bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of tissue factor on human monocytes by cisplatin and adriamycin
Authors: Walsh John , Wheeler Helen R. , Geczy Carolyn L.
Published: March 2008

Coagulation disorders have been associated with the use of chemotherapeutic drugs. Pharmacological doses of cisplatin and adriamycin directly induced low levels of procoagulant on normal human blood monocytes and on a human myelomonocytic cell line, RC2a. Activity was maximal after 24 h and was not due to cell lysis as increasing drug doses which decreased cell viability were less effective. Procoagulant induction was markedly enhanced in the presence of bacterial lipopolysaccharide (LPS), with as little as 10–100 pg/ml LPS potentiating the cisplatin response by 2–5‐fold and more than doubling the adriamycin response. Greater than 90% of the procoagulant activity was membrane‐bound tissue factor as indicated by the factor VII‐dependent generation of factor Xa by viable cells and by the neutralization of this activity by a monoclonal antibody to tissue factor. Tissue factor antigen was measured simultaneously by immunohistochemical staining and by cell ELISA. Blood monocytes activated with LPS expressed high levels of tissue factor antigen; by contrast, adriamycin and cisplatin did not appear to induce antigen expression, but to enhance the specific activity of that already present. Results suggest that membrane alterations which occur following treatment with DNA/RNA intercalating drugs, may result in a highly active form of monocyte/macrophage tissue factor which may contribute to the complications caused by activated coagulation. Secondary Gram‐negative infection or cyto‐kines released by an active immune response to a tumour may contribute to the procoagulant potential of these cytotoxic drugs.
Analysis of the TNF receptors on human leukaemia cells by affinity cross‐linking
Authors: Elbaz Osama , Mahmoud Lotfy A. , Touw Ivo P. , Löwenberg Bob
Published: March 2008

Tumour necrosis factor (TNF) affects the growth of human leukaemic cells by different modes of action depending on the type of leukaemia involved. We have analysed the structure of TNF receptors on cells from different types of leukaemia, including acute lymphoblastic leukaemia (ALL), acute myeloblastic leukaemia (AML), chronic lymphocytic leukaemia (CLL), and chronic myelocytic leukaemia (CML) either in chronic phase (CML‐CP) or blastic crisis (CML‐BC). The affinity crosslinking technique showed the existence of TNF receptors on cells from all the leukaemic cases studied with similar receptor structures. The TNF receptor showed a molecular weight of 76 kD when examined by sodium dodecyl sulphate–polyacrylamide gel electro‐phoresis. In conclusion, we provide evidence for existence of TNF receptors on several types of human leukaemia cells with an apparent molecular weight of 76 kD. Apparently, the discrepancy of TNF actions on the leukaemic growth are not related to the structure of TNF receptors.
HLA‐DR expression by platelets in acute idiopathic thrombocytopenic purpura
Authors: Boshkov Lynn K. , Kelton John G. , Halloran Philip F.
Published: March 2008

Induction of expression of MHC class II antigens on the surface of cells that do not ordinarily express these proteins has been implicated in the pathogenesis of autoimmunity in diabetes mellitus and autoimmune thyroiditis. Platelets express class I but not class II HLA antigens. In this report, we describe a child with acute idiopathic thrombocytopenic purpura who at the time of the thrombocytopenic episode had class II (HLA‐DR) antigens on his platelets. Following recovery, the HLA‐DR antigens were no longer present on the platelets. We postulated that class II had been induced on his megakaryocytes by a cytokine such as interferon gamma, and that the induced expression of class II antigens contributed to the autoimmune disorder. To substantiate this possibility we next studied class I and II antigen expression on an erythroleukaemia cell line (HEL), which has many megakaryocytic features. Following treatment of HEL cells with interferon gamma, class I expression was increased and HLA‐DR antigens were induced. These observations suggest that cytokine‐mediated induced HLA‐DR expression may contribute to the pathogenesis of a subset of thrombocytopenias.
The spectrum of β thalassaemia in Burma
Authors: Brown Jennifer M. , Thein Swee Lay , Weatherall David J. , Mar Khin Mar
Published: March 2008

The molecular defects causing β thalassaemia have been analysed in 85 unrelated Burmese patients. The patients included 14 with homozygous β thalassaemia, 70 with HbE/β thalassaemia and one with HbS/β thalassaemia. Using a combination of allele‐specific oligoprobe hybridization and direct sequencing of genomic DNA amplified by the polymerase chain reaction, 95/99 of the β‐thalassaemia alleles have been characterized. Six mutations have been identified of which three, the G‐T at IVS‐1 position 1, the G‐C at IVS‐1 position 5 and the deletion of TCTT in codons 41/42, accounted for 85% of the alleles. Despite the diversity of ethnic groups in Burma, the number of β‐thalassaemia alleles in Burma is relatively small. Thus, diagnosis of the majority of the β thalassaemias would be possible using a limited number of oligonucleotide probes.
Rheological changes in the prodromal and established phases of sickle cell vaso‐occlusive crisis
Authors: Akinola N. O. , Stevens S. M. E. , Franklin I. M. , Nash G. B. , Stuart J.
Published: March 2008

A rheological study has been made in 20 patients with sickle cell anaemia in the steady state and in the prodromal and established phases of 12 vaso‐occlusive crises. Rheology of sickle cells was studied by discontinuous density gradient fractionation and by filtration through pores of 5μm diameter. The prodromal phase of crisis (day 1), when compared with mean steady state values, was associated with the development of a sub‐population of poorly deformable dense cells. This sub‐population appeared 1 or more days before the acute‐phase rise in C‐reactive protein, orosomucoid, fibrinogen, plasma viscosity and leucocytes, and before the rise in serum lactate dehydrogenase. As crisis evolved, the sub‐population decreased to steady‐state values, or below, by days 6–7. Identification of the prodromal phase of sickle cell crisis has allowed the detection of rheological changes of potential aetiological significance.
The influence of iron deficiency on erythrocyte deformability
Authors: Reinhart Walter H.
Published: March 2008

