bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence and significance of anticardiolipin, anti‐β2 glycoprotein I and anti‐prothrombin antibodies in chronic hepatitis C
Authors: Leroy Vincent , Arvieux Josiane , Jacob Marie‐Christine , Maynard‐Muet Marianne , Baud Maryline , Zarski Jean‐Pierre
Published: March 2003

Antiphospholipid antibodies have been demonstrated in chronic hepatitis C, but their clinical and pathogenetic significance remains elusive. We prospectively studied 115 patients (85 men, mean age 36.9 years) with chronic hepatitis C without cirrhosis and treated by α‐interferon (α‐IFN). Antiphospholipid determinations comprised anticardiolipin (ACA), anti‐β2‐glycoprotein I and anti‐prothrombin antibodies of the IgG and IgM classes. At entry, 24 patients (21%) were found to possess low to moderate ACA levels (18 IgG, two IgM and four both isotypes) compared with only 4/115 age‐ and sex‐matched control subjects (3.5% P = 0.001). ACA positivity rate increased to 31% (P = 0.01) after a 6‐month course of α‐IFN treatment. In contrast, the prevalence of anti‐β2‐glycoprotein I and anti‐prothrombin antibodies was not significantly different from controls at either time point. The presence of ACA correlated with that of antinuclear antibodies (P = 0.0002), but was not associated with parameters such as histological activity, viral burden and response to α‐IFN, nor with a history of thrombosis or pregnancy loss. However, a non‐significant trend of higher incidence of mild thrombocytopenia among ACA‐positive patients was observed. We conclude that low‐titre ACA positivity is a common finding in patients with chronic hepatitis C, especially following α‐IFN treatment, but does not select a category with different clinical features. These data are in keeping with the absence of associated anti‐β2GPI and anti‐prothrombin antibodies, and do not support a role for HCV infection in the pathogenesis of the antiphospholipid syndrome.
A comparison of artificially‐depleted, lyophilized coumarin and fresh coumarin plasmas in thromboplastin calibration
Authors: Poller  , Van Den Besselaar  , Jespersen  , Tripodi  , Houghton
Published: March 2003

Artificially‐depleted lyophilized plasmas and lyophilized coumarin plasmas were prepared and compared with fresh coumarin plasmas to assess their comparative reliability in local thromboplastin calibration using the manual prothrombin time (PT) technique. Their certified PT values were inserted in turn on the vertical axis in place of the PT obtained with fresh coumarin plasmas. PT results were obtained at eight ECAA national laboratories (‘test centres’) and inserted on the horizontal axis. The resulting thromboplastin calibration slopes were compared with conventional fresh coumarin plasma calibration slopes at the same ‘test centres’. When 60 artificially‐depleted plasmas were substituted for 60 fresh plasmas, the mean calibration slopes with the human plain International Reference Preparation (IRP) were 4.2% higher. For comparison with 20 lyophilized coumarins, three sets of 20 artificially‐depleted plasmas were selected in sequential order from the 60. The lyophilized coumarin plasmas gave a mean deviation of 9.6% from the fresh plasma calibration slopes compared with values of 2.0%, 6.1% and 11.7% for the three sets of 20 depleted plasmas.

Although both types of lyophilized plasma calibration slopes give measurable differences from conventional fresh plasmas, these may be regarded as acceptable in clinical terms.
Gain of chromosome 7 marks the progression from indolent to aggressive follicle centre lymphoma and is a common finding in patients with diffuse large B‐cell lymphoma: a study by FISH
Authors: Bernell  , Jacobsson  , Liliemark  , Hjalmar  , Arvidsson  , Hast
Published: March 2003

Gain of chromosome 7 represents one of the most frequent cytogenetic findings in B‐cell lymphomas with a follicular growth pattern. We used fluorescence in situ hybridization (FISH) and a probe specifying chromosome 7 on lymph node imprints and/or bone marrow (BM) and peripheral blood (PB) smears from six consecutive patients with follicle centre lymphomas (FCLs) grade I or II (low‐grade lymphomas), four patients with FCLs grade III and 11 patients with diffuse large B‐cell lymphomas (DLBCLs) (high‐grade lymphomas). We found gains of chromosome 7 in 14/18 successfully analysed cases (i.e. 2/6 FCLs grade I–II, 3/3 FLCs grade III and in 9/9 DLBCLs) using lymph node imprints. Moreover, the FISH technique demonstrated gains of chromosome 7 in 1/4 BM and 0/4 PB samples from FCLs grade I–II, in 2/4 BM and 2/4 PB specimens from FCLs grade III and in 4/9 BM and 2/9 PB samples from the DLBCLs. In contrast, morphologically recognizable lymphoma cells were seen in only 1/4 BM and 0/4 PB samples from the FCLs grade III and in 1/11 BM and 1/11 PB samples from the DLBCLs. We conclude that: (i) gain of chromosome 7 marks the progression from indolent to aggressive FCL and would appear to be a common finding in patients with FCLs grade III and in DLBCLs, (ii) clonal lymphoid cells occur frequently in BM and PB in high‐grade lymphomas, making traditional staging by cytomorphology uncertain, and (iii) using gains of chromosome 7 as a marker of lymphoma cells, FISH is a useful method to detect minimal residual disease in FCLs grade III and DLBCLs.
Genetic analysis of splenic lymphoma with villous lymphocytes: a Groupe Français d'Hématologie Cellulaire (GFHC) study
Authors: Troussard  , Mauvieux  , Radford‐Weiss  , Rack  , Valensi  , Garand  , Vekemans  , Flandrin  , Macintyre
Published: March 2003

