
Published: March 2003

We studied extracellular accumulation in buffy‐coat‐derived platelet pools suspended in platelet additive solutions (PAS). 20 platelet pools were prepared from buffy coats of 60 donors and stored in either PAS I (n = 10) or PAS II (n = 10). Samples were drawn from the donors to determine the normal range of histamine, plasminogen activator inhibitor 1 (PAI‐1), myeloperoxidase (MPO) and interleukin (IL)‐6. After preparation samples were collected on days 0, 5 and 7 from the 20 platelet pools respectively, to determine extracellular accumulation of the same substances. Extracellular histamine, PAI‐1 and MPO were detected in significant levels (P < 0.0001) immediately after preparation of the platelet pools. Only the extracellular level of PAI‐1 increased significantly during storage from day 0 to day 7 (P < 0.01). After preparation and storage to day 7 IL‐6 was found in only three of the 20 platelet pools.

Even though platelets were prepared by the buffy‐coat method, to produce a low leucocyte count and thereby low cytokine (IL‐6) content, there was still significant contamination by other bioactive substances.
FISH detection of chromosome 14q32/IgH translocations: evaluation in follicular lymphoma
Authors: Rack Katrina A , Salomon‐Nguyen Florence , Radford‐Weiss Isabelle , Ocana Gil Maria , Schmitt Christian , Belanger Coralie , Nusbaum Sylvie , Vekemans Michel , Valensi Françoise , Macintyre Elizabeth A
Published: March 2003

A FISH strategy capable of detecting chromosome 14q32 rearrangements involving the IgH locus, including in interphase nuclei, was developed using Ig variable and constant region cosmids from the extremities of the locus in a dual hybridization approach, using signal splitting as evidence of rearrangement. The large size of the locus (1.3 Mb) and the propensity for internal deletion due to physiological VDJ recombination and isotype switching complicate analysis of this locus. We used the Ig10 cosmid, which hybridizes to Cε and Cα2 at the 3′ end of the constant region, in order to minimize deletion and/or splitting of the constant region probe. Cos Ig10 and the IgV18 VH probes were compared with a specific IgH‐BCL2 FISH dual hybridization approach in follicular lymphoma (FL). Both were capable of detecting the t(14;18) in interphase nuclei, including in cases with no apparent abnormality by classic karyotype analysis, although the sensitivity of the IgH approach was slightly lower. We have also successfully applied these probes to whole cell cytospin preparations, rendering analysis of cryopreserved material possible, although interpretation should be limited to frequent events, particularly following cell manipulation. Analysis of flow cytometric sorted bone marrow fractions from three FL patients by FISH and FICTION showed that the t(14;18) was present in a much lower proportion of CD34 positive than negative cells but that the higher level of background hybridization limits use of these techniques for the reliable quantification of rare events.
Detection of the t(2;5)‐associated NPM/ALK fusion cDNA in peripheral blood cells of healthy individuals
Authors: Trümper  , Pfreundschuh  , Bonin  , Daus
Published: March 2003

The translocation t(2;5), which leads to the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to the receptor kinase ALK on chromosome 2p23, is found in CD30+ anaplastic large cell lymphomas and some cases of B‐cell lymphoma. Hodgkin's disease (HD) is a malignant lymphoma characterized by large multinucleated tumour cells, Hodgkin and Reed‐Sternberg (H&RS) cells, surrounded by a dense lymphohistiocytic infiltrate. Our group recently demonstrated NPM/ALK fusion cDNAs by single‐cell RT‐PCR in < 3% of CD30+ tumour cells in 2/9 cases of HD. To further delineate the relevance of this finding for HD, we studied the occurrence of NPM/ALK fusion genes in peripheral blood cells of healthy donors by RT‐PCR. NPM/ALK fusion cDNAs were found by RT‐PCR in 14/29 healthy individuals and confirmed by hybridization with a breakpoint‐specific oligonucleotide. Due to the low rate of NPM/ALK‐positive cells in the peripheral blood of positive individuals, an assignment to a defined cellular subpopulation was not possible. We conclude that NPM/ALK fusion genes are present in peripheral blood cells of healthy donors. After t(14;18) and t(9;22), t(2;5) represents the third example of tumour‐associated translocation products in blood cells of apparently healthy donors. The implications of this finding are discussed.
Home treatment with recombinant activated factor VII in patients with factor VIII inhibitors: the advantages of early intervention
Authors: Santagostino E. , Gringeri A. , Mannucci P. M.
Published: March 2003