Summary The influence of iron deficiency on erythrocyte deformability is controversial. The present study was designed to analyse cell deformability in 14 patients with iron deficiency and controls in a comprehensive way by three different methods, namely erythrocyte filtration, erythrocyte elongation, and measurement of membrane elasticity. Suspensions of washed erythrocytes (haematocrit 0.10) free of leucocytes were used. Erythrocyte deformability measured by filtration was increased by iron deficiency: The relative filtration resistance of a cell in a 3 μm pore was 26.5 ± 6.9 and 75.8 ± 23.8 in iron deficiency and controls, respectively (P<0.0001). In 5 μm pores the values were 2.80 ± 1.23 and 3.46 ± 0.51 (not significant); when the red cell number/volume was adjusted to that in control samples, the value for iron deficiency became significantly lower than in controls (2.32 ± 0.60, P<0.0001). Erythrocyte elongation by centrifugation was unaffected (ratio length/width 1.66 ± 0.11 and 1.60 ± 0.10 in iron deficiency and controls, respectively). Membrane elasticity, as assessed by a filter aspiration technique, was also unchanged (membrane elastic modulus 3.94 ± 0.31 and 3.94 ± 0.37 ± 10‐3 dyn/cm, respectively). It is concluded that iron deficiency does not affect erythrocyte membrane elasticity and that the deformability of whole cells is not impaired, but improved under certain conditions such as the passage of 3 μm pores because of microcytosis with preserved surface/volume ratio. These results are in contrast to earlier studies and they have pathophysiological and clinical implications.
Ouantitation of granulocyte antibodies in sera and determination of their binding sites
Authors: Bux J. , Sohn M. , And R. Hachmann , Mueller‐Eckhardt C.
Published: March 2008

Summary An enzyme immunoassay using eluates was developed for the quantitation of granulocyte antibodies in sera. Incubation of donor granulocytes with sera containing granulocyte alloantibodies (NA1, NA2, NB1, 5b. HLA) or autoantibodies resulted in a significantly higher amount of immunoglobulin G (IgG) per cell than did incubation with control sera. Ultracentrifugation of the sera prior to testing reduced unspecific binding of IgG to granulocytes. In eight of 10 assessed sera from patients with Felty's syndrome eluted IgG decreased from elevated to normal levels. Ultracentrifugation further allowed determination of the binding sites of granulocyte‐reactive alloantibodies. Using sera containing NA1‐and NA2‐specific alloantibodies 173000–188000 binding sites on homozygous and 84000–110000 on heterozygous cells were determined. A similar number was found using a Fc gamma receptor III (FcRIII)‐speciflc monoclonal antibody. Granulocytes from patients with paroxysmal nocturnal haemoglobinuria showed greatly reduced binding sites for NA‐specific alloantibodies. The binding sites for the human alloantibody NB1 ranged from 36000 to 318000 and for the 5b alloantibody from 44000 to 65000. Mean values of 139000 binding sites for HLA antigens and 27000 binding sites for the FcRII were determined using monoclonal antibodies.
Von Willebrand disease family studies: comparison of three methods of analysis of the von Willebrand factor gene polymorphism related to a variable number tandem repeat sequence in intron 40
Authors: Gaucher Christine , And Bernard Mercier , Mazurier Claudine
Published: March 2008

Summary A region with a variable number of tandem ATCT repeats (VNTR) has previously been localized within intron 40 of the von Willebrand factor (vWF) gene. In the present report we describe the use of this polymorphism as a genetic marker to study the inheritance pattern in five families affected with various types of von Willebrand disease (vWD): types I, IIA, IIB, IIC and the newly characterized variant with totally defective FVIII binding. Three means of investigation previously reported, all using polymerase chain reaction (PCR) amplification of this vWF gene region, were compared in terms of informativeness. The two direct single‐step procedures analysing only partial sequences of the VNTR region turned out to be less informative (three studies informative out of five) than the third method characterizing the variability of the whole VNTR sequence. This latter approach, based on the analysis of the Alu I restriction pattern of the VNTR region, was informative in all the families investigated, therefore avoiding the need to combine it with other genetic marker studies for efficient gene tracking. In conclusion, this two‐step (PCR and digestion) method is the most informative for the characterization of the inheritance of the different subtypes of vWD and for the prenatal diagnosis of its severe forms.
Australian β0‐thalassaemia: a high haemoglobin A2β0‐thalassaemia due to a 12 kb deletion commencing 5′to the β‐globin gene
Authors: Motum Penelope I. , Lindeman Robert , Hamilton Tracey J. , Trent Ronald J.
Published: March 2008

Summary A large novel deletional β0‐thalassaemia mutation associated with unusually high levels of haemoglobin A2 in heterozygotes is described in an Australian family. The deletion was characterized by restriction enzyme analysis followed by PCR amplification and sequencing of the breakpoint region. Australian β0‐thalassaemia extends from 835 basepairs (bp) 5’to the cap site of the β‐globin gene downstream for 12.023 kb. This deletion, similar to previously described deletional β0‐thalassaemias associated with high Hb A2, removes sequences 5’to the β‐globin gene promoter and emphasizes the functional importance of the 5’β‐globin region in eliciting the unusually high Hb A2 phenotype.
Detection of activity of P‐glycoprotein in human tumour samples using rhodamine 123
Authors: Ludescher Christof , Thaler Josef , Drach Doris , Drach Johannes , Spitaler Martin , Gattringer Claus , And Heinz Huber , Hofmann Johann
Published: March 2008

