bluuebunny/crossref_metadata_2025 huggingface dataset:
Pretransfused patients with severe aplastic anaemia exhibit high numbers of cytotoxic T lymphocyte precursors probably directed at non‐HLA antigens
Authors: Kaminski E. R. , Hows J. M. , Goldman J. M. , Batchelor J. R.
Published: March 2008

Patients with severe aplastic anaemia (SAA) have a relatively high risk of graft rejection after transplantation of bone marrow from HLA‐identical siblings compared with patients transplanted for leukaemia. This is presumed to be due to pretransplant blood transfusions which sensitize the recipient's immune system to non‐HLA antigens expressed on donor marrow cells. To test this hypothesis, we estimated in the peripheral blood of 18 SAA patients, the frequencies of alloreactive cytotoxic T lymphocyte precursors (CTL‐p) directed against mononuclear cells from syngeneic twins, HLA‐identical siblings or HLA‐matched unrelated individuals. The results were compared with the frequencies of CTL‐p in 13 healthy subjects or leukaemia patients who had not been transfused. Pretransfused patients with SAA had significantly higher frequencies of CTL‐p directed against HLA‐identical siblings (P= 0·002), and against HLA‐matched unrelated individuals (P= 0·004), than did untransfused individuals. In contrast, the frequency of CTL‐p in two pretransfused patients against syngeneic mononuclear cells was very low. We propose that the increased numbers of CTL‐p in the blood of pretransfused SAA patients are directed against non‐HLA antigens on target cells and may be the result of prior blood transfusion.
Immunocytochemical detection of ferritin in human bone marrow and peripheral blood cells using monoclonal antibodies specific for the H and L subunit
Authors: Invernizzi Rosangela , Cazzola Mario , De Fazio Pasqualina , Rosti Vittorio , Ruggeri Giuseppina , Arosio Paolo
Published: March 2008

We have used the monoclonal antibodies 2A4 (specific for the H subunit of human ferritin) and LO3 (specific for the L subunit) for immunocytochemical detection of ferritin in bone marrow and peripheral blood cells from normal subjects and patients with various haematological disorders. Formalin‐fixed slides were stained by the immunoalkaline phosphatase procedure (APAAP). In normal subjects, ferritin could be found only in bone marrow smears and appeared to be largely confined to erythroid precursors and reticuloendothelial cells. The more immature erythroid precursors contained higher concentrations of cellular ferritin. Although evaluation could be only semiquantitative, erythroblast ferritin appeared to be more reactive with the monoclonal 2A4 (15 ± 7% positive erythroblasts) than with the monoclonal LO3 (6 ± 5% positive erythroblasts), indicating that H‐type ferritin was predominant, particularly in proerythroblasts and basophilic erythroblasts. By contrast, the ferritin present in reticuloendothelial cells appeared to be predominantly of L‐type. Patients with iron deficiency showed low levels of positive erythroblast, whereas the reverse was true in patients with transfusional iron overload. Intense positivity for reticuloendothelial cell ferritin was found in patients with anaemia of chronic disease. In myelodysplastic syndromes and acute myeloid leukaemia (AML), ferritin positivity was generally very strong at any stage of erythroblast development, particularly with the monoclonal antibody 2A4. Perls‐positive perinuclear granules of ring sideroblasts were not stained, confirming that mitochondrial iron deposition is not in the form of ferritin. In AML and myelodysplastic syndromes with excess of blasts, ferritin could be detected also in immature myeloid cells. These data indicate that: (a) in normal conditions ferritin is mainly expressed in red cell precursors and reticuloendothelial cells, and this is in keeping with the peculiar role of these cells in iron metabolism; (b) abnormal cell ferritin contents can be observed in both iron overload and malignancy.
Effect of recombinant human erythropoietin on anticancer drug‐induced anaemia
Authors: Matsumoto Tomoko , Endoh Kouichi , Kamisango Keiichi , Akamatsu Ken‐ichi , Koizumi Kinya , Higuchi Masato , Imai Nobuo , Mitsui Hiroki , Kawaguchi Tsutomu
Published: March 2008

Summary. Anaemia was induced in rats with fluorouracil (5‐FU) or cisplatin (CDDP) and the mechanisms of anaemia induction were analysed. Furthermore, the therapeutic effects of recombinant human erythropoietin (rHu Epo) on these anticancer drug‐induced anaemias were investigated.

In 5‐FU‐induced anaemia, marked serum erythropoietin (Epo) elevation was observed in inverse correlation to blood Hb concentration and Hb concentration rapidly recovered to normal levels. On the other hand, in CDDP‐induced anaemia, serum Epo elevation was modest and the lowered Hb concentration persisted longer. Treatment with rHu Epo significantly improved both anticancer drug‐induced anaemias but rHu Epo was more effective on CDDP‐induced anaemia. These results suggest that rHu Epo might be useful for the therapy of anaemia associated with anticancer chemotherapy.
Malignant plasma cells are sensitive to LAK cell lysis: pre‐clinical and clinical studies of interleukin 2 in the treatment of multiple myeloma
Authors: Gottlieb David J. , Prentice H. Grant , Mehta Atul B. , Galazka Andrew R. , Heslop Helen E. , Hoffbrand A. Victor , Brenner Malcolm K.
Published: March 2008

