bluuebunny/crossref_metadata_2025 huggingface dataset:
A study of Protein S antigen levels in 3788 healthy volunteers: influence of age, sex and hormone use, and estimate for prevalence of deficiency state
Authors: C. Dykes Anne , Walker Isobel D. , McMahon Alex D. , Islam S. I. A. M. , Tait R. C.
Published: March 2003

Total Protein S (tPS) and free Protein S (fPS) antigen levels were measured in 3788 healthy blood donors. Men had higher levels of both parameters than women (P < 0·001). Age had no effect on tPS in men, although there was a slight reduction in fPS levels with increasing age. In women increasing age was associated with a significant increase in tPS levels (P < 0·001) but had no effect on fPS after adjustment for menopausal state. Oral contraceptive pill (OCP) use significantly lowered tPS but had no effect on fPS. In post‐menopausal women, hormone replacement therapy (HRT) use had no statistically significant effect on either tPS or fPS. Donors with tPS or fPS levels in the lowest percentile (n = 56) were retested; only nine with repeat low levels were identified, eight of whom had persistently low levels over a 4–7‐year follow‐up. Acquired deficiency was excluded. When possible, family studies were performed, leading to an estimate of prevalence of familial PS deficiency of between 0·03% and 0·13% in the general population.
Aberrant expression of caspase cascade regulatory genes in adult T‐cell leukaemia: survivin is an important determinant for prognosis
Authors: Kamihira Shimeru , Yamada Yasuaki , Hirakata Yoichi , Tomonaga Masao , Sugahara Kazuyuki , Hayashi Tomoni , Dateki Natsuko , Harasawa Hitomi , Nakayama Katsushi
Published: March 2003

Derangement of either apoptosis or cell division is known to play an important role in tumorigenesis. Fas‐mediated apoptosis on normal and leukaemic T cells is finely tuned by inhibitory proteins, such as FAP‐1, FLIP and survivin, and defective caspase isoform which can attenuate the function of its intact caspase as a decoy molecule. However, complex involvement of such inhibitors in tumour biology relating to apoptotic pathology remains unclear in the neoplasms. We report the aberrant expression of FAP‐1, FLIP and survivin mRNAs on leukaemic T cells from adult T‐cell leukaemia (ATL) patients. Among these inhibitors, only survivin was aberrantly expressed in all ATL cases, but not in any normal peripheral blood mononuclear cells (PBMCs). Furthermore, survivin mRNA expression level was characteristic in each subtype of ATL and represented an important determinant for ATL prognosis. However, the apoptotic effector of casp‐8, which is essential in Fas‐mediated signal transduction, was dominant in defective casp‐8 rather than intact casp‐8 in ATL cells, suggesting a favourable biological situation for escape from apoptosis. Taken together, ATL cells probably possess many different regulatory mechanisms in order to attenuate Fas‐mediated signalling and subsequently expand their populations under escape from apoptosis. Among these inhibitors, survivin is a useful bio‐marker to assess tumour biology and may be a potential new target for apoptosis‐based selective therapy in neoplasms as the expression is a general feature of neoplasia, but not normal tissues.
Growth and immortalization of human myeloma cells in immunodeficient severe combined immunodeficiency mice: a preclinical model
Authors: Rème Thierry , Gueydon Elisabeth , Jacquet Chantal , Klein Bernard , Brochier Jean
Published: March 2003

Human multiple myeloma (MM) purified tumour cells readily undergo apoptosis in vitro. Interleukin 6 (IL‐6), a main growth factor of tumour cells, has enabled the development of IL‐6‐dependent MM cell lines. Recently, we developed anti‐gp130 monoclonal antibodies (mAbs), two of which (B1 + I2) were able to dimerize gp130 and replace IL‐6 in vitro. We show here that the injection of B1 + I2 IL‐6 agonistic mAbs via the inguinal subcutaneous (SC) route efficiently produced tumours in severe combined immunodeficiency (SCID) mice grafted with IL‐6‐dependent myeloma cell lines compared with either the intraperitoneal (IP) or abdominal surgical bursa (SB) routes. The SC tumour graft, together with Matrigel and vascular endothelial growth factor (VEGF), leads to a strong vascularization and early detection of serum human immunoglobulins (huIgs). SCID mice treated with B1 + I2 mAbs were injected with fresh MM cells from five patients, four of whom had consistent levels of huIgs, and tumour growth was present in two. For one patient, tumour plasma cells that were passed several times subcutaneously in new SCID mice, still expressed their initial markers after several months. They remained unable to grow in vitro in the presence of B1 + I2 or IL‐6. The nature of the SCID factors involved and the triggered genes are under investigation.
Cloning of the Ly‐6A (Sca‐1) gene locus and identification of a 3′ distal fragment responsible for high‐level γ‐interferon‐induced expression in vitro
Authors: Ma Xiaoqian , Ling Kam‐Wing , Dzierzak Elaine
Published: March 2003

The Ly‐6A and Ly‐6E allelic genes encode the Sca‐1 protein, which is one of the most widely used markers in haematopoietic stem cell isolation procedures. Identification of the specific gene regulatory elements that direct haematopoietic stem cell specific expression of Sca‐1 is of current interest for purposes of stem cell manipulation. Both the Ly‐6E and Ly‐6A alleles have been examined for regions containing DNase I hypersensitive sites thought to be indicative of transcriptional regulatory elements. In these previous studies, the Ly‐6E allele with its flanking regulatory sequences was cloned, and the region responsible for high‐level γ‐interferon (γ‐IFN)‐induced expression was localized to a 3′ distal sequence containing two strong DNase1 hypersensitive sites. Because the Ly‐6A allele is thought to provide higher levels of expression in haematopoietic stem cells, we isolated over 25 kb of the Ly‐6A gene and flanking regulatory regions. We show here that sequences analogous to those in the Ly‐6E allele are responsible for high‐level γ‐IFN‐induced expression in vitro. Furthermore, we show that this 3′ distal Ly‐6A fragment directs high‐level γ‐IFN‐induced expression from a heterologous promoter, suggesting that it is a potent enhancer that could be useful for expression in haematopoietic stem cells.
Activation of natural killer cells with interleukin 2 (IL‐2) and IL‐12 increases perforin binding and subsequent lysis of tumour cells
Authors: Lehmann Christof , Zeis Matthias , Uharek Lutz
Published: March 2003