To evaluate the feasibility, efficacy and safety of home treatment with recombinant activated factor VII (rFVIIa), 10 inhibitor patients (all haemophiliacs except one acquired post‐partum) self‐administered up to four doses of 90 μg/kg rFVIIa every 3 ± 1 h. The response was rated by the patient as effective (haemorrhage stopped or decreased substantially), partially effective (reduced) or ineffective (unchanged or worsened). 45 haemarthroses and eight haematomas were treated within a median time of 1.0 h (range 0.3–11.9) from the onset of bleeding, with a median of two rFVIIa doses per course (range 1–4). rFVIIa was effective in 42 episodes (79%), partially effective in six (11%) and failed in five (10%). Compared with partially effective and ineffective treatments, effective treatments started earlier (median time: 0.6 v 2.7 h, P = 0.02) and required a smaller number of doses (median: 1.5 v 3, P = 0.007). The risk of a partially effective or ineffective treatment was smaller for treatments started within 6 h from the onset of bleeding than for those which started later (OR 0.24, 95% CI 0.09–0.63). Mild side‐effects were reported only after 3/113 self‐infusions (2.6%). Early home treatment with rFVIIa is safe, feasible and effective, inducing and maintaining haemostasis with a small number of doses.
Distribution of myeloma plasma cells in peripheral blood and bone marrow correlates with CD56 expression
Authors: Rawstron Andrew , Barrans Sharon , Blythe David , Davies Faith , English Anne , Pratt Guy , Child Anthony , Morgan Gareth , Jack Andrew
Published: March 2003

There is a wide variation in the degree of marrow and blood involvement between patients with multiple myeloma. Both of these parameters are known to be highly significant prognostic factors, and the differences between patients may be due to variable expression of adhesion molecules. To test this we used three‐colour flow cytometry to study three adhesion molecules associated with myeloma, namely CD38, CD56 and CD138. The level of expression of these molecules was compared with the distribution of myeloma plasma cells in bone marrow (n=59) and peripheral blood (n=26) in patients at presentation or relapse. The extent of marrow infiltration on the trephine biopsy correlated inversely with CD56 expression (Mann‐Whitney U Test, P=0.022); there was no difference in CD38 or in CD138 expression. CD56 expression also correlated inversely with the number of circulating plasma cells (linear regression, R2=0.4268, slope=−0.58, P=0.0003). Peripheral blood plasma cell numbers correlated weakly with bone marrow plasmacytosis, and inversely with CD38 expression. The level of CD56 expression by neoplastic plasma cells was assessed in 37 patients over a median of 11 months (range 6–25). There was no significant change in expression (Wilcoxon Signed Rank, P=0.6271). We conclude that plasma cell CD56 expression is constant over the course of the disease; unlike CD138 expression, it is significantly linked to the degree of both bone marrow and peripheral blood involvement.
The 20210 G → A mutation in the 3′‐untranslated region of the prothrombin gene and the risk for arterial thrombotic disease
Authors: Franco R. F. , Trip M. D. , Ten Cate H. , Van Den Ende A. , Prins M. H. , Kastelein J. J. P. , Reitsma P. H.
Published: March 2003

A sequence variation in the 3′‐untranslated region of the prothrombin (PT) gene (20210 G → A) was recently claimed to be associated with elevated plasma prothrombin levels and an increased risk for venous and arterial thrombosis. We examined the prevalence of the 20210 A allele in the prothrombin gene in 400 healthy controls and in 263 patients with proven premature atherosclerotic disease. In addition, we measured prothrombin, prothrombin fragment 1 + 2, thrombin–antithrombin (TAT) complex and D‐dimer levels in plasma from carrier and non‐carrier patients. The frequency of the variant allele was 1% in the control subjects and 2.7% in the patient group, yielding a relative risk (RR) for the 20210 A allele of 2.7 (95% CI 0.8–9.4). All heterozygotes in the patient group were found to have had a myocardial infarction (MI), yielding a RR for MI of 4.2 (95% CI 1.2–14.6). Plasma prothrombin levels in carriers (126 ± 10) were higher than in non‐carriers (103 ± 1, P = 0.02). The levels of TAT complexes (16 ± 9 v 6 ± 1 μg/ml, P = 0.02) as well as of prothrombin fragment 1 + 2 (1.5 ± 0.3 v 1.0 ± 0.1 nmol/l, P = 0.02) were also elevated in carriers of the mutation. Our findings suggest that the 20210 G → A mutation in the prothrombin gene is a genetic risk factor for MI. In addition, our data provide evidence for an association of the mutation with excessive thrombin generation, which may contribute to the understanding of its role in venous and arterial disease.
Prevalence and persistence of a novel DNA TT virus (TTV) infection in Japanese haemophiliacs
Authors: TAKAYAMA S. , MIURA T. , MATSUO S. , TAKI M. , SUGII S.
Published: March 2003