Summary. To assess the role of the cytokine interleukin 2 (IL2) in the treatment of patients with multiple myeloma, we examined the sensitivity of plasma cell lines and malignant plasma cells from multiple myeloma (MM) patients to cell and cytokine‐mediated killing induced by IL2. Unstimulated peripheral blood mononuclear cells (PBM) from normal donors produced little killing (mean lysis 1.0 ±1.0%, effector: target (ET) ratio 50:1), but cytotoxicity was modestly increased when PBM were incubated with IL2 prior to assay (8.0 ± 2.9%). Unstimulated PBM from patients with MM also failed to kill autologous malignant plasma cells (mean 0.6±0.6%), but after exposure to IL2 they induced substantial lysis of autologous malignant cells (mean 55.3±22.1%). In addition, tumour necrosis factor (TNF) and interferon gamma (IFN‐γ), two cytokines released from mononuclear cells in response to IL2, also reduced the survival and thymidine uptake of malignant plasma cells in culture. To determine whether these potentially beneficial immuno‐modulatory effects could be reproduced by in vivo administration of IL2, we have administered seven courses of IL2 to four patients with MM after autologous bone marrow transplant (ABMT). No serious adverse effects were noted. Increases in natural killer (NK) and lymphokine‐activated killer (LAK) activity of PBM occurred during IL2 infusion, although cells capable of killing autologous MM cells did not circulate. However, IL2 infusions also induced substantial increases in the production of the cytokines TNF and IFN‐γ from peripheral blood lymphocytes. These results suggest that the in vivo administration of IL2 in MM deserves further evaluation. particularly for its potential to control minimal residual disease.
The platelet and plasma pools of plasminogen activator inhibitor (PAI‐1) vary independently in disease
Authors: Simpson A. John , Booth Nuala A. , Moore Norma R. , Bennett Bruce
Published: March 2008

Summary. The relative importance and behaviour of plasma and platelet plasminogen activator inhibitor (PAI‐1) in disease has not hitherto been examined. In this study the concentration of PAI‐1 in the plasma and platelets of patients with a variety of disorders was examined using a specific ELISA and a functional assay. Mean plasma PAI‐1 was elevated in groups of patients with diabetes mellitus, hypertension, alcoholic cirrhosis, angina and myocardial infarction. Plasma PAI‐1 was raised in the post‐operative phase and the PAI‐1 released after surgery was not derived from platelets. In all groups PAI‐1 in the platelet pool reflected the platelet count, except in type II diabetes mellitus and chronic renal failure, where a reduced quantity of PAI‐1 antigen per platelet was found. In severe chronic renal failure, abnormal platelets and diminished platelet PAI‐1 may contribute to the haemorrhagic tendency sometimes seen in this disorder.

Plasma PAI‐1 represents a larger proportion of total circulating PAI‐1 in disease than it does in healthy individuals; PAI‐1 per platelet is abnormal only in a minority of disorders. Plasma and platelet pools of PAI‐1 vary independently in disease and both merit consideration in evaluating the importance, if any, of PAI‐1 in thrombosis or haemorrhage.
The role of fibronectin in platelet aggregation
Authors: Thurlow P. J. , Kenneally D. A. , Connellan J. M.
Published: March 2008

Summary. A monoclonal antibody (anti‐Fn2) was prepared which was reactive with both plasma fibronectin and fibronectin located within the platelet alpha granule. Immunoblotting analysis, on thermolysin digestion fragments of fibronectin, identified two immunoreactive fragments of Mr 145 kDa and 155 kDa which are known to contain a cell and DNA binding region. Anti‐Fn2 was found to inhibit binding of fibronectin to platelets and DNA. Functional platelet studies, measuring platelet aggregation and 14C‐serotonin release in washed platelet systems, demonstrated the ability of anti‐Fn2 to totally inhibit low dose thrombin and low‐dose collagen induced platelet aggregation and serotonin release. Anti‐Fn2 partially inhibited platelet aggregation induced by ADP (10 μM) and arachidonic acid, but had no effect on platelet aggregation induced by high‐dose thrombin or by the calcium ionophore A23187.

These studies indicate that fibronectin participates in platelet aggregation and release induced by a range of agonists and suggest that it has a more important involvement in platelet function than previously described.
The platelet glycoprotein Ia‐IIa‐associated Br‐alloantigen system is expressed by cultured endothelial cells
Authors: Giltay J. C. , Brinkman H.‐J. M. , Vlekke A. , Kiefel V. , van Mourik J. A. , von dem Borne A. E. G. Kr.
Published: March 2008

Summary. To obtain information on the immunological relationship between the endothelial and platelet glycoprotein (GP)Ia‐IIa (VLA‐2) complex, we studied whether endothelial GPIa‐IIa was able to express the platelet GPIa‐IIa‐associated Br‐alloantigen system. Therefore, we tested antisera to both allelic forms of the Br system (Bra and Brb) on platelets (by an assay based on monoclonal antibody‐specific immobilization of platelet antigens, MAIPA) and on cultured umbilical vein endothelial cells (by immunoprecipitation experiments) from the same individual. Endothelial cells from a platelet Br(a + b +), and from a platelet Br(a ‐ b +) individual were studied. Our results indicate that endothelial GPIa‐IIa is indistinguishable from platelet GPIa‐IIa in its ability to express the Bra and Brb alloantigens. The association of Br alloantigens with endothelial GPIa‐IIa was confirmed by the results of an assay based on monoclonal antibody‐specific immobilization of endothelial antigens (MAIEA). These data further illustrate the structural and immunologic similarity of platelet and endothelial cell GPIa‐IIa (VLA‐2).
Evaluation of a new UVB source for irradiation of platelet concentrates
Authors: van Prooijen H. C. , van Marwijk Kooy M. , van Weelden H. , Aarts‐Riemens M. I. , Borghuis L. , Akkerman J. W. N.
Published: March 2008