Summary Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P‐glycoprotein (P‐gp), we developed a functional flow cytometric assay for the detection of multidrug‐resistant (MDR) cells. Using drug sensitive cell lines (KB‐3–1) and MDR mutants (KB‐8–5, KB‐C1) experimental conditions were established that enabled demonstration of significant differences in Rhl23 efflux and accumulation. Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples. Using two‐colour flow cytometry, the leukaemic blast cells of six patients suffering from acute myeloid leukaemia (AML) were analysed. In three cases the blast cells showed a rapid and marked Rh123 efflux. In the presence of MDR inhibitors these cells retained Rh123. To determine whether the efflux of Rhl23 was associated with P‐gp expression, the leukaemic cells were stained with the monoclonal antibody MRK‐16. In addition extracted RNA was analysed by polymerase chain reaction to evaluate the expression of mdr 1 mRNA. In all three Rh123+ cases mdr 1 mRNA was detectable whereas only one AML case expressed P‐gp. In comparing Rh123 with daunorubicin, which also allows the detection of MDR cells, accumulation studies proved Rh123 to be the more sensitive drug for flow cytometric MDR screening. Additionally, two‐colour flow cytometry was much easier to perform with Rh123 than with daunorubicin.

Our results indicate that flow cytometric measurement of Rh123 accumulation/efflux proves applicable to detect MDR cells in heterogenous clinical samples.
Second transplants for patients with chronic myeloid leukaemia in relapse after original transplant with T‐depleted donor marrow: feasibility of using busulphan alone for re‐conditioning
Authors: Cullis J. O. , Schwarer A. P. , Hughes T. P. , Hows J. M. , Franklin I. , Morgenstern G. , Goldman J. M.
Published: March 2008

Summary. Between July 1986 and March 1991, 16 patients who had relapsed after T‐lymphocyte depleted bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML) received a second transplant using unmanipulated marrow cells from the same HLA‐identical sibling donor. The median numbers of days from first BMT to haematological relapse and to second BMT were 557 (range 273–1543) and 1211 (range 476–2310) respectively. 11 patients were in uncomplicated chronic phase at time of second BMT, and five had more advanced disease. As conditioning for second BMT, eight patients received various combinations of cytotoxic drugs, and eight received high‐dose busulphan alone. Eight (50%) patients survive at a median of 424 d post‐second BMT (range 158–1789) and all are free of leukaemia by conventional criteria: five had been conditioned with high‐dose busulphan alone. Causes of death in the eight patients who died included relapse (n= 2), graft‐versus‐host disease (n= 2). interstitial pneumonias (n= 2), and infection (n= 2). We conclude that patients relapsing into chronic phase after BMT with T‐lymphocyte depleted donor marrow may be offered the option of second BMT with unmanipulated marrow from the original donor. Conditioning with high‐dose busulphan alone may be safer than use of more intensive schedules.
Mononuclear phagocyte assays, AutoAnalyzer quantitation and IgG subclasses of maternal anti‐RhD in the prediction of the severity of haemolytic disease in the fetus before 32 weeks gestation
Authors: Garner S. F. , Wiener E. , Contreras M. , Nicolini U. , Kochknour N. , Letsky E. , Rodeck C. H.
Published: March 2008

Summary. In 40 cases of haemolytic disease of the fetus due to RhD immunization where the fetus required intrauterine transfusion, fetal packed cell volume was compared with the following parameters of maternal anti‐D: (a) concentration, (b) IgG subclass, (c) activity in a macrophage binding assay, and (d) activity in a monocyte antibody‐dependent cell mediated cytotoxicity (ADCC) assay. The anti‐D concentration exceeded 4 iu/ml in all cases, correctly indicating the risk of haemolytic disease. A relationship between IgG subclass composition of the anti‐D and severity of anaemia was not observed; IgGl, IgG3 and IgG 1 + 3 antibodies were all detected. The AüCC assay gave the best correlation between assay results and fetal packed cell volume: high results correctly indicated fetal anaemia in 95% of cases. Macrophage binding assay results were only considered as high in 70% of cases. Overall, these results indicate that serological tests and bioassays in highly immunized mothers may not generate any information that proves more useful than ultrasonography and their previous obstetric history.
Recombinant human granulocyte‐macrophage colony‐stimulating factor after autologous bone marrow transplantation for malignant lymphoma: a British National Lymphoma Investigation double‐blind, placebo‐controlled trial
Authors: Khwaja A. , Linch D. C. , Goldstone A. H. , Chopra R. , Marcus R. E. , Wimperis J. Z. , Russell N. H. , Haynes A. P. , Milligan D. W. , Leyland M. J. , Winfield D. A. , Hancock B. W. , Newland A. , Dürrant S. T. , Devereux S. , Roitt S. , Collins M. , Hudson G. Vaughan
Published: March 2008