In order to characterize the genetic diversity in splenic lymphoma with villous lymphocytes (SLVL), we have undertaken cytogenetic and molecular analyses of CCND1 expression and BCL1‐IgH PCR rearrangement in 76 cases diagnosed predominantly on morphological criteria. Cytogenetic abnormalities were detected in 19/44 (43%) of cases, including in 16/25 (64%) of cases with an absolute lymphocytosis. Abnormalities included those involving chromosome 14q32 (9/19, 47%), predominantly t(11;14)(q13;q32) (5/19, 26%), chromosome 3 (26%), predominantly 3q, chromosome 17p (26%) and trisomy 12 (3/19, 16%) and were thus suggestive of pathogenetic diversity. CCND1 was expressed in 8/30 (27%) cases, including in all t(11;14) cases, 5/10 (50%) CD5‐positive cases and also in 3/20 (15%) CD5‐negative cases. Three CCND1‐positive SLVL demonstrated immunophenotypic features similar to mantle cell lymphoma (MCL) but the majority differed in their CD5 negativity or CD23 positivity. BCL1‐IgH rearrangement was only seen in 1/62 (2%) of cases overall and in none of the t(11;14) cases, which demonstrated FISH breakpoints both centromeric and telomeric to the BCL1/MTC, suggesting that, if genomic clustering exists in t(11;14) SLVL, it differs from MCL. Although CCND1 expressing SLVL more commonly had marked lymphocytosis, they did not demonstrate a more aggressive clinical course than their negative counterparts, demonstrating that the detection of CCND1 expression or of a t(11;14) should not suffice to alter diagnostic classification in the absence of other criteria.
Inhibitory effect of simvastatin on the proliferation of human myeloid leukaemia cells in severe combined immunodeficient (SCID) mice
Authors: Clutterbuck Robyn D. , Millar Barbara C. , Powles Ray L. , Newman Ann , Catovsky Daniel , Jarman Michael , Millar John L.
Published: March 2003

SCID mice were inoculated intravenously with cells from the human HL60 myeloblastic leukaemia cell line and then treated with the 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, simvastatin, by subcutaneous continuous infusion. The effect of the drug was measured by subsequent colony formation of surviving HL60 cells in vitro and flow cytometry. The number of clonogenic HL60 cells was reduced in the bone marrow of mice that received simvastatin compared with control mice by 65% and 68% in two separate experiments. The number of clonogenic, normal, murine, bone marrow progenitor cells concomitantly exposed to simvastatin in vivo, was not affected in either experiment. Flow cytometric analysis of bone marrow and spleen cells confirmed these results by showing that simvastatin had reduced the percentage of human leukaemia cells in these tissues by 70% and 88% respectively.  The data show that the reported selective effect of simvastatin against acute myeloid leukaemia cells in vitro, can be extended to this in vivo model. HL60 bears an N‐ras mutation. In further in vitro studies, ketoconazole, an inhibitor of cholesterol biosynthesis post farnesyl pyrophosphate synthesis, had a similar effect to simvastatin on HL60 colony development. Furthermore, the clonogenicity of a population of N‐ras mutated, primary acute myeloid leukaemia (AML) cells was no more sensitive to simvastatin than a population without the mutation. The data suggest that the inhibition of AML cell proliferation by simvastatin may be independent of the RAS signalling pathway.
The use of the anti‐malaria drug Fansidar® (pyrimethamine and sulphadoxine) in the treatment of a patient with autoimmune lymphoproliferative syndrome and Fas deficiency
Authors: Van Der Werff Ten Bosch Jutte E. , Demanet Christian , Balduck Nadine , Bakkus Marleen H. C. , DE Raeve Hendrik , Desprechins Brigitte , Otten Jaques , Thielemans Kris
Published: March 2003

Fas is a protein that plays a major role in the apoptotic mechanism of several cell types, including white blood cells (WBC). Mutations of the Fas gene in humans are known to lead to autoimmune lymphoproliferative syndrome (ALPS). Glucocorticoids or cytostatic drugs are sometimes used to treat the lymphoproliferation in these patients. When treated with the anti‐malaria drug Fansidar®, a patient with ALPS showed a marked shrinkage of the lymph node masses, decrease in peripheral blood lymphocytes (PBL) and an increase in neutrophil numbers. In addition, an increased Fas expression was seen on all types of leucocytes.
Dexamethasone plus retinoids decrease IL‐6/IL‐6 receptor and induce apoptosis in myeloma cells
Authors: Smith Mitchell R. , Xie Tao , Joshi Indira , Schilder Russell J.
Published: March 2003

Interleukin 6 (IL‐6) is the most important known growth factor for multiple myeloma, and IL‐6 signalling pathways are potential targets for therapy. We hypothesized that interfering with the IL‐6 signalling pathway at more than one level would be more effective than a single block in inhibiting proliferation of myeloma cells. Accumulating data support the concept that glucocorticoids down‐regulate IL‐6, whereas retinoic acid derivatives (RA) down‐regulate IL‐6R in myeloma. We found that all‐trans RA (ATRA), 13‐cis‐RA and 9‐cis‐RA each similarly inhibited growth of RPMI 8226 myeloma cells and that addition of dexamethasone (DEX) added to RA growth inhibition. The major effects of retinoids were to reduce the proliferative fraction and induce apoptosis whereas DEX increased the apoptotic fraction. When combined, apoptosis was enhanced. Effects of RA + DEX were also least able to be overcome by exogenous IL‐6. RA decreased IL‐6R levels and addition of DEX to RA delayed recovery of IL‐6R levels compared with RA alone. Since RPMI 8226 cells have undetectable IL‐6, we investigated U266B1 cells and found that RA and DEX decreased both IL‐6 secretion and IL‐6 RNA levels. Mechanistically, IL‐6R down‐regulation by RA was enhanced by DEX, whereas IL‐6 protein and RNA levels were reduced by DEX and by RA. In summary, combinations of RA + DEX were not only more effective in inhibiting myeloma cells growth by the dual mechanisms of decreasing proliferative fraction and increasing apoptotic fraction, but were also less able to be overcome by IL‐6.
Modulating the oxygen affinity of human fetal haemoglobin with synthetic allosteric modulators
Authors: Papassotiriou Ioannis , Kister Jean , Griffon Nathalie , Stamoulakatou Alexandra , Abraham Donald J. , Marden Michael C. , Loukopoulos Dimitris , Poyart Claude
Published: March 2003