Summary. The application of ultraviolet B (UVB) radiation has been proposed as a new technology to decrease immunogenicity of leucocytes in platelet transfusions. UV radiation also induces platelet aggregation, which occurs most effectively at wavelengths between 240 and 280 nm and falls off sharply above 300 nm. In order to minimize the effects of UV energy on the platelets we evaluated in this study the expected benefit of a new narrow‐band UVB source, emitting a narrow peak around 312 nm. Exposure of platelet or lymphocyte suspensions to this source induced in the platelets both aggregation and functional defects at a dose of 12 J/cm2 and in the lymphocytes inhibition of in vitro function at a dose of 2 J/cm2. A conventional UVB source, emitting a broad spectrum between 280 and 340 nm, was more deleterious for the cells and induced similar defects in the platelets at a dose of 6 J/cm2 and inhibition of lymphocyte function at a dose of 1 J/cm2. These data indicate no benefit for the new UVB source, since the ratio of the doses to induce platelet defects and inhibition of lymphocyte function is identical for each of the two sources.

Absorption of UV energy by plasma and the plastic material of platelet containers is another criterium for selection of UV sources. In view of the better transmission characteristics of long wavelength UV energy we propose that there is a preference for the new narrow‐band UVB source.
Interaction of monocytes and T cells in the regulation of normal human megakaryocytopoiesis in vitro: role of IL‐1 and IL‐2
Authors: Hamaguchi Hiroyuki , Takano Naoto , Saito Kenji , Enokihara Hideo , Furusawa Shimpei , Shishido Hideo
Published: March 2008

Summary. Autologous or allogeneic peripheral blood T cells can stimulate the human megakaryocyte progenitor cell (CFU‐Meg)‐derived colony formation in a dose‐dependent fashion in agar cultures of nonadherent (NA), T cell‐depleted (NT) bone marrow (BM) cells. Low concentrations of monocytes and T cells can collaborate in the stimulation of CFU‐Meg colony formation or in the production of megakaryocyte colony stimulating factor (Meg‐CSF) by T cells in the presence of mitogens or IL‐2. Monocytes alone can produce only negligible Meg‐CSF under any conditions. When monocyte conditioned medium (CM) was added to T cell‐stimulated NA, NT BM cell cultures, CFU‐Meg colony growth was appreciably increased compared with that stimulated by T cells alone. Dose‐dependent increase in CFU‐Meg colony growth was noted when varying concentrations of IL‐1 were added to T cell‐stimulated NA, NT cell cultures, although IL,‐1 itself could support no CFU‐Meg colony growth in the absence of T cells. These data suggest that a synergistic interaction between T cells and monocytes during the production of Meg‐CSF by T cells could be partly mediated by IL‐1. IL‐2 was found to stimulate Meg‐CSF production by T cells in the presence or absence of mitogens. IL‐2‐stimulated Meg‐CSF production by T cells was augmented by the addition of monocytes. Although IL‐2 itself had no stimulatory effect on CFU‐Meg colony growth, dramatic augmentation in the CFU‐Meg colony number was noted when IL‐2 was added to T cell‐stimulated NA, NT cell cultures. High concentrations of monocytes and prostaglandin E (PGE) inhibited the CFU‐Meg colony formation. These results suggest that IL‐1 and IL‐2 may play a stimulatory role on the normal human in vitro megakaryocytopoiesis and may be involved in the development of reactive thrombocytosis and bone marrow mega‐karyocytic hyperplasia in various inflammatory diseases.
The utility of a HindIII polymorphism of factor VIII examined by rapid DNA analysis
Authors: Graham John B. , Kunkel Glenna R. , Fowlkes Dana M. , Lord Susan T.
Published: March 2008

Summary. A previously described HindIII restriction fragment length polymorphism (RFLP) of factor VIII (FVIII) has its polymorphic site in the unsequenced nineteenth intron. We have located the polymorphic site, as well as an invariant site, by amplifying and sequencing IVS 19 using the polymerase chain reaction (PCR). The oligonucleotide primers were synthesized from known FVIII sequence on either side of the 19–20 splice junction. The amplified product was cloned into a plasmid and sequenced by the dideoxy chain termination method. The polymorphic HindIII site was 103 bp and the invariant site 184 bp from the 3’end of the nineteenth exon.

The frequency of the polymorphism was determined in 457 subjects (643 chromosomes) of seven ethnic groups on whom frequency of the BclI RFLP of IVS 18 was also assessed. The HindIII site is highly polymorphic in all groups, ∼ 0.25:0.7 5, the expected heterozygosity averaging 37.6%. and the observed number of heterozygotes did not differ significantly from expectation. The (+):(‐) allelic ratio is similar in all groups, except African‐Americans in whom it is reversed. Strong allelic association (linkage disequilibrium) is present between the HindIII polymorphism of IVS 19 and the BclI polymorphism of IVS 18.
Lack of useful clinical predictors of response to splenectomy in patients with chronic idiopathic thrombocytopenic purpura
Authors: Juliá A. , Araguás C. , Rosselloa J. , Bueno J. , Domenech P. , Olona M. , Guardia R. , Petit J. , Flores A.
Published: March 2008

SummaryWe set out to identify clinical or analytical variables that might predict the response to splenectomy in patients with chronic idiopathic thrombocytopenic purpura (ITP).

We retrospectively examined 138 mostly adult patients with chronic ITP, treated with splenectomy. Information was compiled from five Public Health Hospitals from a questionnaire and chart review. Sixty‐one potentially prognostic variables were analysed by univariate and multivariate statistical methods. After multivariate analysis, age (relative risk=1.02; CI 1–1.03) and a severity of the bleeding diathesis (relative risk = 1.6; CI 1.13–2.22) were independent prognostic factors for a sustained response to splenectomy.