Summary. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is active in enhancing the production of mature myeloid cells in vitro and several phase I/II clinical trials have suggested that its administration may accelerate neutrophil recovery after autologous bone marrow transplantation (ABMT). We have conducted a multicentre randomized double‐blind placebo controlled trial in patients with poor prognosis malignant lymphoma receiving an identical high‐dose combination chemotherapy regimen with ABMT. 61 patients were entered and 29 in each arm of the trial were evaluated. Treatment with GM‐CSF did not affect the period of severe neutropenia (absolute neutrophil count (ANC) of <0.1×109/1) but accelerated recovery to an ANC of 0.5 ×109/1(median 14d v 20 d in controls, P= 0.001). There was no significant difference in platelet recovery between the groups (GM‐CSF group platelet dependent for 25 d v control 19 d, P= NS). The number of positive blood cultures was similar in both groups (GM‐CSF 14 v placebo 13) and there were no differences in days of fever > 37.5°C (median 8 v 6) or days on parenteral antibiotics (11 v 10). Patients receiving GM‐CSF had a median period of hospitalization following BMT of 24 d (control 25). No significant major toxicity attributable to GM‐CSF administration was detected. We have confirmed in a randomized trial that GM‐CSF accelerates neutrophil but not platelet recovery following ABMT. We were unable to demonstrate any accompanying changes in clinical outcome and believe that further trials are necessary to assess the clinical value of GM‐CSF in BMT.
Re‐emergence in remission of primary clone in acute myelogenous leukaemias with multiple chromosomal aberrations at diagnosis
Authors: Heim Sverre , Sørensen Anne‐Grethe , Christensen Bjarne Egelund , Pedersen Niels Tinggaard
Published: March 2008

Summary. We describe the clinical, haematological and cytogenetic features of three patients who had acute myelogenous leukaemia (AMD with complex bone marrow karyotypes when first cytogenetically examined. Induction chemotherapy led to remission from the acute leukaemia. However, neither clinically nor morphologically did this remission mean a return to normal haematopoiesis. The two patients who displayed myelodysplastic features before and when AML was diagnosed, again developed myelodysplasia. and the third patient, who had a long history of polycythaemia vera, returned to this myeloproliferative condition. Nor was cytogenetic normalization achieved: instead, abnormal cell clones were found in which all but one of the karyotypic aberrations present at acute leukaemia diagnosis had disappeared. The solitary anomalies that were detected in these reemerging clones must correspond to the primary cytogenetic aberrations of the patients pre‐leukaemic diseases. They were del(5) (q11q33) and del(17) (p11) in the two myelodysplastic cases, and der(18)t(9;18) (p11;p11) in the patient with long‐standing polycythaemia vera. The other, secondary, aberrations were probably the leukaemogenic changes, and with the eradication or reduction of the subclones containing them, the leukaemic phenotype disappeared. The three cases add cytogenetic evidence to the growing understanding that the remission obtained in some AMLs is actually a return to a preleukaemic, myeloproliferative or myelodysplastic. syndrome.
Biological characteristics of CD7 positive acute myelogenous leukaemia
Authors: Eto Tetsuya , Akashi Koichi , Harada Mine , Shibuya Tsunefumi , Takamatsu Yasushi , Teshima Takanori , Niho Yoshiyuki
Published: March 2008

Summary. We studied the biological characteristics of CD7+ acute myelogenous leukaemia (AML). We diagnosed nine out of 88 consecutive AML cases as CD7+ AML based on myeloperoxidase positivity and surface antigen expression. In eight of these nine cases more than 20% of leukaemic blasts were found to coexpress both CD7 and a myeloid‐associated antigen, CD33, by a two‐colour flowcytometric assay, while in the remaining case more than 90% of blasts were positive for CD7 and myeloperoxidase. CD7+ AML was most frequently observed in M1 among AML subtypes according to the FAB classification. An early stage‐specific antigen, CD34 was also expressed on leukaemic blasts from eight of these nine cases. Neither the T‐cell receptor (TcR)‐β nor the TcR‐γ gene was clonally rearranged in any of the cases. We then studied the proliferative responses to stimulation by various growth factors. Among interleukin‐3 (IL‐3), granulocyte macrophage colony‐stimulating factor (GM‐CSF), and granu‐locyte‐CSF (G‐CSF), IL‐3 showed the strongest stimulatory effect on DNA synthesis and leukaemic blast colony formation in 8/9 and 6/8 CD7+ AML cases examined, respectively. On the other hand, the strongest stimulatory effect exerted by IL‐ 3 on blast colony formation was observed in only six out of the 33 CD7− AML cases examined. Furthermore, CD7+ AML blasts could proliferate in response to stem cell factor (SCF); SCF alone showed stimulatory effects on blast colony formation (7/8 cases), and in 5/7 SCF‐responding cases, stimulatory effects of SCF were more potent than those of IL‐3. In addition, SCF enhanced blast colony formation synergistically with IL‐ 3 in four of these seven cases. These data suggest that progenitor cells of CD7+ AML may possess the biological properties characteristic of immature haematopoietic stem cells.
Modified adriamycin‐vincristine‐dexamethasone (m‐VAD) in primary refractory and relapsed plasma cell myeloma: an NCI (Canada) pilot study
Authors: Browman George P. , Belch Andrew , Skillings Jamey , Wilson Kenneth , Bergsagel Daniel , Johnston Dianne , Pater Joseph L.
Published: March 2008