Improving the delivery of oxygen to the tissues by decreasing the oxygen affinity of haemoglobin has been a major aim of several laboratories over recent years because this may reduce the consequences of anaemia and/or improve tissue oxygenation in cases of decreased blood perfusion. Within the same context, lowering the oxygen affinity may prove valuable in the application of native or recombinant haemoglobin solutions as a blood substitute. The shift of the oxygen equilibrium curve to the right is obtained by various modulators. Among them, the bezafibrate derivatives are considered as a most interesting group. These principles are of the utmost importance in thalassaemia and other haemoglobinopathies where the beneficial effects of the compensatory synthesis of fetal haemoglobin are diminished by the increased oxygen affinity of this pigment. In this paper we present the results of a study initiated to determine whether a potent oxygen affinity modifier, RSR‐4, could satisfactorily decrease the oxygen affinity of fetal haemoglobin, thus improving tissue oxygenation. The experiments were carried out on whole blood and on purified haemoglobin solutions and showed that the effector markedly decreased the oxygen affinity of HbF (from 18.7 to 37.3 mmHg in whole blood). At the same time the cooperativity index (n50) and the oxygen saturation levels remained within normal limits under the conditions of the main experiment. These observations have important implications for the potential application of oxygen affinity modifiers in vivo.
Prolonged but reversible neutrophil dysfunctions differentially sensitive to granulocyte colony‐stimulating factor in children with acute lymphoblastic leukaemia
Authors: Lejeune Marylene , Ferster Alina , Cantinieaux Brigitte , Sariban Eric
Published: March 2003

Treatment of average‐risk acute lymphoblastic leukaemia (ALL) in children consists of 6 months of intensive chemotherapy followed by 18 months of maintenance therapy. Polymorphonuclear leucocyte (PMN) functions from children with ALL were studied in order to evaluate and compare the toxicity of the initial intensive treatment with the toxicity of the subsequent less intensive maintenance treatment. H2O2 and O−2 production, evaluated by chemiluminescence, were significantly decreased during the intensive period but returned to normal values when maintenance therapy began. In contrast, bactericidal activity against Gram‐positive and Gram‐negative micro‐organisms remained at low levels throughout the treatment but returned to normal values in patients off chemotherapy. PMN from patients on maintenance therapy exhibited an excess of morphological changes associated with apoptosis. This was confirmed by standard two‐colour flow cytometry which revealed an increase in the number of hypodiploid cells, and increased expression of membrane phosphatidylserine together with a drastic reduction in the expression of the Fcγ receptor IIIB (CD16). These defective PMN were differentially sensitive to the effects of granulocyte colony stimulating factor (G‐CSF): G‐CSF induced similar increase in chemiluminescence in control and patient PMN; GSF partially corrected the defective bactericidal activity; G‐CSF did not affect the accelerated PMN apoptosis. These observations indicate that ALL children undergoing chemotherapy present PMN defective functions which are partially sensitive or even resistant to G‐CSF.
Paediatric Hodgkin's disease in Spain: association with Epstein‐Barr virus strains carrying latent membrane protein‐1 oncogene deletions and high frequency of dual infections
Authors: SantÓn Almudena , MartĹn Carmen , Manzanal Ana Isabel , Preciado MarĹa Victoria , Bellas Carmen
Published: March 2003

The present report analyses the distribution of 30‐base pair (bp) latent membrane protein‐1 (LMP‐1) oncogene deletions in 24 cases of Epstein‐Barr virus (EBV)‐positive paediatric Hodgkin's disease (HD) and 39 normal controls.

The 30 bp deletion was identified in 19/24 paediatric HD cases (79.2%), of which seven (29.2%) showed the deleted fragment alone, whereas in the remaining 12 (50%) it was accompanied by the nondeleted fragment. Conversely, the deletion was found in 8/22 (36.4%) EBV‐positive healthy children, in two (9.1%) of whom the deleted fragment was alone, and was coinfecting with the nondeleted fragment in the other six (27.3%). The LMP‐1 deletion was significantly associated with paediatric HD, both including dual infections (P = 0.006) or excluding them (P = 0.01).

Type 2 EBV was carried by 25% of HD children, whereas all controls harboured type 1 EBV. The 30 bp deletion was present in all the paediatric HD specimens that contained type 2 EBV, suggesting that a deleted type 2 EBV strain may be more tumourigenic than a nondeleted type 2 EBV strain.

These findings indicate that EBV strains carrying a 30 bp deletion in the third exon of the LMP‐1 oncogene may have a more important role in the pathogenesis of paediatric HD than full‐length EBV strains. Dual infection by LMP‐1 deleted and nondeleted EBV strains is a frequent event both in healthy children and in the paediatric HD population.
Molecular, haematological and clinical studies of a silent β‐gene C → G mutation at 6 bp 3′ to the termination codon (+1480 C → G) in twelve Greek families
Authors: Maragoudaki  , Vrettou  , Kanavakis  , Traeger‐Synodinos  , Metaxotou‐Mavrommati  , Kattamis
Published: March 2003