Natural killer (NK) cells can lyse a variety of different tumour cells by exocytosis of perforin, subsequent binding of perforin to the target cell membrane and formation of lytic pores. Some tumour cells, however, are resistant to cellular cytotoxicity. Using the NK‐resistant tumour cell lines ML‐2, MONOMAC‐1, RPMI and L540Cy, we demonstrated that activation of NK cells with interleukin 2 (IL‐2) and IL‐12 resulted in significant lysis of these tumour targets. To investigate the underlying mechanisms, we isolated the cytotoxic granules from non‐activated and IL‐2‐/IL‐12‐activated NK cells and compared the killing of K562 leukaemia cells (sensitive to NK cell‐mediated lysis) and ML‐2 leukaemia cells (resistant to NK cell‐mediated lysis). In contrast to K562 cells, which were easily killed by NK‐cell granules, ML‐2 cells were resistant to granules from non‐activated NK cells. However, granules from NK cells activated with IL‐2 and IL‐12 were able to induce significant tumour cell lysis. Cell death of both K562 and ML‐2 cells by granules from activated NK cells was completely blocked by anti‐perforin antibodies, indicating that perforin mainly accounts for the lysis induced by NK granules. Comparing granules from non‐activated and IL‐2‐/IL‐12‐activated NK cells, the increased cell death of ML‐2 cells was caused by an improved binding of perforin to the target cell membrane. Functional assays, however, indicated that the differences in perforin binding were not as a result of an augmented production of perforin by activated NK cells. We conclude that activation of NK cells results in an increased binding of perforin and subsequent lysis of tumour cells.
X‐linked cerebellar ataxia and sideroblastic anaemia associated with a missense mutation in the ABC7 gene predicting V411L
Authors: Maguire Andrew , Hellier Kate , Hammans Simon , May Alison
Published: March 2003

Two brothers with X‐linked ataxia (XLA) were found to have hypochromic red cells and increased erythrocyte protoporphyrin despite normal iron stores. The mother was unaffected by ataxia and had normal iron stores but showed evidence of some red cell hypochromia with heavy basophilic stippling that stained positive for iron. Bone marrow biopsy confirmed the presence of ring sideroblasts in one of the brothers. The absence of mutations in the ALAS2 gene and the predominance of zinc over free protoporphyrin led to a search using a combination of DNA and cDNA analysis for the presence of mutations in the ABC7 gene. ABC7 encodes a mitochondrial half‐type ATP Binding Cassette transporter involved in iron homeostasis. The published cDNA sequence was used to search databases for the genomic sequence of which 12 exons spanning 23·4 kb were mapped leaving the most 5′ nucleotides unaccounted for. The identified exons and their exon–intron boundaries were amplified from DNA while the most 5′ sequence including the initiation codon was amplified from cDNA of peripheral blood cells. Direct sequencing revealed hemizygosity in the brothers and heterozygosity in the mother for a G→C transversion at position 1299 of the published cDNA. This predicts a V411L substitution at the beginning of the last of six putative transmembrane regions of the protein. Restriction enzyme digestion confirmed the presence of this mutation in the three family members but could not detect it in 200 normal alleles. An uncle affected by ataxia also carried this mutation. This study supports the recently hypothesized involvement of the ABC7 gene in XLSA/A and highlights a protein structure region of importance to this syndrome.
Unrelated cord blood transplantation for adult patients with myelodysplastic syndrome‐related secondary acute myeloid leukaemia
Authors: Ooi Jun , Iseki Tohru , Nagayama Hitomi , Tomonari Akira , Ito Kiyoshi , Shirafuji Naoki , Tojo Arinobu , Tani Kenzaburo , Asano Shigetaka
Published: March 2003

Seven adult patients with myelodysplastic syndrome (MDS)‐related secondary acute myeloid leukaemia (AML) were treated with total body irradiation (TBI), cytosine arabinoside (Ara‐C) and cyclophosphamide (CY), followed by unrelated human leucocyte antigen (HLA)‐mismatched cord blood transplantation (CBT). Granulocyte colony‐stimulating factor (G‐CSF) was infused continuously from 12 h before until the end of Ara‐C therapy to enhance the antileukaemia effect of Ara‐C. Five patients are alive and free of disease at 7–31 months after transplantation. These preliminary results suggest that adult MDS‐related secondary AML patients without suitable related or unrelated bone marrow donors should be considered as candidates for CBT.
Purification of cytomegalovirus‐specific CD8 T cells from peripheral blood using HLA–peptide tetramers
Authors: Keenan R. D. , Ainsworth J. , Khan N. , Bruton R. , Cobbold M. , Assenmacher M. , Milligan D. W. , Moss P. A. H.
Published: March 2003