Summary. The purpose of this single arm phase II study was to test a modified version of the three drug combination vincristine, adriamycin and dexamethasone (m‐VAD), in which intravenous vincristine (0·4 mg/d) and adriamycin (9 mg/m2 per day) infusions are administered for only 2 h on days 1–4 of each 28 d cycle, in patients with refractory multiple myeloma. In addition, only two 4 d courses of dexamethasone 40 mg/d was given during each cycle. The entry criteria for 44 patients included plasma cell myeloma and a measurable monoclonal peak, either refractory to initial treatment with melphalan and prednisone, or resistant to melphalan and prednisone after initially responding (resistant relapsed disease, 27 patients). Patients treated previously with chemotherapy other than melphalan and predisone were excluded. There were no complete responses. Of the 41 evaluable patients who completed at least one course of therapy 11 had a partial response (27%, 95% C.I. 14–40%). The response rates were 19% for primary refractory disease patients, and 32% for those with resistant relapsed disease. The median duration of response was 4 months. The median survival for all 44 patients was 7·6 months (5·5 months for primary refractory patients, and 10 months for relapsed resistant disease patients). Episodes of documented bacterial infection occurred in 12 patients, and 10 patients had minor viral infection. The dexamethasone dose was reduced in 12 patients. The median neutrophil nadir was 1·2 × 109/l, and median platelet nadir was 147 × 109/l. Five deaths were judged as treatment related and occurred during marrow cytopenia. The results of this modified form of VAD are inferior to that reported previously for 4 d continuous infusions of vincristine and doxorubicin. This could be related to either patient selection factors, or to a reduction of the efficacy of the drug combination produced by either the shortened intravenous infusions and/or omission of one 4 d course of dexamethasone.
Beta thalassaemia in the indigenous British population
Authors: Hall G. W. , Barnetson R. A. , Thein S. L.
Published: March 2008

Summary. We have analysed the molecular basis of β‐thalassaemia in 22 Anglo‐Saxon individuals, all of whom were heterozygous for β‐thalassaemia except for one, who was a compound heterozygote. Using a combination of allele‐specific priming of the polymerase chain reaction (PCR) and direct sequencing of genomic DNA amplied by the PCR, 20/ 23 β‐thalassaemic genes were characterized. Nine different mutations were identified; four are commonly found in the Mediterranean, one in Asia, one has been described previously in both Europe and Asia, and three are rare mutations associated with a dominant β‐thalassaemia phenotype. In three individuals the mutation remains uncharacterized despite sequence analysis of the β2‐globin gene and its immediate flanking regions. We report our findings and discuss the diversity of these mutations.
Polyethylene glycol (PEG) modification of granulocyte‐macrophage colony stimulating factor (GM‐CSF) enhances neutrophil priming activity but not colony stimulating activity
Authors: Knüsli Claudio , Delgado Cristina , Malik Farooq , Dómine Manuel , Tejedor M. Cristina , Irvine Alexandra E. , Fisher Derek , Francis Gillian E.
Published: March 2008

PEG‐modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but, almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG‐proteins, we compared PEG‐modified and unmodified GM‐CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG‐GM‐CSF was unexpectedly more active than GM‐CSF in its ability to prime neutrophils to respond to the synthetic peptide n‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM‐CSF induced thymi‐dine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG‐GM‐CSF was confirmed using FPLC fractionated PEG‐modified GM‐CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG‐modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG‐modified fetal calf serum proteins, carrier proteins and‘irrelevant’cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG‐modification of GM‐CSF. The demonstration that PEG‐modification can partially dissociate two biological activities suggests the feasibility of using PEG‐modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.
Effects of recombinant human granulocyte colony‐stimulating factor on neutrophil functions in aged animals
Authors: Yoshino Takeshi , Tamura Masahiko , Kawabe Mari , Nomura Hitoshi , Imai Nobuo , Ono Masayoshi
Published: March 2008

We have studied the effects of recombinant human granulocyte colony‐stimulating factor (rG‐CSF) on granulopoiesis and neutrophil functions in aged rats and aged mice. We subcutaneously injected rG‐CSF or control vehicle into aged rats (22 months old and 25 months old) for 7 consecutive days. counted the peripheral neutrophils and evaluated the functions of neutrophils isolated from venous blood. The peripheral neutrophil count in aged rats tended to be increased as compared with that in young rats (11 weeks old). However, the neutrophils in aged rats exhibited a decline of superoxide anion (O2‐‐) release and phagocytic activity as compared with young rats. The peripheral neutrophil count in aged rats was significantly increased 5–6‐fold as many as the control value by rG‐CSF treatment, which was accompanied by a significant enhancement of O2‐release and of phagocytic activity being restored to normal levels or better.

In another series of experiments, we subcutaneously injected rG‐CSF or control vehicle into aged mice (24–28 months old) or young mice (8 weeks old) for 7 consecutive days, and evaluated the functions of neutrophils isolated from peritoneal cavity. The peritoneal exudate neutrophils from the aged mice exhibited a decline of phagocytic and chemo‐tactic activity as compared with the young mice. These functions in both young and aged mice were significantly enhanced by rG‐CSF‐treatment, and these functions in rG‐CSF‐treated aged mice were restored to a level higher than the level in control young mice.

These findings demonstrate that rG‐CSF is capable of enhancing granulopoiesis and restoring the age‐related decline of neutrophil functions.
Long‐term treatment results for acute megakaryoblastic leukaemia patients: a multicentre study
Authors: Ruiz‐Argüelles Guillermo J. , Lobato‐Mendizábal Eduardo , San‐Miguel Jess F. , González Marcos , Caballero María D. , Ruiz‐Argüelles Alejandro , Orfao Alberto , Gómez‐Almaguer David , Vidriales Belén , Ruiz‐Reyes Guillermo , López‐Borrasca Antonio
Published: March 2008