We report the clinical, haematological, biosynthetic and molecular data of 12 β‐thalassaemia intermedia patients and their heterozygous parents, all of whom carried a rare C → G mutation at nucleotide position 6 3′ to the termination codon (term. cd +6 C → G) in the 3′ untranslated region (3′ UTR) of the β‐globin gene (+1480 C → G). This mutation has been reported previously in a single β‐thalassaemia intermedia patient of Greek origin. The 12 patients of the present study had the clinical phenotype of mild non‐transfusion‐dependent thalassaemia intermedia, preserving haemoglobin levels around 9 g/dl and haemoglobin F levels < 25%. All were compound heterozygotes for the +1480 C → G mutation and common severe β‐thalassaemia mutations. The haematological parameters of heterozygotes with this mutation were within the normal range with the exception of a slightly raised α/non‐α‐globin chain synthesis (1.2–1.9).

mRNA analysis demonstrated a 20–34% reduction in mRNA levels associated with the +1480 C → G mutation compared to normal β‐globin alleles. These findings confirm that the C → G mutation at position 6 3′ to the termination codon is a mild β‐thalassaemia mutation causing slight reduction in β‐globin mRNA levels and β‐globin chain synthesis. It becomes clinically relevant when co‐inherited with a severe β‐thalassaemia mutation in trans.
Analysis of risk factors for over‐anticoagulation in patients receiving long‐term warfarin
Authors: Panneerselvam Sabapathy , Baglin Caroline , Lefort Wendy , Baglin Trevor
Published: March 2003

A cohort of patients with an INR >7.0 were identified prospectively and compared with a group of patients with stable anticoagulant control. During the study 15 100 INR measurements were recorded and 31 (0.2%) were >7.0. Odds ratios of patient characteristics were calculated as an estimate of relative risk for the development of a high INR. The highest risk factor was a target INR of 3.5 (OR 7.3, 95% CI 2.6–20.2). The second highest risk factor was antibiotic therapy in the 4 weeks preceding the high INR (OR 6.2, 95% CI 1.4–27.7). Bleeding was reported more frequently in the high INR group (OR 5.4, 95% CI 2.1–13.9). Five major bleeds occurred in this group compared to none in the stable group. This analysis identifies risk factors for over‐anticoagulation and hence when to intensify monitoring and when to consider pre‐emptive warfarin dose reductions.
Bone marrow transplantation for adults with acute leukaemia and 11q23 chromosomal abnormalities
Authors: Forrest  , Nevill  , Horsman  , Brockington  , Fung  , Toze  , Conneally  , Hogge  , Sutherland  , Nantel  , Shepherd  , Barnett
Published: March 2003

Adults with acute leukaemia and abnormalities of chromosome 11q23 have a poor prognosis when treated with conventional chemotherapy. To determine whether more intensive therapy can improve outcome for patients with this karyotypic finding, a retrospective analysis of all patients with acute leukaemia and 11q23 abnormalities treated at our centre was performed. 12 patients were treated with conventional chemotherapy alone (CC); 20 patients received high‐dose chemo/radiotherapy (HDCT) with autologous (seven patients) or allogeneic (13 patients) bone marrow transplantation (BMT). The treatment‐related mortality was 25% [95% Confidence Interval (CI) 7–69%] for the CC group and 46% (CI 25–73%) for the BMT group (P = 0.69). Cumulative risk of leukaemia progression was 89% (CI 61–100%) in the CC patients and 38% (CI 12–69%) in the BMT patients (P = 0.001). The 2‐year event‐free survival for patients treated with CC was 8% (CI 0–31%) and for patients receiving HDCT and BMT was 34% (CI 14–54%) (P = 0.03). These results confirm that conventional chemotherapy is rarely curative for adults with acute leukaemia and 11q23 abnormalities but that HDCT with BMT can result in long‐term survival in a significant proportion of patients.
Expression of p210 and p190 BCR‐ABL due to alternative splicing in chronic myelogenous leukaemia
Authors: Lichty Brian D. , Keating Armand , Callum Jeannie , Yee Karen , Croxford Ruth , Corpus George , Nwachukwu Bevoline , Kim Peter , Guo Joyce , Kamel‐Reid Suzanne
Published: March 2003

The hallmark of chronic myelogenous leukaemia (CML) is the presence of the Philadelphia chromosome and its resultant fusion message, BCR‐ABL, and fusion protein, p210. Patients with CML in blast crisis, or with Philadelphia positive acute lymphoblastic leukaemia (ALL), can have a smaller BCR‐ABL fusion transcript possessing only the first exon of BCR fused to ABL. This smaller transcript encodes a 190 kD protein which is more strongly transforming than the p210 protein derived from the larger CML‐associated transcript. We performed RT‐PCR on samples from CML patients in chronic phase to determine the frequency and mechanism of p190 and p210 co‐expression and to see if this correlated with clinical indices.

We examined the peripheral blood or marrow of 67 patients with CML and found that 35 of them expressed both transcripts whereas the remainder expressed the p210‐encoding transcript exclusively. Additional PCR products of an intermediate size were also frequently detected and have been isolated and sequenced. Data from two of these products indicate that they are the result of alternative splicing and include variable combinations of BCR exons. We believe that the expression of the p190‐encoding transcript in the chronic phase of CML is also due to alternative splicing. A comparison of patients co‐expressing the p190‐ and p210‐encoding transcripts with those patients who expressed only the p210‐encoding transcript detected significantly higher white blood cell (WBC) counts and blast cell counts at time of testing as well as significantly higher white blood cell counts at diagnosis.
A novel BCR‐ABL fusion gene (e2/1a) in a patient with Philadelphia‐positive chronic myelogenous leukaemia and an aggressive clinical course
Authors: Byrne J. L. , Carter G. I. , Davies J. M. , Haynes A. P. , Russell N. H. , Cross N. C. P.
Published: March 2003