Cytomegalovirus (CMV) reactivation and disease remains an important clinical problem for patients after allogeneic stem cell transplantation. Impaired cellular immune control of viral replication is responsible for viral reactivation, and transfer of CMV‐specific T cells from transplant donors can be effective in providing protection. Recent reports have indicated that the frequency of CMV‐specific CD8+ T cells in the peripheral blood of healthy donors is surprisingly high. Here we demonstrate that by using a combination of human leucocyte antigen (HLA) Class I‐peptide tetramers and magnetic selection it is possible to select CMV‐specific T cells from CMV antibody‐positive individuals to high purity. Reliable purification of CMV‐specific T cells up to 99·8% of CD8+ cells was possible within hours, even when starting with a precursor frequency of < 0·1% of peripheral blood CD8+ T cells. CMV‐specific T cells remained functional after the selection process. This novel form of antigen‐specific T‐cell selection should facilitate the selection of T cells for cellular immunotherapy to treat or prevent CMV disease after transplantation. In addition, this technique could potentially be applied to many antigens including against other infective agents and tumour‐specific antigens.
Semiquantitative reverse transcription polymerase chain reaction analysis for detection of bcr/abl rearrangement using RNA extracts from bone marrow aspirates compared with glass slide smears after 0, 2 and 4 d of storage
Authors: Schoch Robert , Pitako Jurij , Schafhausen Philippe , Jenisch Stefan , Haferlach Torsten , Kneba Michael , Suttorp Meinolf
Published: March 2003

The polymerase chain reaction (PCR) is an established tool for the detection of specific chromosomal aberrations in different haematological malignancies. Owing to fast degradation of RNA, the immediate processing of samples is thought to have a major influence on the reliability of results. Any delay caused by transport may be an obstacle to reverse transcription PCR (RT–PCR)‐based methods in multicentre studies. However, as air‐dried bone marrow smears are usually available, we have improved a method to use smears as a source for routine RT–PCR investigations. We studied whether this source of RNA could overcome problems caused by delayed transport of samples. The aim of the present study was (i) to investigate the influence of a storage period of up to 4 d before processing of a specimen by nested bcr/abl RT–PCR, and (ii) to compare bone marrow aspirates with bone marrow smears stored at room temperature in parallel. Bone marrow aspirates and smears were taken from 11 patients with Ph‐positive chronic myeloid leukaemia (CML). PCR results were semiquantified using a limiting dilution assay. We observed a loss of sensitivity < 1 log in stored bone marrow aspirates, even after 96 h. Results obtained from air‐dried unstained glass slide smears were similar to investigations performed on approximately 1 × 105 cells of a bone marrow aspirate. We conclude that a storage period of up to 96 h has little influence on the detection of a bcr/abl fusion transcript in CML at diagnosis. Glass slide smears were equivalent to bone marrow aspirates in 8 out of 11 cases as a source for RT–PCR analysis when nested PCR was performed.
Assessment of antimalarial effect of ICL670A on in vitro cultures of Plasmodium falciparum
Authors: Goudeau Carmen , Loyevsky Mark , Kassim Olakunle O. , Gordeuk Victor R. , Nick Hanspeter
Published: March 2003

We tested in vitro the antimalarial properties of ICL670A, a newly developed iron chelator for the long‐term oral treatment of iron overload. Ring‐stage synchronized cultures of Plasmodium falciparum cultured in human erythrocytes were exposed to different concentrations of ICL670A and the conventional iron chelator, desferrioxamine B (DFO), for 48 h. Malarial growth was measured by incorporation of [3H]‐hypoxanthine. ICL670A at 30 µmol/l had marked antimalarial activity that was observable by 6 h after beginning the exposure of ring‐stage parasites to the agent. Over 48 h of culture, malarial growth was significantly lower with ICL670A than with DFO at concentrations of both 30 µmol/l (P = 0·008) and 60 µmol/l (P = 0·001). At 48 h, growth relative to control was 53% with ICL670A and 83% with DFO at concentrations of 30 µmol/l, and 20% with ICL670A and 26% with DFO at concentrations of 60 µmol/l. Standard 50% inhibitory concentrations (IC50s) were similar for ICL670A and DFO. Precomplexation with iron completely abolished the inhibitory effect of ICL670A, indicating that this new agent, like DFO, probably inhibits parasite growth via deprivation of iron from critical targets within the parasite. Further studies to address the question of the antimalarial potential of ICL670A in combination with classic antimalarials would be of interest.
High expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood acute lymphoblastic leukaemia
Authors: Crazzolara Roman , Kreczy Alfons , Mann Georg , Heitger Andreas , Eibl Günther , Fink Franz‐Martin , Möhle Robert , Meister Bernhard
Published: March 2003

Childhood acute lymphoblastic leukaemia (ALL) is a malignancy with the potential to infiltrate the liver, spleen, lymph nodes and brain. Such extramedullary presentation is important for understanding the biology of childhood ALL and also for developing new prognostic parameters. A potential mechanism in the trafficking of leukaemia cells is the interaction of the chemokine receptor CXCR4, which is expressed on ALL cells, and its ligand stromal cell‐derived factor‐1 (SDF‐1), produced by stromal cells in bone marrow and extramedullary organs. Functionality of CXCR4 was demonstrated by a high correlation between cell surface density of CXCR4 and transendothelial migration of leukaemia blasts towards a gradient of SDF‐1 (r = 0·73, P = 0·001). Inhibition of SDF‐1‐induced migration by an anti‐CXCR4 monoclonal antibody (78·33 ± 23·86% inhibition) evidenced the specificity of CXCR4 to SDF‐1. In order to evaluate clinical significance of CXCR4 expression, lymphoblasts from the bone marrow of 73 patients with and without extramedullary organ infiltration were compared. Multiparameter flow cytometry revealed that lymphoblasts from patients with high extramedullary organ infiltration, defined as ultrasonographically measured enlargement of liver or spleen, expressed the CXCR4 receptor at higher fluorescence intensity (median 66·12 ± 66·17) than patients without extramedullary organ infiltration (median 17·56 ± 19·29; P < 0·001). Consequently, high expression of CXCR4 was strongly predictive for extramedullary organ involvement, independently of the peripheral lymphoblast count. Highest CXCR4 expression was seen in mature B ALL (median 102·74 ± 92·13; P < 0·003), a disease characterized by a high incidence of extramedullary bulky disease. As high expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood ALL, we suggest that CXCR4 and its ligand play an essential role in extramedullary invasion.
The −1185 A/G and −1051 G/A dimorphisms in the von Willebrand factor gene promoter and risk of myocardial infarction
Authors: Di Bitondo Rosa , Cameron Cherie L. , Daly Martina E. , Croft Stuart A. , Steeds Rick P. , Channer Kevin S. , Samani Nilesh J. , Lillicrap David , Winship Peter R.
Published: March 2003