The prognosis and long‐term results of a group of 57 acute megakaryoblastic leukaemia (M7‐AML) patients was analysed from a multicentre perspective. Ages ranged from 4 to 83 years, median 49 years: 30 were males and 27 were females. The median follow‐up time was 7 months, range 1–24 months. Early exits occurred in 12 cases, their median age being 71 years. Forty‐five patients were treated with combined aggressive chemotherapy (CT) (n= 26) or low‐dose cytarabine (LD‐AraC) (n= 19). The following results were obtained with combined CT or AraC, respectively. Complete remission (CR) rates were 73% and 84%. 12‐month survival (SV) were 37% and 26%, 24‐month SV were 12% and 11%. median SV 10 and 4 months, and relapse rates (RR) were 68% and 94%. These differences were not statistically significant. Irrespective of the treatment modality, the results were better for children (n = 10) than for adults (n = 35): RR rates were 90% and 74%, median SV: 7 and 5 months, 12‐month SV: 40% and 22%, 24‐month SV: 30% and 9%, and RR: 78% and 81%. respectively: these differences also were not statistically significant. In addition, a literature review of 42 patients from 18 previous reports is presented, including seven cases treated with allogeneic bone marrow transplantation (BMT). The best results were obtained with BMT: 12 and 24 month SV was 86% and the RR was 0%. On the above‐mentioned basis, we feel that children and young adults with M7‐AML should be offered BMT. In patients over 60 years old or not eligible for aggressive chemotherapy or BMT, an interesting possibility would be the use of LD‐AraC which allows a high CR rate, followed by a classical consolidation regimen in order to prevent early relapses.
Follicular dendritic cells in non‐Hodgkin‐lymphoma express adhesion molecules complementary to ligands on neoplastic B‐cells
Authors: Petrasch Stephan , Kosco Marie , Schmitz Jörn , Heinrich Wacker Hans , Brittinger Günter
Published: March 2008

In non‐Hodgkin‐lymphoma (NHL) with nodular growth patterns, follicular dendritic cells (FDC) form a spherical network which contains neoplastic B‐cells. In order to dissect the basis of this close FDC/B cell association, the antigenic profile of adhesion molecules expressed by individual FDC and NHL‐B‐lymphocytes was evaluated. FDC isolated from NHL were found to express C3bi receptors (CD11b), the very late antigen (VLA) alpha‐5– and alpha‐6‐chain (CD49e. CD49f), and the intercellular adhesion molecule‐1 (ICAM‐1; CD54). Only a percentage of the FDC population was positive for the VIA beta‐1– and alpha‐3‐chain (CD29. CD49c). the vitronectin receptor (CD51) and the vascular cell adhesion molecule‐1 (VCAM‐1). B‐cells obtained from the lymph nodes of patients with centroblastic‐centrocytic lymphoma expressed several ligands complementary to the adhesion molecules detected on FDC. These include LFA‐1 alpha‐ and beta‐chains (CD11a, CD18), and ICAM‐1 (CD54). Surprisingly. monoclonal lymphocytes in the peripheral blood of patients with a leukaemic course of this lymphoma entity were devoid of these antigens. It seems likely then that neoplastic B‐cells without CD11a, CD18 and CD54 surface molecules are unable to associate with FDC and now invade other compartments. Thus, the adhesive interactions which do occur between FDC and NHL‐B‐cells may directly influence the peculiar growth pattern and spread of centroblastic‐centrocytic lymphoma.
Adhesion efficiency, platelet density and size
Authors: Polanowska‐Grabowska Renata , Raha Sanghamitra , Gear Adrian R. L.
Published: March 2008

We have previously shown that adhesion of human platelets to immobilized collagen is extremely rapid, with initial rates approaching 3% of single particles adhering per 10 ms. Here, we have investigated adhesion efficiency to collagen as a function of platelet density. Platelet subpopulations: low‐density (1.040d< 1.065 g/ml), intermediatedensity (1.065 d< 1.070 g/ml) and high‐density (1.070 d< 1.080 g/ml) were separated by Percoll density gradient centrifugation. They constituted 24%, 47% and 29% of the total platelet population and had mean volumes of 6.01, 7.37 and 8.21 fl, respectively. Using a continuous‐flow, micro‐affinity column, we found that the most dense (large) platelets exhibited initial rate of adhesion 4 times greater than the least dense (small) platelets. They were also less sensitive to inhibition by prostacyclin (PGIJ. In contrast, there was no significant difference in aggregation induced by high doses of ADP and collagen, indicating that the most dense platelets were not preferentially involved in aggregation induced by high doses of agonists. These results suggest that normal circulating platelets can be distinctly heterogeneous in their ability to adhere to collagen under arterial‐flow conditions. The greater efficiency of high‐density platelets may be related to increased content of the glycoprotein Ia/IIa (GPIa/IIa) complex.
Molecular characterization of a novel 10.3 kb deletion causing β‐thalassaemia with unusually high Hb A2
Authors: Craig Jamie E. , Kelly Susan J. , Barnetson Rebecca , Thein Swee Lay
Published: March 2008

A family of Asian‐Indian descent has a variant form of β‐thalassaemia characterized by unusually high levels of Hb A2 in the heterozygous state. The propositus who is homozygous for the mutation has thalassaemia intermedia. Restriction endonuclease mapping suggested the presence of a 10.3 kilobase (kb) deletion removing the whole of the β‐globin gene. Subsequently, molecular analysis was performed by directly sequencing a specifically amplified region of genomic DNA. A 10329 basepair deletion was precisely defined which results in the loss of the 5’β promoter region and the entire β‐globin gene. The deletion extends from 3011 bp 5’to the mRNA cap site to an L1 repeat element downstream of the β‐globin gene and is very similar to the 12.6 kb deletion of Dutch β‐thalassaemia. In common with four other mutations, both these deletions remove the 5’promoter region of the β gene and all are associated with unusually elevated levels of Hb A2 in the heterozygous state.
Immunosuppressive property of a very high purity antihaemophilic preparation: a low molecular weight component inhibits an early step of PHA induced cell activation
Authors: Vermot‐Desroches Claudine , Rigal Dominique , Blourde Christian , Bernaud Jeanine
Published: March 2008