To clarify the clinical implication of a newly discovered ‘TT virus (TTV)’, we assayed TTV DNA in sera from 50 haemophiliacs by a seminested‐PCR. TTV DNA was detected in 75% (35/50), which was a much higher prevalence than for HBV (HBc‐Ab), HCV RNA, or HGV RNA. In particular, TTV DNA was found in 44.4% (4/8) of patients who had been treated only with virally inactivated factor VIII concentrates. Elevated ALT levels were observed in patients with HCV RNA and TTV DNA; however, the elevation in TTV DNA was obtained from patients co‐infected with HCV RNA (62.9%, 22/35). There was no significant difference in ALT levels between TTV DNA‐positive and DNA‐negative in patients without HCV RNA. 85.3% (35/41) of TTV DNA‐positive sera in 1990 were again positive for TTV DNA in 1995. These findings suggest that many haemophiliacs have been infected with TTV. Although TTV infection was not associated with serum ALT elevation, persistent TTV infection may contribute to cryptogenic hepatic failure in haemophiliacs.
Successful treatment of a non‐haemophilic patient with inhibitor to factor VIII by double‐filtration plasmapheresis
Authors: SUNAGAWA TAKASHI , UEZU YOSHINAO , KADENA KEIZO , TOKUYAMA KIOYUKI , KINJO FUKUNORI , SAITO ATSUSHI
Published: March 2003

Factor VIII (FVIII) inhibitors appear in 3–20% of haemophilia A patients after injection of FVIII concentrates. However, autoantibodies to FVIII are also reported in non‐haemophiliacs. In these patients FVIII inhibitor disappears spontaneously or diminishes in response to immunosuppressive therapy. However, a few patients show resistance to immunosuppressive therapy. We describe a non‐haemophilic elderly patient with acquired FVIII inhibitor who failed to respond to prednisolone. He was treated with double‐ filtration plasmapheresis (DFPP) which resulted in a very rapid reduction in inhibitor levels and resolution of symptoms.
Central venous catheter‐associated thrombosis in severe haemophilia
Authors: VIDLER VICKY , RICHARDS MIKE , VORA AJAY
Published: March 2003

Significant subclavian vein thromboses associated with indwelling fully implanted (port‐a‐cath) devices are described in two boys with severe haemophilia A and factor VIII inhibitors. Investigations were prompted by prominent chest wall veins in one case, whereas the thrombosis was a chance finding in the other case during investigation of mechanical dislocation of the catheter tubing. Extensive collateral venous circulations were demonstrated by venography in both instances indicating that the thrombus had been present for some time. Possible contributing factors to the thromboses included desensitization therapy (both patients), high‐dose FEIBA (in one patient) and use of lower doses of heparin for line flush than that recommended by some authors. Neither patient had a familial or non‐familial predisposition to thrombosis.
Up‐regulation of adhesion and MHC molecules on splenic monocyte/macrophages in adult haemophagocytic syndrome
Authors: Keréveur A. , McIlroy D. , Samri A. , Oksenhendler E. , Clauvel J. P. , Autran B.
Published: March 2003

Haemophagocytic syndrome (HPS) has been associated with the abnormal activation of mono/macrophages and increased cytokine production. However, neither the phenotype of haemophagocytic monomacrophages nor the cellular origin of cytokine production have been described. We studied splenic monomacrophages and lymphocytes from five patients with HPS (two HIV− and three HIV+) and from controls without HPS (three normal HIV− and two pathological HIV+). Using flow‐cytometry, we observed a marked increase in the expression of MHC class I and II, M‐CSF‐receptor and adhesion molecules LFA‐1, LFA‐3, ICAM‐1 (P < 0.05) on HPS+ splenic monomacrophages compared to HPS−, which was independent of their HIV status. A high percentage of CD8+ lymphocytes from 4/5 HPS+ patients produced TNFα and IFNγ, but no IL‐6 upon in‐vitro activation. In a fifth patient CD4+ but not CD8+ lymphocytes produced these cytokines. Although other cytokines might be involved in the pathophysiology of HPS as suggested by the high expression of M‐CSF‐receptor, these results suggest that TNFα and IFNγ secretion by T cells might play a role in the up‐regulation of adhesion and MHC molecules on monomacrophages from HPS.
Diamond‐Blackfan anaemia in the Italian population
Authors: Ramenghi U. , Garelli E. , Valtolina S. , Campagnoli M. F. , Timeus F. , Crescenzio N. , Mair M. , Varotto S. , D'Avanzo M. , Nobili B. , Massolo F. , Mori P. G. , Locatelli F. , Gustavsson P. , Dahl N. , Dianzani I.
Published: March 2003

Diamond‐Blackfan anaemia (DBA) is a congenital disease characterized by defective erythroid progenitor maturation; 30% of patients have congenital malformations. The link between these malformations and defective erythropoiesis is unclear: a defect in a molecule acting both on embryo development and haemopoiesis has been proposed. Inheritance is autosomal dominant in most familial cases, but recessive families have also been reported. Many cases are sporadic. A DBA locus has been mapped on chromosome 19q13.2 (Gustavsson et al, 1997), but several families unlinked to this locus have also been reported (Gustavsson et al, 1998).