Elevated plasma von Willebrand factor (VWF) levels are associated with coronary artery disease, although the precise mechanism for this is unclear. Recently, four linked dimorphisms in the VWF gene promoter were demonstrated to influence plasma VWF level. We conducted a case–control study of 525 acute myocardial infarction (MI) cases and 451 control subjects, all aged  75 years, to assess the potential contribution of two of these dimorphisms (−1185 G/A and −1051 A/G) to the risk of MI. The frequency of the −1185A/−1051G haplotype, associated with elevated VWF levels, was similar in the case and control groups, yielding a haplotypic odds ratio for MI of 0·93 (95% CI 0·77, 1·12, P = 0·43), and there was no significant association between the −1185A/−1051G haplotype and the risk of MI in any subgroup analysed. We therefore conclude that possession of the −1185A/−1051G haplotype does not confer an increased risk for MI.
Laboratory control of oral anticoagulant treatment by the INR system in patients with the antiphospholipid syndrome and lupus anticoagulant. Results of a collaborative study involving nine commercial thromboplastins
Authors: Tripodi Armando , Chantarangkul Veena , Clerici Marigrazia , Negri Barbara , Galli Monica , Mannucci Pier Mannuccio
Published: March 2003

Because of the variable responsiveness of thromboplastins to lupus anticoagulants (LA), concerns have been raised about the validity of the prothrombin time–International Normalized Ratio (PT–INR) in monitoring oral anticoagulant treatment in patients with the antiphospholipid syndrome (APS) and LA. To date, few studies have been performed, numbers of patients investigated are relatively small and results are conflicting. We report on a multicentre study organized to investigate further this clinically relevant issue. Each of nine thrombosis centres was asked to collect plasma samples from patients with APS who were on oral anticoagulants (cases) and patients without APS who were on oral anticoagulants (controls). Nine thromboplastins (three human recombinant, one from human placenta and five from rabbit brain) were calibrated at the co‐ordinating centre according to World Health Organization guidelines. Measurements of the INR and factor X amidolytic activity for all frozen plasmas were performed centrally. The numbers of patients investigated were 58 cases and 57 controls. Between‐reagent variability of the INR was higher in cases [coefficient of variation (CV) = 12·4%] than in controls (CV = 6·7%), but this was because of one of the thromboplastins only (Thromborel R, human recombinant), which measured considerably higher INR values than the others in cases but not in controls. In conclusion, our data indicate that LA interference on the PT–INR measured with the majority of commercial thromboplastins is not enough to cause concern if insensitive thromboplastins, properly calibrated to assign them an instrument‐specific International Sensitivity Index are used. New thromboplastins, especially those made of relipidated tissue factor, should be checked for their responsiveness to LA before they are used to monitor oral anticoagulant treatment in patients with APS.
Early elevation of serum thrombopoietin levels and subsequent thrombocytosis in healthy preterm infants
Authors: Matsubara Kousaku , Baba Kunizo , Nigami Hiroyuki , Harigaya Hidekazu , Ishiguro Akira , Kato Takashi , Miyazaki Hiroshi
Published: March 2003

To verify pathophysiological mechanisms underlying thrombocytosis in low‐birth‐weight (LBW) preterm babies, we evaluated kinetic changes in platelet counts and thrombopoietic cytokines including thrombopoietin (TPO), interleukin 6 (IL‐6) and IL‐11 in 24 uncomplicated preterm infants. Platelet counts in cord blood (CB) (265 ± 64 × 109/l) were similar to adult levels, increased by d 14 (473 ± 140 × 109/l), and then remained fairly constant. Thrombocytosis (> 500 × 109/l) was observed in 9/24 (38%) subjects. Mean TPO level in CB was 5·11 ± 1·51 fmol/ml, peaked at d 2 (7·64 ± 3·28 fmol/ml), decreased at d 5 (3·93 ± 1·67 fmol/ml), and thereafter kept fairly constant during the remaining neonatal period. Compared with term infants, mean TPO levels of preterm infants in CB and at d 2 were significantly higher (P < 0·01). There was an inverse correlation between platelet counts and TPO levels (r = 0·45, P < 0·001, n = 88). Preterm neonates with thrombocytosis had significantly higher TPO values in CB than those without thrombocytosis (P < 0·05). There was no significant relationship between platelet counts and IL‐6. IL‐11 was not detectable. These results suggest that an early elevation of serum TPO levels is related to the subsequent thrombocytosis in LBW preterm infants.
Beriplex P/N reverses severe warfarin‐induced overanticoagulation immediately and completely in patients presenting with major bleeding
Authors: Evans Gillian , Luddington Roger , Baglin Trevor
Published: March 2003

In an open non‐randomized study, 10 patients with major bleeding and an Internationalized Normal Ratio (INR) greater than 14 were treated with 5 mg of intravenous vitamin K and 30 iu/kg of a single concentrate containing factors II, VII, IX and X (Beriplex P/N; Aventis Behring). The levels of these factors before and immediately after treatment were 4·7, 1·6, 8·5 and 1·1 iu/dl and 94, 30, 66 and 91 iu/dl respectively. The median INR before treatment was greater than 20 and, after treatment, 1·1. All patients had a satisfactory clinical response with immediate cessation of bleeding, and no thromboembolic complications occurred.
Functional monocyte‐derived dendritic cells can be generated in chronic lymphocytic leukaemia
Authors: Vuillier Françoise , Maloum Karim , Thomas Elaine K. , Jouanne Colette , Dighiero Guillaume , Scott‐Algara Daniel
Published: March 2003