Immune deficiency has been reported in haemophiliac patients receiving antihaemophilic factor VIII preparations, but the mechanisms involved in the immunosup‐pression are not fully understood. By using the proliferative response of peripheral blood mononuclear cells to phyto‐haemagglutinin (PHA) as a test system, we investigated the inhibitory influence of a very high purity antihaemophilic factor (AHF) preparation on T cell proliferation and on T lymphocyte activation molecules. We observed that this preparation reduced significantly the PHA‐induced mono‐nuclear cell proliferation, independently of the monocyte concentration. The AHF preparation did not act through a cytotoxic mechanism or a steric hindrance of PHA. The AHF preparation had no effect on the immediate expression of T lymphocyte activation molecules such as CD54 (ICAM‐1). In contrast, the very high purity AHF reduced the induced expression of two early T cell activation molecules: CD25 (interleukin‐2 receptor) and CD71 (transferrin receptor). The very high purity AHF also had the capacity to inhibit the up‐regulation of two late activation antigens, CD38 and CD11a/CD18, and to inhibit the induced expression of HLA‐DR molecule, defined also as a late T cell activation molecule. The CD45R expression level, used as a control marker, was not changed after AHF exposure. The very high purity AHF therefore influenced an early step of cell proliferation.

We have also shown that the immunoregulatory properties of the preparation were not restricted to the factor VIII itself, but resulted from the presence of dialysable and low molecular weight components in the preparation.
Epidermolysis bullosa acquisita induced by GM‐CSF: a role for eosinophils in treatment‐related toxicity
Authors: Ward Jeffery C. , Gitlin Jeffrey B. , Garry Daniel J. , Jatoi Aminah , Luikart Sharon D. , Zelickson Brian D. , Dahl Mark V. , Skubitz Keith M.
Published: March 2008

A patient treated with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) developed eosinophilia and epidermolysis bullosa acquisita. The bullae were subepi‐dermal, and filled with an inflammatory infiltrate composed predominantly of eosinophils. Immunofluorescence studies disclosed linear deposition of IgG, IgA and C3 at the basement membrane zone and immunoelectron microscopy demonstrated antibody deposition in the lamina densa and sub‐lamina densa region; however, the patient's serum did not contain circulating antibody to basement membrane zone antigens. Staining with monoclonal antibodies revealed dense deposits of both eosinophil peroxidase and eosinophil major basic protein at the dermal–epidermal junction. The eosinophilia and skin lesions resolved upon discontinuation of GM‐CSF. This case provides evidence for two hypotheses: (1) GM‐CSF induced proliferation and activation of eosinophils may contribute to some of the toxicities of GM‐CSF treatment, and (2) activated granulocytes, including eosinophils, may mediate blister formation in epidermolysis bullosa acquisita.
Remission with morphological myelodysplasia in de novo acute myeloid leukaemia: implications for early relapse
Authors: Nagai Kazuhiro , Matsuo Tatsuki , Atogami Sunao , Moriuchi Yukiyoshi , Yoshida Yoshiharu , Kuriyama Kazutaka , Tomonaga Masao
Published: March 2008

Myelodysplastic features of remission bone marrow were investigated in 46 adults with de novo acute myeloid leukaemia (AML) according to the FAB morphological criteria for myelodysplastic syndromes (MDS). Compared with a group of 18 patients with the common type of acute lymphoblastic leukaemia in remission, micromegakaryo‐cytes (30·4%v. 5·6%, P<0·05), multi‐separated nuclear megakaryocytes (45·7%v. 0%, P<0·01), degranulated neutrophils (39·1%v. 5·6%, P<0·05), and neutrophils with hyposegmented nuclei (34·8%v. 0%, P<0·01) were significantly more common in the AML patients. In contrast, dyserythropoietic changes had a similar incidence in both groups. When compared with the clinical features at initial diagnosis in AML cases, the dysmegakaryocytic changes in remission marrow were found to be significantly more frequent in patients with monocytic involvement (mainly M4) or trilineage myelodysplasia (T‐MDS AML). Disease‐free survival was significantly shorter in patients with micro‐megakaryocytes (P<0·05) or neutrophils with hyposegmented nuclei (P<0·05) than in those without these features. Overall survival was also significantly shorter in patients with micromegakaryocytes (P<0·05). These findings may help in developing new strategies for the post‐remission therapy of AML, and also suggest that myelodysplastic changes in the remission marrow of de novo AML patients may be related to haematopoietic recovery by a preleukaemic clone which eventually leads to early leukaemic relapse. Dysplastic marrow, however, was not necessarily associated with peripheral pancytopenia in the present series, warranting basic research on such putative clonal remissions.
Comparison of chromosome analysis and BCL‐1 rearrangement in a series of patients with multiple myeloma
Authors: Fiedler W. , Weh H. J. , Hossfeld D. K.
Published: March 2008