This paper presents clinical, epidemiological and molecular data for DBA in the Italian population. Segregation analysis of 19q markers in patients with DBA showed exclusion of this locus in 5/12 families with inherited DBA. There was evidently locus heterogeneity for DBA in this population. A new microdeletion was identified in one patient. Other families, in which DBA segregates concordantly with the 19q critical region, suggest incomplete penetrance and expressivity of the DBA gene.
Rapid genotyping of human platelet antigen 1 (HPA‐1) with fluorophore‐labelled hybridization probes on the LightCyclerTM
Authors: Nauck Markus S. , Gierens Hedi , Nauck Matthias A. , März Winfried , Wieland Heinrich
Published: March 2003

Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post‐transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single‐tube method for HPA‐1 genotyping that combines rapid‐cycle PCR with allele‐specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescently‐labelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real‐time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele‐specific manner as the detection probe, which was fully complementary to the HPA‐1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post‐PCR sample manipulation, thereby eliminating the risks of end‐product contamination and sample tracking errors. The genotypes determined with the LightCyclerTM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA‐1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.
Intron 22‐specific long PCR for the Xba I polymorphism in the factor VIII gene
Authors: El‐Maarri Osman , Oldenburg Johannes , Çağlayan S. Hande
Published: March 2003

The Xba I polymorphic site in the factor VIII gene is present in the int22h‐1 region which is found in two other copies (int22h‐2 and int22h‐3) distal to the gene. Previously the polymorphic status of the Xba I locus was studied by either Southern blot or PCR that amplified all three copies. Here we report the use of a long PCR that specifically amplifies the intragenic site in intron 22, making use of this marker an easy and reliable assay. Moreover, about 25% of previously uninformative Turkish haemophilia A families examined with three markers proved to be informative for linkage analysis, using the Xba I polymorphism.
High‐dose therapy with autologous haemopoietic stem cell support for Waldenström's macroglobulinaemia
Authors: Desikan R. , Dhodapkar, M. , Siegel D. , Fassas A. , Singh J. , Singhal S. , Mehta J. , Vesole D. , Tricot G. , Jagannath S. , Anaissie E. , Barlogie B. , Munshi N. C.
Published: March 2003

Standard doses of alkylating agents or purine analogues effect response rates of up to 50% in Waldenström's macroglobulinaemia (WM); however, complete responses are infrequent and there are no cures. We have evaluated the feasibility, safety and efficacy of high‐dose chemotherapy with peripheral blood stem cell support in six patients aged 45–69 years (median 51.5) with WM; four patients relapsed after prior therapy inclusive of purine analogues and two patients proceeded with transplant after minimal therapy. Four patients mobilized adequate numbers of stem cells; however, two patients with more extensive fludarabine therapy failed to mobilize and required a second attempt at stem cell collection. Five patients were treated with melphalan 200 mg/m2, including one patient who received tandem transplants and one patient who received melphalan 140 mg/m2 with added total body irradiation (TBI). There was no treatment‐related mortality and non‐haematological toxicities were manageable. Engraftment was prompt except in one patient with extensive prior use of fludarabine. All the six patients achieved at least partial response (PR), including one who achieved complete response (CR). Five patients are alive and four are event‐free at 52+, 15+, 12+ and 2+ months post transplant. This pilot study suggests safety and efficacy of high‐dose therapy in WM and suggests that the peripheral blood stem cells should preferably be procured prior to extensive use of purine analogues.
DNA image cytometry in MDS bone marrow smears
Authors: Widell Susanne , Hast Robert , Auer Gert , Cox Christopher , Bennett John M.
Published: March 2003

The nuclear DNA content, defined as DNA index (DI) in blasts/promyelocytes (bla/pro), were determined on Feulgen‐stained bone marrow smears from 39 patients with myelodysplastic syndromes (MDS) and eight control subjects by the use of image cytometry (ICM). The DI in patients was compared to that of corresponding normal cell types, and to cytogenetic data available in 32/39 patients. The mean DI in bla/pro of patients with MDS was significantly (P < 0.01) lower compared to corresponding cell types in control subjects. By ICM, a DNA aneuploidy in bla/pro was found in 67% of the MDS patients, and 59% expressed DNA hypodiploidy. By cytogenetics, an abnormal karyotype was found in 31%, and 6/9 MDS patients with a ‘hypodiploid’ abnormal karyotype showed DNA hypodiploidy. Of patients with a normal karyotype (69%), seven (32%) showed a normal, 12 (55%) a lower, and three (14%) a higher DI compared to controls. No difference between groups of MDS patients was found. DNA hypodiploidy is suggested to be a common feature in MDS without a relationship to cytogenetics.
De novo CD5‐positive diffuse large B‐cell lymphoma: clinical characteristics and therapeutic outcome
Authors: Yamaguchi Motoko , Ohno Toshiyuki , Oka Kouji , Taniguchi Masanori , Ito Motohiro , Kita Kenkichi , Shiku Hiroshi
Published: March 2003

