bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of advanced acute leukaemia with allogeneic bone marrow transplantation from unrelated donors
Authors: Schiller Gary , Feig Stephen A. , Territo Mary , Wolin Maurice Waurice , Lill Michael , Belin Tom , Hunt Lynne , Nime Stephen , Champlin Richard , Gajewski James
Published: March 2008

Summary. Bone marrow transplantation from a histocompatible donor may produce complete remission in patients with induction failure or relapsed acute leukaemia. Through the National Marrow Donor Program, histocompatible bone marrow from unrelated donors has become available for high‐risk patients. In this study we analyse the results of matched unrelated bone marrow transplant in 55 patients with highly advanced acute myelogenous and acute lymphoblastic leukaemia. 28 patients with advanced acute lymphoblastic leukaemia and 27 patients with advanced acute myelogenous leukaemia, age 2–51, were treated withy high‐dose chemoradiotherapy and transplantation of of 6/6 HLA matched (n= 46) or one antigen mismatched (n= 9) unrelated donor bone marrow. After a median follow‐up of 36 months, 13 patients remain alive 17–24 months after transplant for a 2‐year actuarial disease‐free and overall survival of 23 ± 12% (median disease‐free survival 3.5 months). The actuarial risk of relapse is 24 ± 16% at 1 year. Moderate to severe graft‐versus‐host disease occurred in 27/47 evaluable patients (57%). Significant prognostic factors for poor leukaemia‐free survival include age >21, abnormal karyotype, and active leukaemia at the time of transplant. Other pretreatment characteristics such as gender or type of leukaemia were not significant prognostic factors. Our results show that matched unrelated bone marrow transplant for patients with advanced acute bone marrow transplant for patients with advanced acute leukaemia may provide long‐term leukaemia‐free survival, but transplant‐related complications produce a sigbificant impact on survival with older age and adverse disease characteristics predicting for poor prognosis
Estimatetd 8‐year survival of more than 40% in a population‐based study of 79 adult patients with acute lymphoblastic leukaemia
Authors: Evensen S. A. , Brinch L. , Tjønnfjord G. , Stavem P. , Wislösff F.
Published: March 2008

Summary. We report 11 years experience with a modified version of a chemothearpy programme in use at the MRC Leukaemia Unit from 1982 to 1984, supplemented by allogeneic bone marrow transplantation in first relapse or second or later remission from 1985. 79 consecutive patients aged 15–60 years with newly diagnosed acute lymphoblastic leukaemia (ALL) were given induction chemotherapy. This included a stadard DAT course (aunorubicin, cytarabine and thioguanine) applied as In acute myelogenous leukaemia approximately midway in the induction programme. A 3‐year rotating maintenance programme consisted of combinations of cytotoxic drugs used in the induction therapy. CNS prophylaxis did not include CNS irrasiation. Allogeneic BMT was not performed in first remission.

The overall complete remision (CR) rate was 82% (65/79). 26 patients relapsed (seven first in the CNS). Seven patients underwent allogeneic BMT of whom six are alive and well with a mean observation time of 32 months (range 4–99 months) after transplantation. Three patients died in first CR. Estimated 5‐and 8‐year overall survival was 51% (95% confidence interval (CI) 39–63) and 47% (CI 33–61). For patients who reached CR, the corresponding figures were 63% (CI 50–76) and 57% (CI 41–73). Estimated disease‐free survival in the remitters was 54% (CI 40–68) at 5 years and 44% (CI 28–60) at 8 years. patient age below 25 years and white cell count below 15 * 109/1 at presentation were both found to improve the chance of overall survival.
Acute lymphocytic leukaemia in the elderly: characteristics and outsome with the vincristine‐adriamycin‐dexamethasone (VAD) regimen
Authors: Kantarjian Hagop M. , O'Brien Susan , Smith Terry , Estey Elihu H. , Berran Miloslav , Preti Alejandro , Pierce Sherry , Keating Michael J.
Published: March 2008

To analyse the pretreatment characteristics, responseto therapy, and overall prognosis of elederly patients with acute lymphocytc leukaemia (ALL). 268 consecutive adults with newly‐diagnosed ALL who received the vincristine‐adria‐mycin‐dexamethasone (VAD) regimens were reviewed; Pretreatment fharacteristics, response to VAD therapy, and overall outcomewere analysed in patients 60 years or older, and compared to younger patients.