Chronic lymphocytic leukaemia (CLL) remains an incurable disease. Although modern available treatments are able to induce disease regression, relapse almost inexorably occurs. Therefore, novel therapeutic strategies aimed at reducing the disease relapse rate are very much needed. Among these, the induction of tumour‐associated antigen‐specific cytotoxic T lymphocytes (CTL), through either DNA vaccines or injection of idiotype pulsed dendritic cells (DCs), has been actively investigated with encouraging preliminary results in B‐cell malignancies. As the CLL B lymphocyte characteristically expresses low amounts of surface immunoglobulin (Ig) and T cells from these patients have been reported to display impaired functional activity, there are concerns related to the possibility of generating specific cytotoxic antitumoral T cells in this disease. In addition, no information is presently available regarding the functional ability of CLL‐derived DCs. In the present work, freshly purified monocytes from CLL patients and normal donors were induced to differentiate in granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4 serum‐free medium and compared for their morphological, phenotypic and functional characteristics. Our results demonstrate that: (1) functional DCs can be generated from CLL patients with similar phenotype and function to those observed from normal donors; (2) in contrast to normal control subjects, monocyte‐derived DCs from CLL patients spontaneously secrete endogenous IL‐10; and (3) interferon (IFN)‐γ in combination with CD40L plays a major role in priming DCs from CLL patients for IL‐12 and IL‐15 production. Overall, these results indicate that it is possible to derive functionally competent DCs from circulating monocytes in CLL patients.
Generation of cytokine‐induced killer cells from leukaemic samples with in vitro cytotoxicity against autologous  and allogeneic leukaemic blasts
Authors: Linn Y. C. , Lau L. C. , Hui Kam M.
Published: March 2003

Summary. Cytokine‐induced killer (CIK) cells are CD3+CD56+ non‐major histocompatibility complex (MHC)‐restricted immune effector cells. The present report demonstrates that it was possible to expand CIK cells obtained at diagnosis from patients with acute leukaemia. The percentage of CD3+CD56+ CIK cells generated following culture ranged between 7·6% and 65% (median of 35·3%) and these cells were able to kill the human natural killer target K562 cells. Although the same effector cells were able to lyse autologous acute myeloid leukaemia (AML) target cells, they were not able to lyse autologous acute lymphoblastic leukaemia target cells. Pre‐absorption of the CIK effector cells by K562 cells did not completely abrogate the cytotoxicity of CIK cells against autologous blasts in 9 out of 12 samples tested. Moreover, it was observed that the cytotoxicity generated by the CIK effector cells against allogeneic leukaemic blasts was similar to that against autologous blasts. The present study suggests the potential application of CIK cells in the immunotherapy of AML, either in minimal disease state, as donor lymphocyte infusion in relapse post allogeneic transplant, or in cases of chemotherapy refractory leukaemia.
A pilot study of the recombinant soluble human tumour necrosis factor receptor (p75)‐Fc fusion protein in patients with myelodysplastic syndrome
Authors: Maciejewski Jaroslaw P. , Ristiano Antonio M. , Sloand Elaine M. , Wisch Laura , Geller Nancy , Barrett John A. , Young Neal S.
Published: March 2003

Summary. Laboratory observations suggest that, in some myelodysplastic syndromes (MDS), immune mechanisms may contribute to the impaired blood cell production. Tumor necrosis factor α (TNF‐α), a potent inhibitor of haematopoiesis, has been hypothesized to mediate suppressive effects in MDS: TNF‐α levels are elevated and correlated with marrow apoptosis and cytopenia. Inhibition of TNF‐α production using the soluble TNF receptor (Enbrel) has been successful in rheumatoid arthritis, and we have now applied the same principle to MDS. We determined spontaneous TNF‐α production by marrow cells in MDS; TNF‐α production was elevated (> mean + 2 × SD of controls) in > 1/3 of patients, but did not correlate with clinical parameters. Sixteen patients participated in a 3‐month pilot study of Enbrel. The drug was well tolerated and 15 patients were evaluable. Of these, one became temporarily (14 weeks) transfusion independent. In another patient, absolute neutrophil count (ANC) rose from 0·5 × 109/l to 0·84 × 109/l. Serious infections were seen in two out of six neutropenic patients. Progression to refractory anaemia with excess blasts in transformation (RAEBt) or leukaemia was observed in three patients. When the effects of Enbrel on haematopoietic colony formation were studied, no significant increase was seen in MDS and there was no correlation with TNF‐α levels. Although anti‐TNF therapy with Enbrel was well tolerated at the dosages used in MDS, its efficacy as a single agent appears low.
Mycophenolate mofetil for the treatment of refractory auto‐immune haemolytic anaemia and auto‐immune thrombocytopenia purpura
Authors: Howard Jo , Hoffbrand A. Victor , Prentice H. Grant , Mehta Atul
Published: March 2003

Summary.  The treatment of both auto‐immune haemolytic anaemia (AIHA) and auto‐immune thromobocytopenic purpura (AITP) remains unsatisfactory in those refractory to first‐line management. Mycophenolate mofetil (MMF) is an immunosuppressive agent originally used to prevent acute rejection of solid organ transplants and used more recently in the management of auto‐immune conditions. We report its use in four patients with AIHA and six patients with AITP. All four patients with AIHA and five of the six patients with AITP showed a complete or good partial response to treatment with MMF, confirming a possible role in the treatment of these conditions.
Three parameters, plasma thrombopoietin levels, plasma glycocalicin levels and megakaryocyte culture, distinguish between different causes of congenital thrombocytopenia
Authors: Van Den Oudenrijn Sonja , Bruin Marrie , Folman Claudia C. , Bussel James , De Haas Masja , Von Dem Borne Albert E. G. KR.
Published: March 2003