To determine the clinical significance of CD5 expression in diffuse large B‐cell lymphoma (DLBL) without a clinical history of low‐grade B‐cell lymphoma, we have reviewed the clinical features and therapeutic outcome of 25 patients with de novo CD5‐positive DLBL, and compared the results with those of 87 patients with CD5‐negative DLBL and 22 patients with mantle cell lymphoma (MCL). The patients with de novo CD5‐positive DLBL had clinical characteristics of elderly onset (median age 63, range 37–91), and female predominance (male/female 10/15). 21 (84%) of these patients had extranodal involvement at presentation, with great variation in the sites. In comparison with the patients with CD5‐negative DLBL, the treatment outcome for the patients with de novo CD5‐positive DLBL was very poor with frequent relapse. The failure‐free survival curve was almost identical to that of patients with MCL, showing that standard chemotherapy for DLBL was not effective for most of the patients with de novo CD5‐positive DLBL. These findings suggest that de novo CD5‐positive DLBL forms a distinct subgroup of DLBL.
Erythroblasts from Friend virus infected‐ and phenylhydrazine‐treated mice accurately model erythroid differentiation
Authors: Hodges Vivien M. , Winter Paul C. , Lappin Terence R. J.
Published: March 2003

The dynamics of gene expression during terminal erythroid differentiation have been examined in three murine models; the erythroleukaemia cell line HCD‐57 and splenic erythroblasts isolated from mice treated with either the anaemia‐inducing strain of Friend virus (FVA cells) or the haemolytic agent phenylhydrazine (PHZ cells). In response to erythropoietin (EPO) and haemin, HCD‐57 cells proliferated and synthesized haemoglobin, but failed to complete terminal differentiation as indicated by lack of change in both gene expression and morphological appearance. In contrast, EPO‐induced terminal differentiation in FVA and PHZ cells in vitro was accompanied by increases in haemoglobin positivity, morphological maturation and a shared pattern of gene expression. EPO receptor (EPO‐R) mRNA levels peaked before globin gene expression which was maximal at 24 h. Peak GATA‐1 and EKLF mRNA levels also preceded the globin gene peak, but the highest NF‐E2 levels coincided with maximal globin levels, suggesting a role for NF‐E2 in the maintenance, rather than the initiation of globin gene expression. Peak expression of δ‐aminolaevulinic acid synthase (ALAS) coincided with peak globin expression. FVA and PHZ cells represent more effective models than the HCD‐57 cell line for the investigation of erythroid gene expression during EPO‐regulated terminal erythropoiesis.
Def‐2, ‐3, ‐6 and ‐8, novel mouse genes differentially expressed in the haemopoietic system
Authors: Hotfilder Marc , Baxendale Sarah , Cross Michael A. , Sablitzky Fred
Published: March 2003

To identify developmentally regulated genes during myeloid differentiation, a self‐inactivating retroviral gene‐trap vector carrying a β‐galactosidase‐neomycin (SA/lacZ/neo) fusion gene was constructed and used to infect myeloid progenitor cells (FDCP‐Mix A4). G418‐resistant and β‐galactosidase positive cell lines (gene‐trap integration [GTI] clones) were established and induced to differentiatein vitrointo either macrophages or granulocytes. Expression of the trapped loci was monitored at a single‐cell level by analysing the mature cell types for β‐galactosidase activity. All 37 GTI clones tested showed down‐regulation either during granulocyte or both granulocytic and macrophage differentiation. The endogenous coding regions fused to the SA/lacZ/neo reporter gene were isolated from eight clones. Molecular analysis revealed that half of them represented novel mouse genes (def‐2, ‐3, ‐6 and ‐8) which we confirmed to be differentially expressed in primary haemopoietic tissues. Database searches revealed no significant similarities for def‐2 (associated with haemopoietic progenitors) and def‐8 (expressed most strongly in peripheral leucocytes). Def‐6, which is down‐regulated upon the differentiation into myeloid as well as erythroid lineages, was found to be closely related but not identical with the recently described B‐cell‐specific switch recombinase SWAP‐70. Def‐3, which is down‐regulated upon differentiation into granulocytes but expressed in progenitor cells and macrophages, defines a novel family of RNA binding proteins.
Additional translocation (8;21)(q22;q22) in a patient with Philadelphia‐positive chronic myelogenous leukaemia in the blastic phase
Authors: Kojima Kensuke , Yasukawa Masaki , Ishimaru Fumihiko , Dansako Hiromichi , Matsuo Yoshinobu , Kimura Yukio , Nawa Yuichiro , Hara Masamichi , Harada Mine
Published: March 2003

We report a case of Philadelphia‐positive chronic myelogenous leukaemia in blastic phase with the additional translocation (8;21)(q22;q22), which is frequent in acute myeloid leukaemia but not in chronic myelogenous leukaemia. The t(8;21) was not detected in the chronic phase, and was the only additional chromosomal anomaly in the blastic clone. Reverse transcription‐polymerase chain reaction revealed the AML1/ETO fusion transcript in the cells of blastic phase but not in those of chronic phase. Regarding t(9;22), the breakpoint on chromosome 22 occurred in the μ‐BCR region of the BCR gene, resulting in hybrid BCR/ABL mRNA with an e19a2 junction. Our findings provided molecular evidence that t(8;21) can occur as an additional genetic change in Philadelphia‐positive chronic myelogenous leukaemia.
Production and characterization of anti‐Kell monoclonal antibodies using transfected cells as the immunogen
Authors: Chu Te‐Hua Tearina , Yazdanbakhsh Karina , Øyen Ragnhild , Smart Elizabeth , Reid Marion E.
Published: March 2003