Fifty‐two patients (19%) were 60 years or older. Compared with younger patients, elderly patients with ALL had a significantly worse performance status at presentation (P < 0.001); higher incidences of FAB L2 morphology (P=0.03). thrombocytopenia (P < 0.01), hypoalbuminaemia (P < 0.001) and renal dysfunction (P=0.03); and trends for worse anaemia (P = 0.06) and a higher rate of myeloid markeers positivity (P= 0.06). With VAD induction therapy, elderly patients had a lower CR rate than younger patients following two induction courses (CR rate 58% rate 58% v 82%; P < 0.01), and overall (CR rates 65% v 90%; P <).01). The response rate to VAD chemotherapy, however, appeared superior to the 30–35% CR rates reported from other series in elderly ALL patients. The lower CR rate was due to both higher induction mortality (P = 0.02) and more resistant disease (P < 0.01). The longterm CR rate was 20% in elderly patients achieving CR, but the overall survival was poor (< 10% at 3 years). There were strong associations between older age and other poor prognostic features. However, multivariate analyses identified older age to be an independent poot prognostic factor for response to chemotherapy. In summary, elderly patients with ALL have an appreciable CR rate with the VAD regimens. Patients achieving CR may have durable remissions. The myelosuppression‐associated mortality in both remission induction and maintenance may be reduced with the addition of growth factors in both treatment phases, and by using standard maintenance therapy without consolidation‐intensification phases.
Immunoglobulin replacement in patients with chronic lymphocytic leukaemia: a comparison of two dose regimes
Authors: Chapel H. , Dicato M. , Gamm H. , Brennan V. , Ries F. , Bunch C. , Lee M.
Published: March 2008

Summary. Previous studies have shown that intravenous immunoglobulin (IVIg) therapy is useful prophylaxis against infection in patients with secondary hypogammaglobulinaemia due to a low‐grade lymphoproliferative disease. This randomized double‐blind study was undertaken to determine prospectively the dose regime required. 34 such patients received IVIg at either 500 or 250 mg/kg every 4 weeks for 1 year. There was no significant difference in the rates of serious infections between the two groups of patients, which were well matched for disease and laboratory parameters. The rates of infection seen were similar to those in IVIg groups of previous studies and strikingly different from those in the placebo group in the previously randomized placebo‐controlled study.
Persistent polyclonal lymphocytosis with binucleated B lymphocytes: a genetic predisposition
Authors: Troussard X. , Valensi F. , Debert C. , Maynadie M. , Schillinger F. , Bonnet P. , Macintyre E. A. , Flandrin G.
Published: March 2008

Summary. Persistent lymphocytosis is usually associated with a malignant lymphoproliferative disease (MLPD). We report six female patients presenting a chronic, moderate lymphocytosis of 2–16 years duration with atypical binucleated lymphocytes on peripheral blood smears. Further investigation showed a polyclonal increase in serum IgM and HLA‐DR7 phenotype in all patients. The B cells were polyclonal because Southern hybridization of DNA and polymerase chain reaction failed to demonstrate a clonal rearrangement of immunoglobulin heavy chain genes. Peripheral blood examination showed binucleated lymphocytes in a family member of two of the cases; taken together with the association with HLA‐DR7 these data suggest a genetic predisposition. The identification of this benign syndrome is important in order to prevent its misdiagnosis as a MLPD.
A novel β‐thalassaemia mutation in the 5’untranslated region of the β‐globin gene
Authors: Athanassiadou Aglaia , Papachatzopoulou Adamandia , Zoumbos Nicholas , Maniatis George M. , Gibbs Richard
Published: March 2008

Summary. A thymidine deletion at position +10 of the 5’untranslated region of the β‐globin gene was detected in a β‐thalassaemia intermedia patient carrying a βd́; 39 stop codon mutation on the other chromosome; this new mutation, +10(‐T), was detected by automated fluorescent DNA sequencing and verified by dot‐blot allele‐specific hybridizations.