Summary.  Fourteen children with congenital thrombocytopenia were analysed in order to unravel the mechanisms underlying their thrombocytopenia and to evaluate the value of new laboratory tests, namely measurement of plasma thrombopoietin (Tpo) and glycocalicin (GC) levels and analysis of megakaryocytopoiesis in vitro. Three groups of patients were included. The first group (n = 6) was diagnosed with congenital amegakaryocytic thrombocytopenia. They had no megakaryocytes in the bone marrow, three out of four patients showed no megakaryocyte formation in vitro, and all had high Tpo and low GC levels. Mutations in the thrombopoietin receptor gene, c‐mpl, were the cause. The second group of patients (n = 3) had normal Tpo and severely decreased GC levels. In bone marrow, normal to increased numbers of atypical, dysmature megakaryocytes were present. In vitro megakaryocyte formation was quantitatively normal. A defect in final megakaryocyte maturation and subsequent (pro‐)platelets may be the cause of the thrombocytopenia. The patients in the third group (n = 5) had Wiskott–Aldrich syndrome (WAS). They had normal Tpo and GC levels and normal megakaryocyte formation both in vivo and in vitro. This corresponded with the generally accepted hypothesis that thrombocytopenia in WAS is due to increased platelet turnover. In conclusion, different causes of congenital thrombocytopenia can be distinguished using three parameters: Tpo and GC plasma levels and in vitro analysis of megakaryocytopoiesis. Therefore, these parameters may be helpful in early diagnosis of different forms of congenital thrombocytopenia.
Large experience with a factor VIII binding assay of plasma von Willebrand factor using commercial reagents
Authors: Caron Claudine , Mazurier Claudine , Goudemand Jenny
Published: March 2003

Summary. The present diagnostic assay for type 2N von Willebrand disease (VWD) is based on the in vitro measurement of the capacity of plasma von Willebrand factor (VWF) to bind exogeneous factor VIII (VWF:FVIIIB). We report a method using only commercially available reagents that is easy to perform. This method has been validated in a cohort of 144 patients with FVIII/VWF ratios < 0·6 using a plasma control mixture representative of intermediate VWF:FVIIIB. In total, 15 patients were diagnosed with markedly decreased VWF:FVIIIB and five patients were shown to have moderately decreased VWF:FVIIIB. Specific type 2N mutations were identified in all these patients.
Low risk of thrombosis in family members of patients with hyperhomocysteinaemia
Authors: Martinelli Ida , Bucciarelli Paolo , Zighetti Maddalena L. , Cafro Anna , Mannucci Pier M.
Published: March 2003

Summary. Mild to moderate hyperhomocysteinaemia, a metabolic disorder due to genetic and/or acquired factors, is associated with an increased risk of venous and arterial thrombosis. To establish whether measuring homocysteine in members of families of hyperhomocysteinaemic patients is warranted, we investigated 169 relatives of patients diagnosed with hyperhomocysteinaemia after they developed arterial or venous thrombosis. The prevalence of hyperhomocysteinaemia was 16·6%; the relative risk of thrombosis in relatives with hyperhomocysteinaemia compared to those without was 1·2 (odds ratio; 95% CI 0·24–4·2), with similarly low absolute annual incidences of thrombosis (0·28% and 0·24%). The low prevalence of hyperhomocysteinaemia among relatives of patients with this metabolic disorder, and their low risk of thrombosis, do not justify family screening.
Evidence that continued remission in patients treated for acute leukaemia is dependent upon autologous natural killer cells
Authors: Lowdell Mark W. , Craston Rose , Samuel David , Wood Marion E. , O'Neill Elena , Saha Vaskar , Prentice H. Grant
Published: March 2003

Summary. Although it has been known for more than 40 years that allogeneic immune responses cure leukaemias after bone marrow transplantation, autologous leukaemia‐specific immunity remains controversial and its impact upon survival has not been established. Here we have tested 25 patients with de novo acute leukaemias, while in remission at completion of their anti‐leukaemia therapy, for evidence of autologous cytolytic immunity to their leukaemic cells taken and cryopreserved at disease presentation. We have measured this degree of cell‐mediated cytotoxicity in vitro and termed it ‘leukaemia cytolytic activity’ (LCA). Patients whose disease ultimately relapsed had significantly lower LCA than those who remained in remission beyond 2 years (P < 0·001); the absence of LCA when in remission predicted subsequent relapse within 2 years with a sensitivity of 100% and specificity of 77%. LCA was mediated in vitro by CD56+/CD8α+/CD3– natural killer cells. We propose that it is this immune response, rather than the chemotherapy per se, which is responsible for continued remission and that measurement of LCA in patients at completion of therapy may be used as an indicator of risk of subsequent relapse. Patients lacking this response will require further treatment, either with an allogeneic donor transplant or an alternative immunotherapeutic strategy.
Combination immunotherapy with rituximab and interleukin 2 in patients with relapsed or refractory follicular non‐Hodgkin's lymphoma
Authors: Friedberg Jonathan W. , Neuberg Donna , Gribben John G. , Fisher David C. , Christine Canning  , Koval Margaret , Poor Christine M. , Green Luke M. , Daley John , Soiffer Robert , Ritz Jerome , Freedman Arnold S.
Published: March 2003