A novel variant BCR‐ABL mRNA transcript was detected by reverse transcription polymerase chain reaction (RT‐PCR) in a patient with Philadelphia positive (Ph+ve) chronic myelogenous leukaemia (CML) who had an aggressive clinical course. Sequence analysis of the amplified cDNA fragment revealed an in‐frame fusion with part of BCR exon e2 joined to part of ABL exon 1a. PCR of genomic DNA from this patient demonstrated that this unusual chimaeric mRNA resulted from breakpoints that fell within each of these exons. This is the first report of breakpoints occurring within both ABL and BCR exons and the first fusion to involve ABL exon 1a.
R411C mutation of the ALAS2 gene encodes a pyridoxine‐responsive enzyme with low activity
Authors: Furuyama Kazumichi , Uno Ritsuko , Urabe Akio , Hayashi Norio , Fujita Hiroyoshi , Kondo Masao , Sassa Shigeru , Yamamoto Masayuki
Published: March 2003

A R411C missense mutation of the erythroid‐specific δ‐aminolaevulinate synthase (ALAS2) gene was identified in a pedigree with X‐linked pyridoxine‐responsive sideroblastic anaemia (XLSA). The normal and the mutant cDNAs were expressed in E. coli, and the enzyme protein was purified. ALAS activity of the mutant enzyme was 12% and 25%, when incubated in the absence and the presence of pyridoxal 5′‐phosphate, respectively, compared with that of the wild‐type enzyme. These findings suggest that the R411C mutation accounts for low ALAS activity and a partial pyridoxine‐responsiveness of the disease in the patient.
Characterization of HIV‐1‐specific antibodies and HIV‐1‐crossreactive antibodies to platelets in HIV‐1‐infected haemophiliac patients
Authors: Chia Wah Kiam , Blanchette Victor , Mody Meera , Wright J. Fraser , Freedman John
Published: March 2003

Sera from HIV‐1‐infected haemophiliacs were examined for human immunodeficiency virus (HIV) specific antibodies and for platelet crossreactive antibodies. Using HIV sepharose 4B affinity columns for serum absorption, antibodies against various HIV antigens, including HIV lysate, HIV‐p24 and HIV‐gp120, were eluted either by low or by high pH buffer. The eluates were examined by ELISA for HIV specificity and by flow cytometry for platelet crossreactivity. Two types of HIV antibodies could be eluted, i.e. acid‐sensitive and alkaline‐sensitive antibodies. HIV antibodies were obtained in 26/29 acid eluates and in 25/29 of the alkaline eluates from HIV‐lysate columns; 96% (25/26) of the acid‐eluted antibodies were HIV‐specific but 48% (12/25) of the alkaline‐eluted antibodies also showed crossreactivity to platelets. Of the 20 alkaline‐eluted HIV‐p24 antibodies, 40% (8/20) reacted specifically with HIV‐p24 and 60% (12/20) were platelet crossreactive. In contrast, of the alkaline‐eluted HIV‐gpl20 antibodies (n=17), 88% (15/17) were HIV gpl20‐specific and only 12% (2/17) were platelet crossreactive. Western blot analysis of platelets demonstrated that the anti‐p24 antibodies recognized three bands with approximate molecular weights of 72 000 to 95 000. 69% of the serum antiplatelet antibodies showed platelet glycoprotein IIbIIIa specificity. Anti‐HIV antibodies could be eluted from platelets. Hence, platelet crossreactive antibodies in HIV infection are primarily alkaline‐sensitive and are associated predominantly with HIV p24 antibody; these antibodies may play a role in the immune thrombocytopenia of HIV‐infected haemophiliacs.
Prothrombotic changes in children with sickle cell disease: relationships to cerebrovascular disease and transfusion
Authors: Liesner  , Mackie  , Cookson  , McDonald  , Chitolie  , Donohoe  , Evans  , Hann  , Machin
Published: March 2003

Vascular occlusion has a central role in the pathophysiology of sickle cell disease (SCD) and, although there is little evidence that thrombosis alone is responsible, patients with sickle cell disease are known to have an ill‐defined but increased thrombotic risk. The most serious complication of this in childhood is stroke which occurs in 7–10% of children and a further 14% have asymptomatic cerebrovascular disease (CVD) on imaging. We have performed a comprehensive profile of coagulation inhibitors and markers of thrombin generation in 96 children (83 non‐transfused [NTx] and 13 transfused [Tx]) with steady‐state SCD and 18 healthy sibling controls. The levels of protein S (free and total) and heparin cofactor II were reduced in both the NTx and Tx groups compared to controls and protein C and APC resistance ratios were reduced in the NTx group only. Antithrombin levels were not different from controls. Thrombin–antithrombin complexes and prothrombin fragment F1+2 were increased in both patient groups. In the NTx subgroups with or without CVD there were no differences for any of the parameters measured except for lower haemoglobin levels and higher white cell counts in those with asymptomatic CVD. We conclude that children with SCD have a reduction in levels of the majority of the coagulation inhibitors and increased thrombin generation in the steady‐state and these are only partially reversed by transfusion. However, these abnormalities do not appear to play a primary role in the development of cerebrovascular disease.
The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) on megakaryopoiesis in patients with aplastic anaemia
Authors: Brereton Michelle L. , Adams Julie A. , Briggs Mark , Liu Yin John A.
Published: March 2003