The +10(‐T) mutation is a ‘silent carrier’, is associated with a reduced amount of steady‐state β‐globin mRNA, and establishes a connection between the 5’untranslated region of the β‐globin gene and the regulation of its expression.
γ‐mRNA and Hb F levels in β‐thalassaemia
Authors: Efremov D. G. , Dimovski A. J. , Sukarova E. , Schiliro G. , Zisovski N. , Efremov G. D. , Burrone O. R. , Huisman T. H. J.
Published: March 2008

Summary. The Hb F levles in β‐thalassaemia can be affected by factors both linked and unlinked to the β‐globin gene cluster. We have recently analysed a group of patients with a homozygosity for the IVS‐1–6 (T → C) mutation, showing a wide variation in Hb F levels (2–47%) which could not be accounted for by any sequence variation within regulatory elements of the β‐globin gene cluster. In order to further investigate factors underlying this phenotypic difference we have developed a competitive reverse trascription/polymerase chain reaction procedure and used this method to determine the relative amounts of γ‐and β‐mRNAs in 10 patients with the IVS‐1–6 homozygosity and 15 heterozygous parents, two IVS‐I‐6δβ‐thalassaemia compound heterozygotes, five homozygotes for the βd́; IVS‐I‐110 (G → A) mutation, and in two with a homozygosity for the β† codon 39 (C → T) mutation. Three heterozygotes were also included. The percentages of γ/(γ+β) mRNA were 10–73% in the IVS‐I‐6 homozygotes and <2% to 10% in their heterozygous parents. A direct relationship existed between the level of mRNA and the % Hb F. However, the relative γ‐mRNA levels in the IVS‐I‐6 homozygotes were higher than their Hb F levels, indicating a possible competition between the γ and β transcripts for translational factors with a less effcient initiation of protein synthesis on the γ‐mRNA or a preferential survival of cells with mainly β‐globi gene expression at the post‐reticulocye stage. The γ‐mRNA levels in the two IVS‐I‐6/δβ‐thalassaemia compound heterozygotes were 71% and 62%, similar to their Hb F levels (63% and 59%), and averaged 82% (range 65–91%) in the five IVS‐I‐110 homozygotes and 97.5% in the two codon 39 homozygotes. The correlation between these values and the % Hb F could not be evaluated because of the transfusion regimens; however, the levels, of γ‐mRNA were as expected for patients with these β‐thalassaemia alleles.
Retroviral‐mediated gene transfer into human myeloma cells
Authors: Björkstrand Bo , Dilber M. Siracl , Smith C. I. Edvard , Gaharton Gösta , Xanthopoulos Kleanthis G.
Published: March 2008

Summary. We have studied retroviral‐mediated gene transfer into human myeloma cells, Bone marrow cells were obtained from four patients with advanced myeloma, where was heavily infiltrated with myelomatous plasma cells. Myloma cells were isolated by immunomagnetic separation, using the high‐affinity B‐B4 monoclonal antibody. Following separation, cells were transduced with the LN retroviral vector, which carries the gene for neomycin phosphotransferase, by incubation in cell‐free supernatant with or without a growth‐factor combination of IL‐3, IL‐6 and SCF. After infection, the cells were cultured for 9 d in RPIMI‐1640 and 10% FCS. either in the presence or absence of the neomycin analogue G418.

Transduction efficiency was 1.5–3.8%, when compared to the number of cells at initiation of the culture, and 5.0–50.0% when compared to the mumber of surviving infected cells cultured without G418. The gene transfer rate was similar whether or not growth factors were present durig the retorviral infectoin.