Summary.  Rituximab has significant activity as a single agent in the treatment of follicular non‐Hodgkin's lymphoma (NHL). Interleukin 2 (IL‐2) is a lymphokine that increases effector cell number. In an effort to augment antibody‐dependent cell‐mediated cytotoxicity (ADCC) associated with rituximab therapy, low‐dose IL‐2 was added to a standard rituximab regimen and patients were evaluated for safety and efficacy. Twenty patients with relapsed or refractory follicular NHL were treated with IL‐2 (1·2 MIU/m2/d for 56 d subcutaneously) as outpatients. Rituximab (375 mg/m2) was given on d 15, 22, 29 and 36. The regimen was well tolerated and only three patients required dose adjustments in IL‐2. Infusional toxicity associated with rituximab was not exacerbated by IL‐2. Peripheral blood immunophenotyping demonstrated significant increases in circulating CD8+ and CD56+ lymphocytes in all evaluable patients (P = 0·0002). Increases in total eosinophil number were observed in all patients. Eleven patients responded to therapy, for an overall response rate of 55%. Four additional patients had stable disease. For these 15 patients, the median time to progression exceeded 13 months. We conclude concomitant cytokine therapy to enhance ADCC with monoclonal antibody therapy was well tolerated and did not exacerbate antibody‐related infusional toxicity. Further studies of this rational combination are warranted and ongoing.
Possible role of Marcks in the cellular modulation of monocytic tissue factor‐initiated hypercoagulation
Authors: Chu Arthur J. , Lin Shi‐Hua , Piasentin Evano
Published: March 2003

Summary.  The enhanced extrinsic tissue factor (TF)‐initiated coagulation, often resulting from sepsis, could lead to disseminated intravascular coagulation presenting cardiovascular complications. Using model human leukaemia THP‐1 monocytes, we studied monocytic TF (mTF) hypercoagulation and its regulation. After an 8 h exposure to bacterial endotoxin [lipopolysaccharide (LPS); 100 ng/ml], mTF activity was significantly upregulated as the result of the enhanced mTF synthesis. Thereafter, LPS induction declined, exhibiting a ‘quiescent‐desensitizing’ phenomenon. Such diminished LPS induction was,however,associated with sustained LPS‐enhanced mTF synthesis, revealing the possible occurrence of a post‐translationaldownregulation. It was noted that LPS desensitization was accompanied by the increased expression of myristoylated alanine‐rich C kinase substrate (Marcks). In contrast, A23187 (20 µmol/l) or Quin‐2AM (20 µmol/l) drastically activated mTF activity without detectable effect on mTF synthesis; both of which showed that sustained functional upregulation during 24 h culture did not enhance Marcks expression. These inverse correlations between mTF activity upregulation and Marcks expression suggested that Marcks could be inhibitory. Marcks phosphorylation site domain (151–175) (Marcks PSD) readily inhibited mTF‐dependent FVII activation and diminished FVIIa formation in LPS‐challenged cells. As a result, Marcks PSD offset LPS‐induced mTF hypercoagulation upon inclusion in the single‐stage clotting assays. The anticoagulant activity was confirmed by showing that Marcks PSD significantly blocked rabbit brain thromboplastin (rbTF) procoagulation and inhibited rbTF‐dependent FVII activation as well as FVIIa formation. Our study suggests that Marcks expression plays a role in a novel cellular modulation to downregulate mTF hypercoagulation.
Retroviral interleukin 1α gene transfer in bone marrow stromal cells in a primate model: induction of myelopoiesis stimulation
Authors: De Revel Thierry , Becard Nicolas , Sorg Tania , Rousseau Sandrine , Spano Jean Philippe , Thiebot Hugues , Methali Magid , Gras Gabriel , Le Grand Roger , Dormont Dominique
Published: March 2003

Summary. Effects of interleukin 1‐α (IL‐1α), a proinflammatory cytokine with pleiotropic activity, in the myelopoietic setting, is mainly linked to its ability to increase haematopoietic growth factor production by bone marrow stromal cells. In order to minimize systemic effects of IL‐1α therapy, we proposed a model of retroviral IL‐1α gene transfer within bone marrow stromal cells in the macaque cynomolgus. Invitro, 10–15% of bone marrow stromal cells was effectively transduced by retroviral vector (murine Moloney leukaemia virus‐derived) expressing IL‐1α/LacZ, or LacZ alone as control marker, as assessed by βGal staining. IL‐1α gene expression was upregulated [semiquantitative reverse transcription polymerase chain reaction (RT‐PCR)] within the transduced cells and the cell supernatant showed an increased production of granulocyte colony‐stimulating factor (G‐CSF) and granulocyte–macrophage (GM)‐CSF (enzyme‐linked immunosorbent assay) and an increased clonogenic activity (colony‐forming cell assay). Ex vivo autologous expanded IL‐1α/LacZ transduced bone marrow stromal cells were reinfused in two macaques (and two control animals for LacZ alone as controls), without clinical systemic toxicity; LacZ expression by RT‐PCR was detected in one animal of each group between d 4 and 9. A slight increase of the peripheral blood leucocyte counts (both polymorphonuclear cells and monocytes) of the two animals transduced with IL‐1α/LacZ was observed within 10 d, indicating stimulation of myelopoiesis.
Incidence and clinical relevance of heparin‐induced antibodies in patients with deep vein thrombosis treated with unfractionated or low‐molecular‐weight heparin
Authors: Lindhoff‐Last Edelgard , Nakov Roumen , Misselwitz Frank , Breddin Hans‐Klaus , Bauersachs Rupert
Published: March 2003