Recombinant human megakaryocyte growth and development factor (rHuMGDF), a truncated form of the Mpl ligand, stimulates megakaryopoiesis both in vitro and in vivo. We describe the in vitro effect of pegylated recombinant human MGDF (PEGrHuMGDF) alone and in combination with other haemopoietic growth factors (G‐CSF, GM‐CSF, IL3, IL6, erythropoietin, SCF) on megakaryopoiesis in bone marrow from 11 normal subjects and 19 patients with aplastic anaemia (AA). We used semi‐solid cultures to assess megakaryocyte colony growth (CFU‐Mk) and 7 d suspension cultures to assess production of platelet glycoprotein IIIa (CD61) positive cells. CFU‐Mk growth from normal marrow increased 3–4‐fold and CD61+ve cells in suspension culture increased 8–10‐fold with the addition of 10 ng/ml PEGrHuMGDF. In normal subjects growth factor combinations further increased responses in suspension culture, PEGrHuMGDF + SCF, PEGrHuMGDF + IL3 and PEGrHuMGDF + SCF + IL3 + Epo (P < 0.05). IL6, GM‐CSF, G‐CSF or Epo added with PEGrHuMGDF did not consistently give this increase. CFU‐Mk growth from AA marrow remained very low in the presence of PEGrHuMGDF, with or without the addition of other growth factors. CD61+ve cells in suspension culture were, however, increased in the presence of PEGrHuMGDF alone in 12/19 AA cases. Of the 12 patients responsive to PEGrHuMGDF, nine were tested with additional growth factors and further responses were seen in six. In the AA cases PEGrHuMGDF+SCF and PEGrHuMGDF+SCF+IL3+Epo gave the highest responses. These data suggest that PEGrHuMGDF, alone or in combination with SCF and/or IL3, can enhance megakaryocyte proliferation in some patients with aplastic anaemia and may therefore have a role in the treatment of thrombocytopenia in these cases.
Plasma thrombopoietin concentrations in thrombocytopenic and non‐thrombocytopenic patients in a neonatal intensive care unit
Authors: Sola Martha C. , Calhoun Darlene A. , Hutson Alan D. , Christensen Robert D.
Published: March 2003

Thrombocytopenia is a frequent occurrence in the neonatal intensive care unit (NICU), but the role of thrombopoietin (Tpo) in the pathophysiology is unknown. We obtained serial plasma Tpo concentrations in 20 thrombocytopenic neonates in our NICU, and performed bone marrow studies in 15. The initial Tpo levels ranged from undetectable (<41 pg/ml) to 1112 pg/ml and did not correlate with gestational age or platelet count. Neonates with decreased marrow megakaryocytes did not have plasma Tpo levels as high as those reported in adults, particularly in small for gestational age infants (Tpo < 300 pg/ml). In 14/15 neonates followed until resolution, the Tpo concentration decreased as the platelet count increased.
Megakaryocyte precursors, megakaryocytes and platelets express the HIV co‐receptor CXCR4 on their surface: determination of response to stromal‐derived factor‐1 by megakaryocytes and platelets
Authors: Kowalska M. Anna , Ratajczak Janina , Hoxie James , Brass Lawrence F. , Gewirtz Alan , Poncz Mortimer , Ratajczak Mariusz Z.
Published: March 2003

Thrombocytopenia is a late complication of human immunodeficiency virus (HIV) infection. The chemokine receptor CXCR4 has been shown to be a co‐receptor for lymphocyte‐tropic HIV‐1 strains. CXCR4 is also a natural receptor for the chemokine SDF‐1. We have previously shown that CXCR1 and CXCR2 are present on megakaryocytes and platelets. Although interleukin‐8 (IL‐8) and other chemokines that bind to these two receptors do not activate platelets, they are able to inhibit megakaryocytopoiesis, presumably through these receptors. We therefore examined whether CXCR4 is present on developing and mature megakaryocytes and on platelets. Reverse transcription‐polymerase chain reaction (RT‐PCR) demonstrated the presence of CXCR4 message. Immature and mature αIIbβ3+ megakaryocytes, and platelets were also positive for CXCR4 by flow cytometric studies using a CXCR4‐specific antibody. We then tested whether SDF‐1 can affect the biology of these cells. CD34+ cells and immature αIIbβ3+ cells responded to SDF‐1 as indicated by Ca2+ mobilization and chemotaxis. However, mature megakaryocytes failed to demonstrate either of these responses, in spite of their continued ability to bind 125I‐SDF‐1. Further, SDF‐1 failed to inhibit megakaryocyte colony growth. Platelets bound 125I‐SDF‐1 with a KD similar to the affinity seen for CXCR4 on other cells, yet SDF‐1 did not aggregate washed platelets nor augment aggregation by low‐dose ADP or thrombin. SDF‐1 also failed to stimulate Ca2+ mobilization, granular release or expression of P‐selectin in platelets. Accordingly, although our studies demonstrate that CD34+ precursors, megakaryocytes and platelets all express CXCR4 and bind SDF‐1, biological effects were only demonstrable of SDF‐1 on CD34+ precursors. The potential biological implications of CXCR4 expression on maturing megakaryocytes and platelets in normal individuals and following HIV infection are discussed.
Autografting with CD34+ peripheral blood stem cells: retained engraftment capability and reduced tumour cell content
Authors: Voso Maria Teresa , Hohaus Stefan , Moos Marion , Pförsich Margit , Cremer Friedrich W. , Schlenk Richard F. , Martin Simona , Hegenbart Ute , Goldschmidt Hartmut , Haas Rainer
Published: March 2003

The efficacy of an immunomagnetic purging method and the Isolex 300 devices were assessed for selecting CD34+ cells from leukapheresis products of 29 patients with non‐Hodgkin's lymphoma (NHL), 39 with multiple myeloma and 34 with breast cancer. The mean purity of the CD34+ cell population was 93.6% and the mean recovery was 67.7%. Following enzymatic cleavage by chymopapain the expression of Thy‐1 and Leu‐8 was significantly reduced without affecting haematological recovery. The population of selected CD34+ cells of 4/8 patients with follicular lymphoma became PCR‐negative. A 2.5 log reduction of tumour cells could be achieved in four patients with multiple myeloma as shown by a quantitative PCR assay. There were no tumour cells detectable in any of the 19 CD34+ cell preparations of patients with breast cancer. In 64 patients who received 94 cycles of high‐dose therapy, a mean number of 4.7 × 106 CD34+ cells/kg were autografted. The time needed for platelet reconstitution was different when a comparison was made with 156 patients, who had received unmanipulated leukapheresis products (10 v 12 d, P = 0.006). No significant differences with regard to neutrophil recovery were noted. Five patients had a graft failure. Two of them died (on day 78 and 88 following PBSCT), and three patients were rescued with unmanipulated back‐up transplants. In conclusion, the immunomagnetic selection of CD34+ cells provides autografts with reduced tumour cell content and an engraftment ability similar to that of unmanipulated autografts.
Tumour necrosis factor‐α cytokine promoter gene polymorphism in Hodgkin's disease and chronic lymphocytic leukaemia
Authors: Wihlborg Christel , Sjöberg Jan , Intaglietta Monica , Axdorph Ulla , Pisa Eva K. , Pisa Pavel
Published: March 2003