Theses preclinical data provide evidence that retroviral‐mediated gene transfer into human myeloma cells is feasible, and form part of the basis for current clinical studies of gene marking of bone marrow or peripheral blood progenitor cells before autologous stem cell transplatation in multiple myeloma.
Cytokine response to infection in patients with acute myelogenous leukaemia following intensive chemotherapy
Authors: Ostermann Helmut , Rothenburger Markus , Mesters Rolf M. , van de Loo Jürgen , Kienast Jochen
Published: March 2008

Summary. Septic shock is the major cause of treatmentrelated death in patients with acute myelogenous leukaemia (AML) undergoing intensive chemotheray. Interleukins (IL)‐1β, −6, −8, and tumour nerosis factor α (TNF‐α) have been implicated as mediators of septic shock, with circulating leucocytes being considered a major source for their release. However, Plasma cytokine levels of leucocytopnic patients with evolving sepsis have not been studied. We have prospectively measured plasma cytokines during chemotherapy‐induced leucocytopenia (< 1 × 109/1) in 50 patiens with AML. Cytokine levels in patients with severe sepsis (n=5) or septic shock (n=8) were cmpared to those measured in 13 matched patients with uncmplicated febrile infections. In evolving septic shock, IL‐6, IL‐8 and TNF‐α peaked within 48 h of fever onset at levels reported for nonleucocytopenic patients and distinctively higher than during uncomplicated febrile episodes (P < 0.05). Peak concentrations measured within 48 h after onset of fever were related to fatal outcome. IL‐1β was detected in less than 5% of all samples. Cytokine concentrations were unrelated to leucocyte counts and markers of neopterphil or monocyte activation (elastase and neeopterin levels, respectively). We conclude that cytokine release associated with evolving septic shock in patients with AML does not depend on circulating leucocytes.
High expression of the multidrug resistance‐associated protein (MRP) in chronic and prolymphocytic leukaemia
Authors: Burger Herman , Nooter Kees , Sonneveld Pieter , Van Wingerden Kyra E. , Zaman Guido J. R. , Stoter Gerrit
Published: March 2008

Summary. The expression of the multidrug resistance‐associated protein (MRP), a new glycoprotein involved in drug resistance, was investigated in tumour samples from 80 patients with chronic B‐cell malignancies by a quantitative RNase protection assay. In B‐cell chronic lymphocytic leukaemia (B‐CLL) (n= 32), either treated (n= 18) or untreated (n= 14), a high percentage of patients (20/32: 63%) had relatively high expression levels of the MRP gene (25 U or more). In addition, hyperexpression of the MRP gene was demonstrated in 4/10 (40%) untreated patients with B‐cell prolymphocytic leukaemia (B‐PLL). In Contrast, low MRP mRNA expression levels were detected in hairy cell leukaemia (n= 7), non‐Hodgkin's lymphoma (n= 13) and multiple myeloma (n= 18). Statistical analysis of MRP expression in untreated CLL (mean ±SD 29.2 ± 18.5U) versus treated CLL (mean ±SD 26.7 ± 13.7U) did not show significant differences in MRP expression between the two groups. Southern blot analysis did not reveal amplification of the MRP gene in the leukaemia samples with elevated MRP mRNA levels. We conclude that B‐PLL and B‐CLL frequently display high MRP expression and that this hyperexpression is probably due to transcriptional activation and/or increased mRNA stability.
Thrombin receptor expression and function in large granular lymphocyte proliferative disorders
Authors: Howells Gareth L. , Macey Marion , Murrell Adele M. , Green Anthony R. , Curtis Michael A. , Stone Stuart R.
Published: March 2008

Summary. It has recently been shown that peripheral blood NK‐cells and a fraction of T‐cells which co‐express CD16 and either CD56 or CD57 express the platelet type thrombin receptor. Large granular lymphocytes exhibit a T‐ or NK‐cell phenotype, and therefore these results raise the possibility that thrombin and its receptor may be involved in the biology of large granular lymphocytes in health and disease. It is difficult, however, to perform functional studies using normal blood as a source of large granular lymphocytes because the small fraction of large granular lymphocytes cannot be separated from other lymphocytes in numbers sufficient for most in vitro experiments. Therefore patients with large granular lymphocyte proliferative disorders have been screened in order to identify a population of cells enriched in large granular lymphocytes that express the thrombin receptor. Expression of the receptor was analysed in polyclonal and clonal large granular lymphocyte proliferative disorders. Using flow cytometry, it was found that the proportion of thrombin receptor positive large granular lymphocytes varied from 3% to 86%. Northern analysis indicated a high level of expression of mRNA in a clonal expansion of large granular lymphocytes that stained positively for the receptor by flow cytometry. Thrombin was found to act as a chemotactic stimulus for large granular lymphocytes from a polyclonal expansion with high numbers of thrombin receptor positive cells. At an optimal concentration of 10‐9m the chemotactic response to thrombin was roughly equivalent to that obtained with the potent chemoattractant 1‐oleoyl 2‐acetyl glycerol. These findings suggest that thrombin may play a role in the recruitment of large granular lymphocytes in sites of inflammation.
Long‐term culture and molecular biological studies highlight differences in relative BCR‐ABL expression levels in the peripheral blood and bone marrow of a patient with chronic granulocytic leukaemia
Authors: Smith M. A. , Mills K. I. , Smith J. G.
Published: March 2008