An independent analysis of the postsplenectomy counts showed that an early (days 1–3) thrombocyte count exceeding 156 ± 109/1 cells increases the likelihood of a permanent unmaintained response.

Our data indicate that the response to splenectomy in patients with chronic ITP cannot be adequately predicted on the basis of pre‐splenectomy clinical or analytical variables.
Locus assignment of human a globin mutations by selective amplification and direct sequencing
Authors: Dodé Catherine , And Jacques Rochette , Krishnamoorthy Rajagopal
Published: March 2008

Summary We describe a simple approach for molecular characterization and locus assignment of structural mutants by direct sequencing of enzymatically amplified DNA selective to α1 and α2 globin gene regions. Nucleotide substitution of two structural variants (Stanleyville II α278Lys and J Mexico α254Glu) were determined and their encoding loci were specified. The amplified segment encompasses sequences upstream of the CAAT box to downstream of the Poly(A) addition signal. Hence all of the α globin structural variants and most of the nondeletion α thalassaemic mutants should be characterizable by this approach.
Effects of deficiencies of glycophorins C and D on the physical properties of the red cell
Authors: Nash G. B. , And J. Parmar , Reid M. E.
Published: March 2008

Summary Red cells of the rare Leach phenotype lack the membrane glycophorins C and D, and a proportion of the red cells are elliptocytes. Judging from tests on suspensions of red cell ghosts sheared rotationally in an ektacytometer, it has previously been suggested that these membranes are relatively fragile and poorly deformable. We have carried out analyses of individual red cells to investigate possible factors which underlie the physical changes in these glycophorin‐deficient cells. Micropipette analysis of the red cell membrane showed that the rigidity and viscosity were normal, both for elliptocytes and discocytes, for three donors deficient in glycophorins C and D. Red cell transit times through 5 μm pores, measured electronically for 2000 individual cells, showed no differences from controls. It was confirmed that the index of deformation obtained using an ektacytometer was reduced, but our results suggest that this arises from shape rather than membrane changes. The elliptocytes were found to have a lower volume and surface area than discocytes from the same donor (measured by micropipette aspiration of single red cells), and were rarely found in less dense red cell fractions. No reticulocytes were found to be elliptical. These data suggest that the elliptocytes are older red cells, and are formed from red cells which are initially released into the circulation with normal shape. Their elongated shape might arise from permanent distortion of the unstable membrane by shear forces in the circulation.
Long‐term trial with the oral iron chelator 1,2‐dimethy1‐3‐hydroxypyrid‐4‐one (L1) II. CLINICAL OBSERVATIONS
Authors: Bartlett A. N. , Hoffbrand A. V. , Kontoghiorghes G. J.
Published: March 2008

Summary 1.2‐Dimethyl‐3‐hydroxypyrid‐4‐one (L1) has been given daily for 1‐15 months to 13 transfusion dependent iron loaded patients. No significant change occurred in liver, renal or cardiac function, ECG and radionucleotide angiocardiogram, in audiometry tests and in visual function and electrical retinography. No skin rashes, gastrointestinal symptoms and no neurological changes that could be detected clinically were observed. Two of the patients died of their underlying diseases. One patient had severe cardiac abnormalities before receiving L1 and died of congestive heart failure with infections 5 weeks after stopping a 2‐month course of L1. The other, a patient with myelodysplasia suffered recurring infections due to progression of the disease. Joint and muscle pains occurred in five patients. In two these disappeared despite continuing the drug: another patient developed swollen ankle joints which gradually resolved on stopping L1 therapy: a patient with underlying osteoarthritis complained of mild pain and stiffness in her knees which remained intermittent both on and off the drug while in the fifth patient peripheral small joint swelling and pain present before starting L1 improved with L1 therapy. One patient, with Blackfan Diamond anaemia, developed a L, red cell antibody 6 months after commencing L1. This disappeared on stopping the drug and did not reappear. She then developed severe agranulocytosis and thrombocytopenia 6 weeks after recommencing L1 after 3 months discontinuation of the drug. No other patient showed a change in granulocyte or platelet count.
Micro‐osteoclast resorption as a characteristic feature of B‐cell malignancies other than multiple myeloma
Authors: Rossi Jean‐FRanclois , Chappard Daniel , Marcelli Christian , Laplante Jacques , Commes Therese , Baldet Pierre , Janbon Charles , Jourdan Jacques , Alexandre Christian , Bataille Regis
Published: March 2008

Summary Multiple myeloma (MM) is characterized by the presence of lytic bone lesion and frequent hypercalcaemia. These are due to an excessive osteoclastic resorption in association with a low bone formation, as demonstrated by bone histomorphometry. Conversely. B‐cell malignancies other than MM are rarely associated with lytic bone lesion and/or hypercalcaemia. In this study we have analysed quantitative bone histology in 65 patients with B‐cell malignancies other than MM at diagnosis: chronic lymphocytic leukaemia (CLL, n = 20). non‐Hodgkin's lymphoma (NHL. n= 25), Waldenström's disease (WD. n= 14), hairy cell leukaemia (HCL. n = 6). Fifty patients presented no clinical evidence of increased bone resorption, including no lytic bone lesions radiologically detectable and/or no hypercalcaemia. 80% of these patients (40/50) had increased bone resorption parameter using quantitative bone histology, including 19/29 (65.5%) patients with CLL or WD and 21/21 (100%) patients with NHL or HCL (P<0.01). As a control group seven patients lacking bone marrow involvement on bone sample presented no excessive bone resorption. However, eight patients presented lytic bone lesions and/or hypercalcaemia. All of these patients had increased resorption parameters with high numbers of osteoclasts per surface trabecular bone (mean = 35.3). as opposed to the patients lacking lytic bone lesions and/or hypercalcaemia (mean = 6.6, n= 28) and to normal individuals (mean ± SD = 3.8 ± 1.7 and 6–3 ± 2–6, respectively before and after 60 years). In all the cases, excessive histologic bone resorption was mediated by mononuclear small osteoclasts (mean osteoclast length ± SD =27.3 ± 4.1 as compared to normal range= 35.0 ± 1.0, P<0.001). In different in vitro models, these small mononuclear osteoclasts are considered as progenitors. These data suggest an abnormal osteoclast differentiation in B‐cell malignancies other than MM, probably due to differences in the production of local factors acting on bone remodelling.
Interleukin‐6 is a cofactor for the growth of myeloid cells from human bone marrow aspirates but does not affect the clonogenicity of myeloma cells in vitro
Authors: Montes Borinaga A. , Millar B. C. , Bell J. B. G. , Joffe J. K. , Millar J. L. , Gooding R. , Riches P. , McElwain T. J.
Published: March 2008