Summary. The frequency of heparin‐induced thrombocytopenia (HIT) varies between different clinical treatment settings and remains unknown for patients treated with unfractionated (UFH) or low‐molecular‐weight heparin (LMWH) because of deep vein thrombosis. In this multicentre, open‐label study, 1137 patients with deep vein thrombosis were randomly assigned to UFH for 5–7 d, reviparin, a LMWH, for 5–7 d (short‐treated group) or reviparin for 28 d (long‐treated group). Heparin‐platelet factor 4 antibodies (HPF4‐A) were determined on d 5–7 and d 21. Heparin‐induced thrombocytopenia was defined by clinical evaluation. Two patients in the UFH group (incidence: 0·53%, 95% confidence interval (CI): 0·06–1·91) and two patients in the long‐treated LMWH group (incidence: 0·53%, 95% CI: 0·06–1·92) had HIT, while no HIT was observed in the short‐treated LMWH group. Pulmonary embolism developed in one of the HIT‐patients, who had HPF4‐A and was treated with UFH. On d 5–7 the incidence of HPF4‐A was 9·1% in the UFH group, 2·8% in the short‐treated LMWH group and 3·7% in the long‐treated LMWH group, with a significant increase to 20·7% in the UFH group and to 7·5% in the long‐treated LMWH group on d 21. Therefore the incidence of HPF4‐A and heparin‐induced thrombocytopenia was lower in patients treated with LMWH compared with UFH for the same duration of treatment.
Strontium‐89: a novel treatment for a case of osteosclerotic myeloma associated with life‐threatening neuropathy
Authors: Sternberg Alexander J. , Davies Paul , Macmillan Craig , Abdul‐Cader Aboobucker , Swart Sonia
Published: March 2003

Summary. Osteosclerotic myeloma is a rare disorder characterized by paraproteinaemia and osteosclerosis, and may be associated with a progressive peripheral neuropathy. Patients with widespread osteosclerotic lesions can succumb from neurological complications despite systemic chemotherapy. We present a case of disseminated osteosclerotic myeloma associated with POEMS (peripheral neuropathy, organomegaly, endocrinopathy, M band, skin changes) syndrome, which was complicated by a rapidly progressive, life‐threatening neuropathy. The patient's symptoms remained unchanged in the face of combination chemotherapy. However, a substantial improvement was seen following outpatient treatment with the commonly available radioisotope strontium 89 in combination with steroids.
Low‐dose, single‐fraction, whole‐lung radiotherapy for pulmonary hypertension associated with myelofibrosis with myeloid metaplasia
Authors: Steensma David P. , Hook C. Christopher , Stafford Scott L. , Tefferi Ayalew
Published: March 2003

Summary. Pulmonary hypertension (PH) can complicate myelofibrosis with myeloid metaplasia (MMM), may arise in the absence of evidence for thromboembolic disease and carries a grim prognosis. Four patients with MMM and severe symptomatic PH were treated with whole‐lung external beam radiotherapy in a single fraction of 100 cGy. Within 72 h, each patient noted marked symptomatic improvement and had relief of hypoxia and reduction of oedema and/or ascites. Three of the four patients enjoyed an objective improvement in pulmonary artery systolic pressure as measured by transthoracic Doppler echocardiography. Low‐dose lung radiotherapy may be a useful palliative tool for patients with MMM complicated by PH.
The effect of plasma caeruloplasmin levels on the sensitivity for activated protein C
Authors: De Visser Marieke C. H. , Souverijn John H. M. , Rosendaal Frits R. , Bertina Rogier M.
Published: March 2003

Summary.  The effect of caeruloplasmin levels on the sensitivity for activated protein C (APC), measured by a clotting assay based on the activated partial thromboplastin time, was investigated in a large group of healthy individuals without factor V Leiden. A modest inverse association between caeruloplasmin and normalized APC sensitivity ratio was found (regression coefficient β = −0·33 × 10−2; 95% confidence interval, −0·42 × 10−2 to −0·24 × 10−2). After adjustment for sex and oral contraceptive use, this association weakened (β = −0·19 × 10−2; 95% CI: −0·34 × 10−2 to −0·05 × 10−2). After additional adjustment for factor VIII levels, which are known to influence the assay, the effect of caeruloplasmin on APC sensitivity completely disappeared.
A descriptive study of randomized trials of treatments for childhood acute lymphoblastic leukaemia
Authors: Burrett Julie A. , Clarke Michael J.
Published: March 2003

Summary. This report presents a historical and descriptive account of randomized trials in childhood leukaemia since the earliest such studies in the 1960s. It focuses on trials that began before 1988, making use of the register of trials developed for a systematic review of treatments for acute lymphoblastic leukaemia in children. The number of randomized trials starting each year increased from one or two in the 1960s to an annual average of five or six in the 1980s. However trials remained relatively small, with more than half of all randomizations accruing less than 200 patients and only five having more than 1000. Most trials were published more than once.
CD34+‐enriched peripheral blood progenitor cells from unrelated donors for allografting of adult patients: high risk of graft failure, infection and relapse despite donor lymphocyte add‐back
Authors: Bornhäuser Martin , Platzbecker Uwe , Theuser Catrin , Hölig K. , Ehninger Gerhard
Published: March 2003

Summary. Fifty‐one adults with haematological malignancies were transplanted with CD34+‐selected peripheral blood progenitor cells (PBPC) from unrelated donors. The conditioning protocol contained total body irradiation (n = 17) or combinations of busulphan and other alkylating agents (n = 34). Antithymocyte globulin was infused in all patients. The median number of CD3+ T cells infused with the graft after purification with the Isolex 300® system in the first cohort of 18 patients was 2·1 × 105/kg. Prophylactic donor lymphocyte infusion (DLI) containing 1 × 105 CD3+ T cells was performed on d 21 in the following 33 patients who had received PBPC purified by the CliniMACS® system. Early graft failure occurred in 8/51 patients (16%). After a median follow‐up of 31 months (range 8–60), the probability of disease‐free survival (DFS) was 36% for the whole group. Reasons for death were opportunistic infections (n = 15), graft‐versus‐host disease (GvHD, n = 7) and relapse (n = 4). Pre‐transplant factors with significant impact on DFS were cytomegalovirus status and risk category of underlying disease. The occurrence of graft failure or GvHD was associated with poor outcome. Recipients of CD34+‐selected PBPC from unrelated donors are at high risk of infectious complications, relapse and graft failure which cannot be prevented by early reinfusion of unmodified donor lymphocytes.