Regulation of cytokine levels has been shown to be under genetic control through the coding and promoter sequences of genetic polymorphisms. We elucidated the prevalence of a previously described G to A transition polymorphism at position −308 of the tumour necrosis factor‐α (TNF‐α) promoter region in a population of patients with Hodgkin's disease (HD) (n = 36) and chronic lymphocytic leukaemia (CLL) (n = 49) and healthy volunteers (n = 51). The DNA fragment containing this polymorphism was amplified by PCR and sequenced by solid‐phase minisequencing. The frequency of the TNF‐α promoter polymorphism was not significantly different between CLL patients and HD patients compared to controls.
Abnormal cytogenetic clones in patients with aplastic anaemia: response to immunosuppressive therapy
Authors: Geary C. G. , Harrison C. J. , Philpott N. J. , Hows J. M. , Gordon‐Smith E. C. , Marsh J. C. W.
Published: March 2003

We report the response to immunosuppressive therapy with antithymocyte globulin (ATG) and cyclosporin or oxymetholone in 13 cases of aplastic anaemia (AA) with an abnormal cytogenetic clone detected at or sometime after diagnosis. Blood and bone marrow examination showed no distinctive morphological features of myelodysplasia (MDS) at diagnosis. Haematological response occurred promptly in eight cases; the remainder responded after additional immunosuppression with or without oxymetholone. Three patients had a late relapse of AA, treated successfully by allogeneic bone marrow transplantation in one; the others responded to oxymetholone. Transformation to MDS or acute leukaemia was not observed after a median follow‐up of 4.1 years (range 1.2–11.2). In four patients the cytogenetic clone disappeared after treatment.
A variant of hereditary stomatocytosis with marked pseudohyperkalaemia
Authors: Coles Suzanne E. , Ho Mei M. , Chetty Margaret C. , Nicolaou Anna , Stewart Gordon W.
Published: March 2003

A family with an unusual form of hereditary stomatocytosis is described. The affected members showed a mild, dominantly‐inherited, haemolytic anaemia with intracellular Na and K levels of 41–48 and 44–53 mmol/(l cells) respectively. This anaemia was associated with marked ‘pseudohyperkalaemia’: that is, loss of K from red cells on storage at room temperature. At 37°C, ‘leak’ tracer flux rates (assessed as the ouabain + bumetanide‐resistant K fluxes) showed a roughly 5‐fold acceleration compared to normal, and an abnormal temperature dependence with a shallow slope between 37 and 20°C (mean Q10 (ratio of reaction rates at temperature T and T − 10) over this interval, 1.6; normal 2.2). The pseudohyperkalaemia could be attributed to the disparity between pump and leak at 20°C. This is an identical mechanism to that previously shown for the haematologically trivial condition, ‘familial pseudohyperkalaemia’. No protein or lipid abnormality was found in the membrane of these cells.
Efficient inhibition of macrophage TNF‐α production upon targeted delivery of K48R ubiquitin
Authors: ANTONELLI ANTONELLA , CRINELLI RITA , BIANCHI MARZIA , CERASI AURORA , GENTILINI LUCIA , SERAFINI GIORDANO , MAGNANI MAURO
Published: March 2003

K48R ubiquitin (K48R‐Ub) is an analogue of native ubiquitin that does not form polyubiquitin chain conjugates. Targeted delivery of this recombinant mutant ubiquitin to human macrophages results in an intracellular increase in the ubiquitin analogue. IkBα polyubiquitination and degradation were significantly inhibited in K48R‐Ub targeted macrophages upon stimulation with lipopolysaccharide. The ability to reduce IkBα degradation was also associated with a reduced production of TNF‐α, the gene of which is under NF‐kB control. At a concentration of 0.1 μM, dexamethasone was less effective than K48R‐Ub in preventing IkBα depletion and TNF‐α release. These data suggest that ubiquitin analogues are potent suppressors of TNF‐α release in macrophages.
Identification of the inverted chromosome 16 using chromosome painting
Authors: RAO P. NAGESH , FLEJTER WENDY L. , RAHI SONYA , LI HONG , PETTENATI MARK J.
Published: March 2003

The inverted chromosome 16 is commonly associated with acute myelomonocytic leukaemia (AML) M4 with bone marrow eosinophilia. Cytogenetic identification of the inverted chromosome 16 can be difficult. To help identify the inversion in bone marrow samples from patients referred for the diagnosis of AML‐M4, we applied the molecular cytogenetic technique of chromosome painting using chromosome 16 p‐arm paint. The results were concordant with standard chromosome analyses and clearly allowed for the identification of a pericentric inversion within chromosome 16 even in poor‐quality metaphase spreads.
A prospective molecular study of chimaerism in patients with haematological malignancies receiving unrelated cord blood or bone marrow transplants: detection of mixed chimaerism predicts graft failure with or without early autologous reconstitution in cord blood recipients
Authors: Cimino Giuseppe , Rapanotti Maria Cristina , Elia Loredana , Iori Anna Paola , Guglielmi Cesare , Screnci Maria , Carmini Daniela , De Felice Lidia , Moleti Maria Luisa , Mengarelli Andrea , Mandelli Franco , Arcese William
Published: March 2003