Summary Several groups have claimed that IL‐6 is a growth factor for human myeloma cells in vitro. Bone marrow aspirates from 30 patients at different stages of treatment with VAMP/high dose melphalan, were examined for myeloma colony formation (MY‐CFU,) using a clonogenic assay in vitro. Myeloma cells from 16/30 patients produced MY‐CFUc in our assay system, which uses heavily irradiated HL60 cells as an underlay in soft agar. These heavily irradiated cells were shown to be essential for the inhibition of granulocyte‐macrophage colonies (GM‐CFUc). The addition of recombinant human IL‐6 (10 ng/plate) reduced the number of bone marrow samples which produced MY‐CFUc from 16 to six. Furthermore, the addition of antibody to IL‐6 (1 μg/plate) failed to inhibit MY‐CFUc from 6/7 samples.

Conditioned medium from human peripheral blood mono‐nuclear cells (PBMC‐CM) contains approximately 2 ng/ml IL‐6 and can be used to stimulate the growth and maintenance of the B9 murine IL‐6 dependent hybridoma cell line. Kecombinant human IL‐6 supported the growth of B9 cells in a clonogenic assay and growth was inhibited by anti‐IL‐h in the presence of rhIL‐6 or PBMC‐CM. Mononuclear cells from a second group of myeloma patients were cultured in soft agar in a mixture of PBMC‐CM and fresh growth medium. Nine of the 10 samples produced myeloid colonies which consisted of granulocytes, monocytes and macrophages and the number of colonies was reduced by at least 50% in 6/8 samples when anti‐IL‐6 was added to the cultures. In no instance were MY‐CFUc produced. Also, conditioned medium from the bladder carcinoma cell line 5637. which is used routinely as a source of granulocyte‐macrophage colony stimulating factor (GM‐CSF), contains approximately 4 ng/ml IL‐6. Although rhIL‐6 failed to stimulate GM‐CFUc in the absence of other growth factors, addition of anti‐IL‐6 to cultures containing a suboptimal amount of 5637‐CM reduced the number of colonies by 50%.

These data provide evidence that IL‐6 is a cofactor for the growth of myeloid precursors but does not affect the proliferation of human myeloma cells in vitro.
Studies on the megakaryocytes of a patient with the Bernard‐Soulier syndrome
Authors: Hourdillé Paquita , Pico Marta , Jandrot‐Perrus Martine , Lacaze Denis , Lozano Miguel , Nurden Alan T.
Published: March 2008

Summary We have used monoclonal antibodies AP‐1 (anti‐GP Ibα), AP‐2 (anti‐GP IIb‐IIIa) and FMC 25 (anti‐GP IX) in immunofluorescence and immunocytochemical studies on megakaryocytes (MK) isolated from a Bernard‐Soulier syndrome (BSS) patient whose giant platelets were characteristically deficient in GP Ib‐IX complexes. Electron microscopy showed that the patient's MK were similar in size to normal MK. However, a striking feature was the variable and intermittent nature of the demarcation membrane system which was often vacuolar in appearance. Permeabilized mature MK from normal individuals were strongly positive with AP‐2. AP‐1 and FMC 25. Those from the BSS patient were normal for AP‐2, negative for AP‐1 but weakly positive with FMC 25. Binding of the monoclonal antibodies to the patient's platelets was evaluated using flow cytometry. The results confirmed the absence of GP Ibα from the surface membranes, but showed the presence of small amounts of GP IX distributed throughout the platelet population. Our findings confirm that the membrane lesion in BSS is also to be found in MK and further show that the defect may affect differently individual constituents of the GP Ib‐IX complex.
L1 (1,2‐dimethyl‐3‐hydroxypyrid‐4‐one) for oral iron chelation in patients with beta‐thalassaemia major
Authors: Toundury Petrin , Kontoghiorghes George J. , Ridolfi‐Luuthy Annette , Hirt Andreas , Hoffbrand A. Victor , Lottenbach Anne Marie , Sonderegger Theo , Wagner Hans P.
Published: March 2008