Monoclonal antibodies (Mabs) to blood group antigens are valuable as diagnostic reagents for typing red blood cells (RBCs) in the clinical setting, and for structure–function studies of proteins. Here, we report a powerful system that enabled us to produce Mabs to blood group antigens. A murine erythroleukaemia (MEL) cell line expressing Kell protein, a transmembrane glycoprotein that carries a number of clinically relevant antigens, was used as a novel immunogen. Mabs with different specificities to the Kell protein were produced from a single mouse fusion: an anti‐Jsb (MIMA‐8), and two antibodies (MIMA‐9 and MIMA‐10) with novel specificities, that reacted with RBCs with the common Kell phenotype but not with RBCs with K+k− or Kp(a+b−) or K0 phenotypes. The non‐reactivity with both K+k− or Kp(a+b−) RBCs implied that the epitope was influenced by the molecular changes associated with an absence of the k or Kpb antigens. MIMA‐8 is the first example of a Mab anti‐Jsb and was used in the clinical laboratory for screening donor RBCs for Js(b−) blood and for typing RBCs from patients even when the RBCs were coated with anti‐IgG as is the case in autoimmune haemolytic anaemia. Heavy and light chain variable regions of MIMA‐8 were cloned and the sequence is given. This study illustrates the potential of this novel immunization approach for making monoclonal antibodies to blood group antigens.
The relationship between monoclonal myeloma precursor B cells in the peripheral blood stem cell harvests and the clinical response of multiple myeloma patients
Authors: Mitterer M. , Oduncu F. , Lanthaler A. J. , Drexler E. , Amaddii G. , Fabris P. , Emmerich B. , Coser P. , Straka C.
Published: March 2003

The aim of this study was to determine the presence of monoclonal myeloma precursor B cells in peripheral blood stem cell harvests and to investigate their role in the clinical outcome of multiple myeloma patients. A total of 39 multiple myeloma patients were treated with a sequential therapy including double high‐dose melphalan therapy followed by a double transplant procedure. The apheresis products for the second transplant were purged using a panel of four or five different mouse monoclonal antibodies against B‐cell antigens (CD10, CD19, CD20, CD22 and CD37). In 19/39 patients a tumour‐specific CDR III signal was identified in the diagnostic bone marrow. Gene scan analysis after CDR III PCR of the magnetic bead isolated B‐cell fraction from the apheresis products in these 19 patients revealed three different patterns: 32% of patients had a predominantly monoclonal B‐cell population; 63% of patients had an identifiable monoclonal signal within an oligoclonal B‐cell population. In only 1/19 patients were no monoclonal B cells identified in the B‐cell population of the apheresis product. A correlation between the clonal pattern and the clinical response after sequential chemotherapy was found. Patients with a predominance of monoclonal myeloma or myeloma precursor B cells had an early relapse or achieved a minimal response or a partial remission. Patients with an oligo‐ and/or polyclonal pattern achieved a high percentage of partial as well as complete remissions.
Prevalence of mild hyperhomocysteinaemia and association with thrombophilic genotypes (factor V Leiden and prothrombin G20210A) in Italian patients with venous thromboembolic disease
Authors: De Stefano Valerio , Zappacosta Bruno , Persichilli Silvia , Rossi Elena , Casorelli Ida , Paciaroni Katia , Chiusolo Patrizia , Leone Antonio Maria , Giardina Bruno , Leone Giuseppe
Published: March 2003

Mild hyperhomocysteinaemia is an established risk factor for deep vein thrombosis (DVT); few data concerning its potential interaction with thrombophilic genotypes are available at the present time. We investigated 121 thrombosis‐free individuals and 111 patients with at least one objectively confirmed episode of DVT. A thrombophilic condition (deficiency in antithrombin, protein C and S, factor V Leiden, prothrombin G20210A) was detected in 25.2% of the patients; mutant factor V or prothrombin genotypes were present in 6.6% of the controls. Hyperhomocysteinaemia was found in 14.4% of patients and 3.3% of the controls, with a 3.7‐fold increase in risk for DVT (95% CI 1.1–12.3). Adoption of different cut‐off levels for definition of hyperhomocysteinaemia did not substantially change the magnitude of the risk. Carriership of both hyperhomocysteinaemia and factor V Leiden or prothrombin G20210A was detected in 2.7% of patients for each combination and in none of the controls. An approximate estimate of 30‐fold increased risk in carriers of both hyperhomocysteinaemia and factor V Leiden and 50‐fold increased risk in carriers of both hyperhomocysteinaemia and prothrombin G20210A was calculated, suggesting a synergistic interaction between hyperhomocysteinaemia and such thrombophilic genotypes. Yet statistical analysis is highly unstable due to the small number of individuals with combined defects. Further investigations on large series of patients are needed.
Is there a correlation between degree of splenomegaly, symptoms and hypersplenism? A study of 218 patients with Gaucher disease
Authors: Gielchinsky Yuval , Elstein Deborah , Hadas‐Halpern Irith , Lahad Amnon , Abrahamov Ayala , Zimran Ari
Published: March 2003