Summary. A patient with accelerating Ph+ve chronic granulocytic leukaemia (CGL) was considered for autologous BMT using marrow ‘purged’ by 4 weeks long‐term culture (LTC). Efficacy of purging was determined using reverse transcriptase PCR for BCR‐ABL mRNA transcripts b2a2 and b3a2. Peripheral blood and bone marrow were compared. Three observations emerged: (i) the initial b2a2:b3a2 ratios for unmanipulated blood and marrow were different with values of 9:1 and 2:1 respectively; (ii) both transcripts were successfully ‘purged’ with LTC of blood but not marrow: and (iii) LTC of marrow caused a transient increase in relative levels of b3a2 mRNA and a corresponding reduction in the b2a2 signal. This is the first case where such differences have been demonstrated in association with LTC.
β2 glycoprotein‐I antigen is increased in primary hyperlipidaemia
Authors: McNally T. , Crook M. , Mackie I. J. , Isenberg D. A. , Machin S. J.
Published: March 2008

Summary. In order to determine whether elevated levels of β2 glycoprotein‐I (β2GPI) are associated with increased plasma lipids, we measured plasma β2 GPI antigen levels in 47 patients with primary hyperlipidaemia (20 severe hypercholesterolaemia, nine severe hypertriglyceridaemia, and 18 mixed hyperlipidaemia) and 34 normal healthy subjects. Mean β2GPI levels were significantly increased in each patient group (302.3, 272.9 and 299.1 mg/1. respectively) compared to controls (199.6 mg/1) (p<0.01). Significant correlations were demonstrated between β2GPI levels and triglyceride and total cholesterol levels in the control group (r = 0.387, r =0.559: P<0.5), but were not observed in all patient groups. These results indicate that β2GPI is increased in hyperlipidaemia and that its distribution between plasma lipid fractions is perturbed. Plasma lipid levels should therfore be considered when interpreting results of β2GPI antigen assays.
Short‐term myeloid growth factor mediated expansion of bone marrow haemopoiesis| studied by localized magnetic resonance proton spectroscopy
Authors: Jensen Karl Erik , Hansen Per Boye , Larsen Vibeke Andreé; , Johnsen Hans E. , Nielsen Helle , Karle Hans , Henriksen Ole
Published: March 2008

Summary. Previously we have shown that short‐term myeloid growth factor priming of haemopoiesis prior to bone marrow harvest increased the yield of myeloid progenitors in the graft. The present study is intended to investigate the expansion of haemopoiesis by volume selective proton magnetic resonance spectroscopy (MRS).

Six patients were treated with daily subcutaneous injections of recombinant human granulocyte colonystimulating factor (rhG‐CSF, n = 2) or granulocytemacrophage colony‐stimulating factor (rhGM‐CSF, n = 4) for 5d before marrow harvest. MRS investigations were performed prior to treatment (day 0), day 5 and day 12. Spectroscopic examinations were performed with the stimulated echo acquisition mode (STEAM) method on a 1.5T clinical whole‐body imaging unit. A cubic volume of interest (VOI) was selected in the bone marrow of the left iliac bone.