Summary L1 was given to eight patients with beta‐thalassaemia major who had previously been treated with deferox‐amine (DF) for 4–10 years. The patients’ages ranged from 11 to 27 years. Serum ferritin values ranged from 1.3 to 11.5 ± 103 pg/l. L1 was given twice daily at a daily dose of 55–80 mg/kg body weight and was continued for 10 months in two patients, 9 months in three, 7 months in two patients and 4 months in one patient. As previously observed with DF, each patient's urinary iron excretion (UIE) varied greatly from day to day. The mean UIE of the eight patients ranged from 11 to 49 mg/d (0.2–0.87 mmol/d) on subcutaneous DF and from 16 to 53 mg/d (0.28–0.95 mmol/d) on L1. Two patients excreted significantly more and one patient significantly less iron while on L1. If the UIE was calculated as mmol Fe/mmol creatinine there was no statistically significant difference. Serum ferritin values fluctuated widely in all, with a consistent downward trend in three, no change in four and an increase in one of two non‐splenectomized patients. This patient's splenomegaly and need for transfusions continued to increase while on L1. No toxicities attributable to the drug were detected during the period of study and tolerance of the drug was excellent.
A prospective study comparing vancomycin and teicoplanin as second‐line empiric therapy for infection in neutropenic patients
Authors: Cony‐Makhoul P. , Brossard G. , Pellegrin G. Marit. J. L. , And J. Texier‐Maugein , Reiffers J.
Published: March 2008

Summary In recent years, the most common cause of infection in neutropenic patients has shifted from Gram‐negative to Gram‐positive bacteria. To compare the efficacy and toxicity of teicoplanin and vancomycin in neutropenic patients. we conducted a prospective study of 151 adult leukaemic patients hospitalized for intensive chemotherapy. After chemotherapy, the median duration of granulocyto‐penia (>500/mm3) was 25 d (range 13–49). When the patients became febrile, they received ceftazidime (CTZ) alone (2 g every 12 h. intravenously). If fever persisted more than 48–72 h after administration of CTZ, the patients were randomly assigned to receive CTZ combined with either vancomycin (vanco) (30 mg/kg/d) or teicoplanin (teico) ((3 mg/kg every 12 h on day 1, then daily). When fever persisted further, an aminoglycoside antibiotic and/or amphotericin B were usually added to the previous combination. Of the 151 patients. 116 patients became febrile during the period of aplasia. Fifty‐nine patients had persistent (or recurrent) fever despite administration of CTZ and received either vanco (n = 35) or teico (n = 24). Sixteen of these latter 59 patients had septicaemia (vanco n = 9: teico n = 7) due to Candida sp. (n = 2), Gram‐negative (n = 2) or Gram‐positive (n=12) bacteria. The main characteristics of patients and infection were similar in both arms. The treatment was considered as a success (disappearance of fever within 48 h) in 21/35 patients of the vanco group (60%) compared to 13/ 24 patients of the teico group (54%: P not significant). The percentage of failures for infection due to Gram‐positive bacteria was 2/11 for vanco versus 2/7 for teico (P not significant). Two patients in each group died from infection. The main cause of failure was retrospectively attributed to fungal pathogens. No major toxic effects were found in either group. These preliminary results do not show any difference between vanco and teico as second‐line antibiotic therapy in leukaetnic patients with severe and prolonged granulocytopenia.
Terminal transferase expression in acute myeloid leukaemia: biology and prognosis
Authors: Gucalp Rasim , Paietta Elisabeth , Weinberg Vivian , Papenhausen Peter , Dutcher Janice P. , Wiernik Peter H.
Published: March 2008

Summary Clinical and biological features were assessed in 114 consecutive previously untreated adult acute myeloid leukaemia (AML) patients whose diagnosis was based on FAB criteria and detailed immunophenotyping. All patients received standard intensive chemotherapy. The main purpose of this study was to establish the prognostic value, if any, of terminal transferase (TdT) expression in myeloid leukaemia. TdT positive cells (7–80% of total blast cells) were detected in 40% of the cases. Among clinical characteristics, a low lactate dehydrogenase (LDH) (<250I.U.) (P=0·003), a low initial white blood cell count (<10 × 109/l) (P=0·002), and an absolute neutrophil count (<5 × 109/l) (P=0·02) were associated with TdT‐positivity. FAB classification was not predictive of TdT expression, and there was no difference in the distribution of FAB subtypes between the groups. Multivariate analysis combining clinical and laboratory data indicated that a low expression of the monocytic antigen CD14 was predictive of TdT positivity in AML (P=0·01). Karyotyping showed no difference in the pattern of occurrence of specific abnormalities between the TdT+ and the TdT− group. When clinical and immunophenotype data were included in a prognostic model, the patient's age was highly predictive of response (P<0·001), and only the CDw65 antigen contributed to the response model (P=0·07). TdT+ patients with a low expression of CD11b achieved a higher frequency of response at a borderline level of significance (P=0·06). Frequency of response to chemotherapy, the response duration or overall survival were not influenced by TdT expression.
Characterization of leukaemic basophil progenitors from chronic myelogenous leukaemia
Authors: Teshima Takanori , Kondo Seiji , Harada Mine , Shibuya Tsunefumi , Okamura Takashi , Tamari Yuichi , Kimura Nobuhiro , Akashi Koichi , Okamura Seiichi , Niho Yoshiyuki
Published: March 2008

Summary We describe the unique characteristics of leukaemic basophils from a patient with chronic myelogenous leukaemia (CML). The leukaemic cells were immature basophil‐like blasts and expressed CD4, CD7 and HLA‐DR in addition to CD13 and CD33. Both immunoglobulin and T cell receptor genes were retained in germline configurations. Interleukin‐1 (IL‐1) or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) as well as IL‐3 or IL‐4 enhanced the proliferation and differentiation of leukaemic cells and only basophils were generated from in vitro culture. These results suggest that basophil progenitors expressing CD4, CD7 and HLA‐DR may be involved in the development of basophilic crisis of CML and that both IL‐1 and GM‐CSF may act on basophil progenitors as well as IL‐3 or IL‐4.
Anti‐idiotypic antibodies in patients with monoclonal gammopathies: relation to the tumour load
Authors: Bergenbrant S. , Österborg A. , Holm G. , Mellstedt H. , Lefvert A.‐K.
Published: March 2008