Despite the prevalence of splenomegaly as a sign in many disorders, there have been no studies that correlate the degree of organomegaly with the symptoms generally ascribed to splenic enlargement. The degree of splenomegaly was compared with five overt symptoms of mechanical displacement, i.e. chronic abdominal pain, abdominal discomfort, early satiety, pain while lying on the side, or attacks of acute (colicky) left upper quadrant pains. We have also employed splenomegaly as seen in Gaucher disease as a paradigm to determine whether there is a correlation between the degree of splenomegaly and the parameters of hypersplenism. Although there was a statistically significant correlation between degree of splenomegaly and blood counts, this proved to be clinically negligible. Surprisingly, there was also no correlation between degree of splenomegaly and any of symptoms investigated.
Origin of mutation in sporadic cases of haemophilia A
Authors: Ljung Rolf C. R. , Sjörin Elsy
Published: March 2003

The aim of this study was to define the origin of mutation in sporadic cases of severe haemophilia A. The series was composed of 31 families with sporadic severe haemophilia A in the geographical catchment area of the Malmö haemophilia centre. The mutation was characterized in 29/31 families: inversion type 1 (n = 11), inversion type 2 (n = 3), other inversion (n = 1), small or partial deletion (n = 6), insertion (n = 2), non‐sense mutation (n = 4) and mis‐sense mutation (n = 2). Of 29 probands, eight carried a de novo mutation, whereas the proband's mother was found to carry the mutation in 21/29 families. Of the 21 carrier mothers, 16 had de novo mutations (i.e. the proband's maternal grandfather and grandmother were non‐carriers). Owing to the lack of samples from the grandparents, origin could not be determined in the remaining five families. Polymorphisms of the FVIII gene were used to determine whether the de novo mutation of the carrier mother was of paternal or maternal origin. In 15/16 cases the mutation was of paternal origin and in 1/16 cases of maternal origin. In the series as a whole, mutation frequency was 6‐fold higher in males than in females, but no differences in the ratio of sex‐specific mutations rates was found among different types of mutation.
Abnormalities of the p53 gene in juvenile myelomonocytic leukaemia
Authors: Miyauchi Jun , Asada Minoru , Tsunematsu Yukiko , Kaneko Yasuhiko , Kojima Seiji , Mizutani Shuki
Published: March 2003

Juvenile chronic myelomonocytic leukaemia (JMML) is a rare myeloproliferative disorder of childhood. Fewer than 30% of cases of JMML terminate in a blast crisis; however, its molecular mechanism is unknown. Since mutation and/or deletion of the p53 gene has been reported to be associated with disease progression in a wide variety of human cancers, including adult‐type chronic myelogenous leukaemia, we studied the p53 gene in 20 patients with JMML (16 samples in chronic phase and seven at blast crisis). Exons 4–8 of the p53 gene, which cover all the hot spots of point mutations, were amplified by the polymerase chain reaction (PCR) method and subjected to mutation screening by single‐strand conformation polymorphism analysis. No mobility shift of single‐strand DNA of PCR products in polyacrylamide gel electrophoresis, indicating point mutations, was found in 19/20 patients. DNA of the remaining patient in the chronic phase failed to be amplified by PCR and Southern blot analysis with XbaI‐digested genomic DNA revealed a gross rearrangement (presumed deletion) of the p53 gene. These data indicate that abnormalities of the p53 gene are rare in JMML and not responsible for acute transformation, but could be involved in the pathogenesis of some cases of JMML.
Pathological events in platelets of Wiskott‐Aldrich syndrome patients
Authors: Shcherbina Anna , Rosen Fred S. , Remold‐O'Donnell Eileen
Published: March 2003

The Wiskott‐Aldrich syndrome (WAS) is a severe X‐linked platelet/immunological disorder arising from mutations of the gene WASP. At the clinical level, the major platelet abnormalities are small size and low number, both partially correctable by splenectomy. To identify underlying pathological events, we examined WAS platelets at various stages of their lifetime. In spleen sections from WAS patients, fluorescence microscopy showed dramatic co‐localization of markers of platelets (CD41) and macrophages (CD68) compared to non‐thrombocytopenic controls, suggesting that WAS splenic macrophages are involved in platelet removal. Study of isolated WAS blood platelets by flow cytometry showed substantial enhancement of surface exposure of phosphatidylserine (PS), a signal for engulfment by macrophages. Isolated resting WAS platelets were also aberrantly susceptible to microparticle release, and plasma samples of WAS patients contained > 5 times normal numbers of platelet‐derived microparticles which may explain the small size of circulating platelets. Measurements with the Ca2+ sensitive dye fluo‐3 revealed significantly increased Ca2+ levels, 310 ± 13 nmol/l for WAS platelets versus 106 ± 12 nmol/l for normal platelets, and also prolongation of agonist‐induced Ca2+ flux. Cumulatively, these studies identify abnormal events occurring in WAS platelets: increased Ca2+ levels and enhancement of two Ca2+ dependent processes, PS exposure and microparticle release; these abnormal events may contribute to the in vivo decrease of platelet number and reduction of platelet size in this disease.
Purification, amplification and characterization of a population of human erythroid progenitors
Authors: Freyssinier  , Lecoq‐Lafon  , Amsellem  , Picard  , Ducrocq  , Mayeux  , Lacombe  , Fichelson
Published: March 2003