We prospectively studied the chimaerism status in the bone marrow (BM) and peripheral blood (PB) of 23 patients receiving umbilical cord (UCB, 14 cases) or BM (nine cases) transplants from unrelated donors by PCR amplification of four individual‐specific VNTR genetic loci. Haematological engraftment, with persistent full donor pattern, was observed in 10/14 (72%) patients receiving UCB and in 9/9 (100%) patients transplanted with marrow from an unrelated donor (MUD). In contrast, the remaining four patients converted to an autologous pattern. Three out of these four patients had an early autologous haematological reconstitution reaching a neutrophil level >0.5 × 109/l at days 27, 33 and 37 after transplant, respectively. In all three of these patients, chimaerism analysis demonstrated an early appearance of donor cells (i.e. within 35 d after UCB transplant) showing a transient full donor (one case) or mixed chimaerism condition (two cases). Despite the early autologous haemopoietic reconstitution, one of the three patients died of GVHD at day 60, which was explained by the demonstration of low levels of donor lymphoid cells. In the MUD group all nine patients converted to a persistent full donor pattern with haematological reconstitution, accompanied in two of them by transient mixed chimaerism lasting to days 60 and 270 after transplant. Our data show that monitoring of chimaerism may predict graft failure with or without early autologous haemopoietic reconstitution in patients receiving unrelated UCB transplants. Furthermore, chimaerism analysis may identify, in patients with autologous reconstitution, those at risk of severe GVHD in whom immunosuppressive therapy should not be discontinued.
Studies of platelet volume, chemistry and function in patients with essential thrombocythaemia treated with Anagrelide
Authors: Bellucci S. , Legrand C. , Boval B. , Drouet L. , Caen J.
Published: March 2003

Anagrelide (imidazoquinazolin derivative) is a new compound proposed for the treatment of myeloproliferative disorders. In this study, Anagrelide was given to patients with essential thrombocythaemia (ET) in a compassionate‐use protocol. The aim of this study was to test the effect of this drug not only on the platelet count but also on platelet volume, chemistry and function, which has not previously been reported. Thus, in ET, different functional or structural platelet abnormalities were reported: a shortening of the bleeding time, hypoaggregation to several agonists, and in particular a lack of response to adrenalin, an increase in the amount of total platelet glycoprotein IV (or CD36), and an abnormal migration of thrombospondin on electrophoresis. These different parameters were studied before and during therapy with Anagrelide. Although the platelet count was corrected, no functional or chemical abnormality was improved. Furthermore, platelet volume was shown to be constantly increased under Anagrelide. Thus, Anagrelide, in reducing the platelet count, may possibly decrease the risk of thrombosis and haemorrhage. Nevertheless, if the risk of thromboses and/or myelofibrosis is related not only to the platelet count but also to the platelet abnormalities, the persistence of a thrombocytopathy in patients treated with Anagrelide must be taken in consideration. Our data suggest that thromboses and myelofibrosis are clinical end‐points which should be included in future large‐scale use of Anagrelide.
Antibody‐mediated inhibition of the human alloimmune response to platelet transfusion in Hu‐PBL‐SCID mice
Authors: Crow Andrew R. , Freedman John , Hannach Barbara , Blanchette Victor , Lazarus Alan H.
Published: March 2003

Severe combined immune deficient (SCID) mice were engrafted with human (Hu) peripheral blood lymphocytes (PBL) from a previously alloimmunized donor and transfused with HLA‐mismatched platelets. We have previously shown this to be a useful model for platelet transfusion. These engrafted mice (Hu‐PBL‐SCID mice) produced high levels of alloantibody in response to standard platelet preparations. However, when the first platelet challenge was presensitized with anti‐HLA antibody and then transfused there was a marked reduction in the amount of alloantibody produced to five subsequent untreated platelet transfusions. Platelets pretreated with platelet‐specific anti‐HPA‐1a (PLA1) sera did not induce a decrease in the anti‐HLA alloantibody response. This demonstrated that platelet‐induced HLA alloimmunization can be blocked by anti‐HLA antibody‐sensitized cells.
Selective modulation of the cyclin B/CDK1 and cyclin D/CDK4 complexes during in vitro human megakaryocyte development
Authors: Bassini Alessandra , Pierpaoli Sabina , Falcieri Elisabetta , Vitale Marco , Guidotti Lia , Capitani Silvano , Zauli Giorgio
Published: March 2003

Mammalian megakaryocyte development is characterized by a progressive accumulation of cells exhibiting a polylobated nucleus with a polyploid DNA content. In this study human megakaryocytes were obtained from CD34+ haemopoietic progenitors by in vitro liquid culture in the presence of 100 ng/ml of recombinant thrombopoietin (TPO). Ultrastructural examination of polyploid megakaryocytes showed the presence of a large number of centrioles, the breakdown of the nuclear envelope, and the progressive chromatin condensation, all aspects characteristic of mitosis. At both indirect immunofluorescence and Western blot analyses, cyclin B and its related cyclin‐dependent kinase (CDK)1, which forms the mitosis promoting factor (MPF), showed an increased expression in maturating megakaryoblasts and megakaryocytes (day 8 of culture) with respect to freshly isolated CD34+ progenitors. This expression tended to decline in fully developed megakaryocytes (day 15 of culture). The amount of cyclin D and of the related CDK4, governing the G1 phase of the cell cycle, increased during megakaryocyte development, maintaining high levels of expression also in mature megakaryocytes. These results indicate that megakaryocyte polyploidization depends on a true, although incomplete, mitotic process, and that cyclin D/CDK4 probably plays a crucial role throughout megakaryocytopoiesis.