Summary The production of anti‐idiotypic antibodies from Epstein‐Barr virus transformed peripheral blood lymphocytes was analysed in 12 patients with multiple myeloma and monoclonal gammopathy of undetermined significance. A low anti‐idiotype production was found in patients with advanced disease (multiple myeloma stage III), whereas the production was high in patients with multiple myeloma stage I and monoclonal gammopathy of undetermined significance (MGUS) (P<0·01). The study supports the existence of an immunological network response in patients with monoclonal gammopathies.
Stochastic versus deterministic in haemopoiesis: what is what?
Authors: Novak J. P. , Stewart C. C.
Published: March 2008

Summary ‘Stochastic’models of haemopoiesis are discussed in juxtaposition to ‘deterministic’concepts. It is argued that certain characteristic features attributed to stochastic models are conjectures, not supported by the observations quoted to provide an experimental basis: for example, an assumption of extrinsic control and another assumption that such control is inaccessible to external influences up to the very latest stages of cell development. Furthermore, it is argued that quantitative relationships between early progenitors (stem cells) and the number of colonies produced in vitro, cited as evidence supporting the proliferation and differentiation scheme of the stochastic model of random restriction, are also compatible with other models, providing that some sufficiently complex mechanism is involved in the control process. A simple model based on the principle of ordered development (usually denoted ‘proliferation tree’) is presented as an example and the calculated distributions are shown to have the same form as the experimental ones.
Developmental process of the T‐cell receptor α and δ gene assembly in B‐cell precursor acute lymphoblastic leukaemia
Authors: Hara Junichi , Kawa‐Ha Keisei , Takihara Yoshihiro , Yumura‐Yagi Keiko , Ishihara Shigehiko , Tawa Akio , Mak Tak W. , Okada Shintaro , Gelfand Erwin W.
Published: March 2008

Summary We analysed the organization of Vδ genes and δ recombining element (δRec) in 27 children with B‐cell precursor acute lymphoblastic leukaemia. Twenty‐two of 54 alleles showed rearrangements of the T‐cell receptor (TCR) δ locus. These rearrangements resulted either from D2Dδ3 (2 alleles) or Vδ2(Dn)Dδ3 (20 alleles) recombinations, and the other Vδ and δRec were not rearranged. Of 23 alleles with deletion of Cδ and rearrangements of Jα. Vδ2, Vδ4 and Vδ5 appeared to rearrange to Jα on five alleles. With regard to the relationship between the rearranged Vα/δ and Jα genes, gene segments 5’to Vδ2 frequently rearranged to Jα more proximal to Cα, whereas Vδ2 and gene segments 3’to Vδ2 showed a tendency to rearrange to Jα distal to Cα. Based on these findings, we suggest that the initial recombination event of the TCR‐α/δ gene may be D2Dδ3 joining, followed by Vδ2 recombination with the D2Dδ3 complex. It was also suggested that use of Vα/δ and Jα/δ may depend on the distance between the involved Vα/δ and Jα/δ at least in B‐lineage cells. These rearrangements in B‐precursor cells appear to be aberrant. However, this recombinational process may be one of the normal differentiation pathways in T‐lineage cells, because cells with a Vδ2(Dn)Dδ3 rearrangement were detected in 0·1–0·01% of normal peripheral mononuclear cells by the polymerase chain reaction.
Expression of coagulation factor V gene by normal adult human hepatocytes in primary culture
Authors: Mazzorana Marlene , Baffet Georges , Kneip Bernard , Launois Bernard , Guguen‐Guillouzo Christiane
Published: March 2008

Summary Normal human adult hepatocytes were examined for their ability to synthesize and secrete factor V using primary culture. The culture medium contained both factor V and factor Va as determined by bioassay and activation experiments. Immunoprecipitation of newly synthesized labelled factor V showed the presence of both native factor V (m.w. 330000) and two fragments of respective molecular weight 300000 and 265000. Northern blot analysis revealed the presence of a single 7 kb factor V mRNA in cultured human hepatocytes as in liver biopsies, together with fibrinogen β and albumin transcripts. Relative levels of factor V, fibrinogen β and albumin mRNAs differed when the cells cultured, suggesting that expression of the three corresponding genes might in part be independently regulated. Furthermore, addition of glucocorticoids enhanced factor V and fibrinogen β mRNA levels 1·6‐ and 5‐fold respectively, but did not significantly increase that of albumin.

These results provide evidence that human hepatocytes actively participate in the synthesis of plasma factor V and constitute a valuable model to study the common and specific regulations involved in the control of the expression of this gene in human liver.
In vitro assessment of marrow ‘stem cell’and stromal cell function in aplastic anaemia
Authors: Marsh J. C. W. , Chang J. , Testa N. G. , Hows J. M. , Dexter T. M.
Published: March 2008

Summary An in vitro model system is described that allows separate assessment of ‘stem cell’and stromal cell function in aplastic anaemia (AA). Seven patients with non‐severe AA, who had responded to immunosuppressive therapy and had haematological evidence of residual marrow function, were studied. Of these, three with otherwise typical AA had an acquired clonal cytogenetic marker. Purified bone marrow haemopoietic progenitors labelled with CD34 monoclonal antibody were positively selected using the fluorescence activated cell sorter (FACS) from both normal subjects and from patients with AA. The generative capacity of the CD34 positive cells was assessed by monitoring the output of granulocyte/macrophage colony forming cells (CFU‐GM) in the non‐adherent layer after inoculation onto irradiated preformed long‐term marrow culture (LTBMC) stromas. Stromal function in AA was assessed by inoculating CD34 positive cells from normal bone marrow onto preformed irradiated stromas from patients with AA. Haemopoietic cell (‘stem cell’) function in AA was assessed by inoculating CD34 positive cells from AA patients onto confluent irradiated normal marrow stromas. Using these crossover/LTBMC experiments, all patients exhibited severe defects in haemopoietic cell function with normal functioning stroma. The proportion of CD34 positive cells present in bone marrow from these patients was reduced compared with controls, they comprised fewer small primitive ‘blast‐like’cells which in normal bone marrow are known to possess marrow repopulating ability, and demonstrated reduced clonogenic potential in short‐term colony assays.
The anti‐mitozantrone monoclonal antibody NO‐1, protects acute leukaemia cell lines from the cytotoxic effects of mitozantrone
Authors: Flavell S. U. , Flavell D. J.
Published: March 2008