Translocation (11;14)(q13;q32) is a recurring chromosome abnormality which is found non‐randomly in non‐Hodgkin's lymphoma, especially of follicular type, as well as in B‐cell chronic lymphocytic leukaemia, Waldenström's macroglobulinaemia and multiple myeloma. To define further the prevalence of this abnormality in multiple myeloma, we studied a series of 17 patients with this disease with concomitant chromosome analysis and Southern blotting, using a probe specific for the major translocation cluster of the BCL‐1 oncogene which is located at chromosome Hq13. Karyotype analysis of 14 evaluable patients revealed three cases with abnormalities of chromosome 11q13, two of them with t(11;14)(q13:q32) and one with del (11)(q13). Southern blot analysis showed no rearrangements in BclI and HindIII digests of DNA from 17 patients including the three patients with anomaly of chromosome 11q13, using a BCL‐1 specific probe. A possible restriction length polymorphism was detected in EcoRI cut DNA of five out of 11 patients studied. Therefore the chromosomal break point in our cases with abnormality of chromosome 11q13 must lie outside the major translocation cluster of the BCL‐1 gene. However, in centrocytic lymphoma rearrangement of the BCL‐1 oncogene has been detected in up to 50% of cases. Location of the break point may therefore be dependent on the differentiation of the transformed B‐cell.
Myelodysplastic syndrome (MDS)‐associated inhibitory activity on haematopoietic progenitor cells: contribution of monocyte‐derived lipid containing macrophages (MDLM)
Authors: Ohmori Masami , Ueda Yasunori , Masutani Hiroshi , Hirama Toshiyasu , Anzai Naoyuki , Yoshida Yataro , Okuma Minoru
Published: March 2008

We studied myelodysplastic syndrome‐associated inhibitory activity (MDS‐IA), which inhibited colony formation in vitro of normal granulocyte‐macrophage progenitors (CFU‐GM). When adherent marrow cells were incubated with fetal calf serum for 21–24 d. monocyte‐derived lipid containing huge macrophages (MDLM) developed. MDLM from MDS marrow (MDS‐MDLMs) and their conditioned medium (MDLM‐CM) consistently suppressed the growth of normal CFU‐GM colony formation. MDS‐IA was active on CFU‐GM during the S‐phase and relatively resistant to heating. Monoclonal antibody against H subunit (acidic) ferritin and polyclonal antibody against placental ferritin neutralized the inhibitory activity of MDS‐MDLMs. In addition, cell lysates of MDS‐MDLMs reacted to both monoclonal anti‐H subunit ferritin and polyclonal anti‐placental ferritin in Western blotting analysis, indicating that the inhibitory activity was predominantly acidic isoferritin.

On the other hand, MDLMs obtained from normal bone marrow had a CFU‐GM enhancing activity. These results suggest that MDS‐MDLMs may be responsible for the suppression of granulopoiesis in patients with MDS and that the suppression may be mediated by soluble factors, notably H subunit isoferritin.
A PCR‐based strategy to detect the common severe determinants of α thalassaemia
Authors: Bowden D. K. , Vickers M. A. , Higgs D. R.
Published: March 2008

A rapid and inexpensive polymerase chain reaction (PCR) based strategy is described which detects the three common, severe α thalassaemia determinants observed in southeast Asia (——SEA) and the Mediterranean (——MED and —(α)20.5). Oligonucleotide primers have been chosen which allow specific identification of both normal (αα) and abnormal (——) chromosomes using identical conditions in either the same or parallel PCR reactions. This strategy should be useful in the development of screening programmes to identify carriers of α thalassaemia (——/αα) and prenatal diagnosis of the Hb Bart's hydrops fetalis syndrome (——/——) for those populations in which this represents a major cause of perinatal death.
Incidence of AIDS in HIV‐1 infected thalassaemia patients
Authors: Costagliola Dominique G. , Girot Robert , Rebulla Paolo , Lefrère Jean‐Jacques
Published: March 2008

To estimate the cumulative incidence of acquired immunodeficiency syndrome (AIDS) in thalassaemia major patients (TMP) human immunodeficiency virus (HIV‐1) infected through transfusion, 79 seropositive TMP were studied. At inclusion, mean age was 12·4 + 6·6 years; 40 were men; 21 were splenectomized. Centers for Disease Control, 1986 (CDC) stages and prescription of zidovudine were noted at least once a year. Cumulative incidence of AIDS and standard error were calculated using non parametric life table method. Age, sex, acute infection and splenectomy associations with progression to AIDS were tested using Breslow statistic. The median follow‐up period was 4 years 11 months. At the end of the study period, 43 TMP were in CDC stage II, 23 in CDC stage III and 13 in CDC stage IV, including seven AIDS cases, of whom three had died. Four subjects died of other causes. Only two patients were treated with AZT prior to the occurrence of AIDS. Rate of progression to AIDS was not associated with acute infection, splenectomy, age, or sex. A cumulative AIDS incidence rate of 1·4% (SE 1·3%) was observed at 3 years and of 9% (SE 4%) at 5 years.
Cellular sites of extrarenal and renal erythropoietin production in anaemic rats
Authors: Schuster S. J. , Koury S. T. , Bohrer M. , Salceda S. , Card J.
Published: March 2008

The cellular origins of erythropoietin were investigated in the rat using a probe derived from a cloned rat erythropoietin cDNA. In anaemic‐hypoxic rat liver, in situ hybridization detected erythropoietin mRNA primarily in hepatocytes and less frequently in nonparenchymal sinusoidal or perisinusoidal liver cells. An RNase protection assay was used to compare the erythropoietin mRNA contents of separated rat liver cell fractions and also suggested that hepatocytes are the major source of extrarenal erythropoietin with nonparenchymal liver cells contributing less than 1% to total hepatic erythropoietin production. In kidney, in situ hybridization localized erythropoietin mRNA in nonepithelial cells, as yet of undefined lineage, in the cortical and outer medullary interstitium. These results indicate that, in the rat, the primary sources of erythropoietin in liver and kidney are different types of cells.