In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum‐free conditions with the cytokine mixture interleukin (IL)‐3/IL‐6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU‐E and 3.4% CFU‐GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2–7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin‐positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood‐derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.
Prognostic evaluation in multiple myeloma: an analysis of the impact of new prognostic factors
Authors: Turesson  , Abildgaard  , Ahlgren  , Dahl  , Holmberg  , Hjorth  , Nielsen  , Odén  , Seidel  , Waage  , Westin  , Wislöff
Published: March 2003

We have analysed the prognostic information for survival of presenting features in an unselected series of 394 myeloma patients. 15 variables with significant prognostic information were identified, among these were some not previously or only recently reported: serum levels of hepatocyte growth factor (HGF), interleukin‐6 (IL‐6), C‐terminal cross‐linked telopeptide of collagen I (ICTP) and soluble interleukin‐6 receptor (sIL‐6R). In a multivariate Cox analysis six variables were significantly and independently associated with poor survival: high age, low W.H.O.‐performance status (PS), high serum levels of calcium, β‐2‐microglobulin (β‐2M), IL‐6 and sIL‐6R. A risk score formed to predict survival for each percentile of the patient population allowed an efficient separation of prognostic groups. The discriminating power of the model compared favourably with three other previously published staging systems applied to the study population. Exclusion of IL‐6 and sIL‐6R from the model only marginally decreased the efficacy of the separation. The predictive value of some variables (sIL‐6R, β‐2M and W.H.O.‐PS) decreased significantly over time. We conclude that formation of a risk score based on independent variables is an efficient way to separate prognostic groups, that the contribution of new and not easily available parameters should be thoroughly evaluated before inclusion in prognostic models for clinical use and that the predictive value of parameters may decrease over time.
Racial background is a determinant of average warfarin dose required to maintain the INR between 2.0 and 3.0
Authors: Blann Andrew , Hewitt Jasvinder , Siddiqui Fouad , Bareford David
Published: March 2003

Warfarin is a commonly used prophylactic agent for the prevention of thromboembolic disease. We hypothesized that racial background influenced warfarin dosage, and tested this by recording the international normalized ratio (INR) in 867 patients aged 40–90 routinely passing through our Anticoagulation Service whose target INR was 2–3. Mean (95% confidence interval) dose was 4.1 (4.0–4.2) mg/d in 737 Caucasians, 5.5 (4.9–6.1) mg/d in 72 Asians, and 6.7 (5.8–7.6) mg/d in 58 Afro‐Caribbeans (P < 0.05) between each group). In a subgroup of 302 (41 Asians, 22 Afro‐Caribbeans, 239 Caucasians), body mass index did not influence warfarin use. Despite small numbers, we conclude that racial background, but not body mass index, is a determinant of warfarin dosage. The reasons for this could be genetic, cultural (diet related), or both.
A novel missense mutation in the human plasmin inhibitor (alpha2‐antiplasmin) gene associated with a bleeding tendency
Authors: Lind Bent , Thorsen Sixtus
Published: March 2003

Heterozygosity for a G → A mutation converting Val384(GTG) to Met(ATG) associated with plasmin inhibitor (alpha2‐antiplasmin) deficiency was identified in three family members with bleeding tendency. The proband had traumatic breast haematoma and per‐/postoperative bleeds. An affected daughter required a blood transfusion after a normal delivery and a son had prolonged bleeding after tooth extraction. The plasma plasmin inhibitor activities of the affected family members were reduced to 49–66% of normal. The antigenic concentrations determined by electroimmunoassay were reduced to 57–68% of normal. Crossed immunoelectrophoresis of plasma from the proband showed a normal pattern. The amino acid Val384 is located eight residues C‐terminal (P8′) of the P1 residue (Arg376) in the reactive site. The P8′ residues of bovine and mouse plasmin inhibitor are both Val. Among other serpins the P8′ residue is unconserved. The mutation was not present in the non‐affected family member or 30 blood donors. In addition to the Val384Met mutation two new polymorphisms Ala‐26(GCG)/Val(GTG) and Arg407(AGG)/Lys(AAG) and one previously reported polymorphism Arg6(CGG)/Trp(TGG) were identified in the plasmin inhibitor gene of the family. The allelic frequencies among 30 blood donors with normal values of plasma plasmin inhibitor (functional) were 0.84/0.16 for C/T in codon −26, 0.81/0.19 for C/T in codon 6 and 0.83/0.17 for G/A in codon 407.