Summary. The monoclonal antibody NO‐1 was raised against the potent anti‐cancer drug mitozantrone by immunization of a BALB/c mouse with a mitozantrone–keyhole limpet haemocyanin conjugate in Freund's complete adjuvant. This antibody was shown to be highly effective in vitro at neutralizing the cytotoxic effects of mitozantrone for the acute leukaemia cell lines ALL‐1 and MOLT4. In order to achieve complete protection, a drug to antibody molar ratio of 1.5:1 was required. The neutralizing effect was specific for mitozantrone, as NO‐1 antibody offered no protection of the MOLT4 cell line to the cytotoxic effects of the anthracycline drug daunorubicin when used at a near identical molar ratio. NO‐1 antibody has already proven a highly successful monoclonal reagent for use in a competitive enzyme‐linked immunosorbent assay for the accurate and sensitive quantitation of mitozantrone in serum. The neutralizing properties of NO‐1 suggest other possible applications for this antibody. These could include a use in the rapid clearance of pharmacologically active mitozantrone from the circulation following very high dose administration prior to bone marrow transplantation and for the construction of bispecific antibodies for targeting mitozantrone to tumour cell populations.
Activation of class I HLA expression by TNF‐alpha and gamma‐interferon is mediated through protein kinase C‐dependent pathway in CML cell lines
Authors: Seong David , Sims Simon , Johnson Elizabeth , Lyding Judith , Lopez Alfredo , Garovoy Marvin , Talpaz Moshe , Kantarjian Hagop , Lopez‐Berestein Gabriel , Reading Christopher , Deisseroth Albert
Published: March 2008

Summary. The combination of tumour necrosis factor alpha (TNFα) and gamma‐interferon induced transcription of class I HLA genes in chronic myelogenous leukaemia (CML) cell lines through the formation of a complex between nuclear proteins and the transcriptional enhancers associated with these genes. Although gamma‐interferon or TNF‐alpha stimulated expression of class I HLA antigens in the EM2 and K562 CML cell lines when used alone, the effect of the combination of TNF‐alpha and gamma‐interferon was greater than that observed with either agent alone. The induction of class I HLA expression by gamma‐interferon and TNF‐alpha was inhibited completely by the isoquinoline sulfonamide H7, an inhibitor of protein kinase C. We conclude that the enhancement of the gamma‐interferon induced transcriptional activation of class I HLA gene expression by TNF‐alpha involves a protein kinase C‐dependent pathway.
A phase II trial of recombinant human granulocyte colony‐stimulating factor in the myelodysplastic syndromes
Authors: Yoshida Yataro , Hirashima Kunitake , Asano Shigetaka , Takaku Fumimaro
Published: March 2008

Summary. We conducted a phase II study of the intravenous administration of a glycosylated recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) for 7–14 d in 41 patients with the myelodysplastic syndromes (MDS). Administration of rhG‐CSF elicited striking rises in both leucocyte and neutrophil counts in the majority of the patients irrespective of the FAB subtypes of MDS. The rises in neutrophil counts were dose dependent and 5 μg/kg/d of rhG‐CSF yielded approximately an 8‐fold increase in neutrophil counts. Leucocytes and neutrophil counts started to increase shortly after the first injection of 5 μg/kg, was maintained at significantly elevated levels during 14 d of treatment, and returned to the pretreatment levels within several days following discontinuation of rhG‐CSF. The action of rhG‐CSF was specific for neutrophils since leucocytosis was due exclusively to neutrophilic increase associated with an increased marrow myeloid maturation. There were no consistent changes in the monocyte, eosinophil, lymphocyte, platelet or reticulocyte counts. After treatment, the percentage of marrow blast cells was reduced in eight of 13 evaluable patients with refractory anaemia with an excess of blasts (RAEB) or RAEB in transformation (RAEB‐t). No patients developed acute leukaemia during the treatment or in the immediate follow‐up period. The treatment was well tolerated with only minimal toxicity. The results suggest that rhG‐CSF is a safe and effective way to promptly improve neutropenia in MDS patients.
Identification of a new haemophilia BM case produced by a mutation located at the carboxy terminal cleavage site of activation peptide
Authors: Solera J. , Magallón M. , Martin‐Villar J. , Coloma A.
Published: March 2008

Summary. We describe a novel point mutation due to C→G transversion at nucleotide 20518 in the exon VI of factor IX gene, resulting in the substitution of glycine (GGG) for arginine (CGG) at position 180 in the polypeptide. This point mutation was found in a patient with a haemophilia BM variant. We designated the altered factor IX produced by this new mutation as factor IXMadrid. This mutation blocks the cleavage site involved in the release of the activation peptide at Arg180–Val181. It also abolishes the Aval site (CTCGGG) in exon VI, which can be directly detected with the enzymatic DNA amplification technique (PCR) and offers the possibility of direct analysis in carrier and prenatal diagnosis in kindreds with this mutation.