The patients responded with a rise in blood absolute neutrophil count from median 3.3 × 109/1 (range 1.3–7.3 × 109/1) before to 15.6 × 109/1 (range 6.8–22.0 x l09/ 1) after treatment. Concomitantly an increase in bone marrow cellularity and myeloid: erythroid ratios documented the stimulation of myelopoiesis. During priming, the light‐density cell proliferation rate in marrow samples increased from median 21.9 (range 4–5–31) x103 cpm to 54–7 (range 13–9–94) x103 cpm and the total number of myeloid progenitors enumerated as day 7/14 GM‐CFUs per volume aspirated marrow increased from median ll/8 x l03 (range 4.0–87.5/2.2–103.0) to 64/76 x l03 (range 28.4–1180.6/23.2–2850.0). MRS detected a significant increase in bone marrow ‘relative water content’ day 12, 1 week after myeloid growth factor treatment was stopped, from median 30.5% (range 16–45) to 79% (range 56–93). In parallel, haemopoiesis was detected in new areas of femur.

In conclusion, the non‐invasive MRS method may be a useful and reliable in vivo examination for expansion of haemopoiesis and a correspondent reduction of fat tissue inbone marrow after priming with recombinant human haemopoietic growth factors.
Changes in light‐scatter profile, membrane depolarization and calcium mobilization of neutrophils induced by G‐CSF in vivo
Authors: Spiekermann Karsten , Emmendoerffer Andreas , Elsner Jörn , Raeder Evelin , Lohmann‐Matthes M.‐L. , Welte Karl , Roesler Joachim
Published: March 2008

Summary. To extend our studies about phenotypical and functional alterations of G‐CSF‐induced neutrophils we have evaluated their light‐scatter profile, mobilization of intracellular calcium ([Ca2+]i) and membrane depolarization after stimulation. A significant increase in the forward scatter signals could be demonstrated in such neutrophils from patients with neutropenias of various origin and from healthy test subjects. This increase began 4 h and returned to normal 96 h after G‐CSF injection in the latter group. We found an impairment of [Ca2+]i mobilization in neutrophils from patients with glycogen storage disease type IB after stimulation of these cells with fMLP, It was even more pronounced than in severe congenital neutropenia (SCN). However, [Ca2+]i fluxes were normal when ionomycin was used. Neutrophils from patients with cyclic neutropenia (cyNP) and chemotherapy‐induced neutropenia (chNP) mobilized [Ca2+]i similar to those from healthy donors. Furthermore, we found a decreased percentage of neutrophils depolarizing after stimulation with fMLP and PMA in patients with SCN, whereas membrane depolarization was normal in patients with chNP and cyNP. All the alterations found here are suggested to be caused by a partial immaturity of the neutrophils, although in vivo activation and a direct effect of G‐CSF on myeloid precursors might be involved.
Isobutyramide, an orally bioavailable butyrate analogue, stimulates fetal globin gene expression in vitro and in vivo
Authors: Perrine Susan P. , Dover George H. , Daftari Pratibha , Walsh Carol T. , Jin YuXin , Mays Abbie , Faller Douglas V.
Published: March 2008

Summary. Butyrate and other short‐chain fatty acids stimulate fetal globin gene expression and have potential for ameliorating the β globin disorders. Butyrate, however, is rapidly metabolized in vivo and reaches only micromolar concentrations in plasma. We report here that a branched‐chain derivative of butyrate, isobutyramide, increases γ globin gene expression in cultured human erythroid progenitors in vitro and stimulates activity from a minimal γ globin gene promoter linked to a reporter gene in stable and transient expression assays, which slightly less activity in these in vitro assays than butyrate. In vivo, administration of isobutyramide to anaemic adult baboons rapidly stimulates fetal globin syntesis and F‐reticulocyte production. Plasma concentrations at millimolar levels are achieved after a single intravenous or oral dose (500–600 mg/kg), and these concentrations are maintained for 90.5–10.5h. These results indicate that although isobutyramide has slightly less activity than butyrate in vitro in enhancing fetal globin expression at the cellular and molecular level, its prolonged in vivo half‐life may provide superior activity as a therapeutic agent for reactivating fetal globin gene expression in vivo.
Treatment of chronic childhood immune thrombocytopenic purpura with intramuscular anti‐D immunoglobulins
Authors: Borgna‐Pignatti Caterina , Battisti Laura , Zecca Marco , Locatelli Franco
Published: March 2008

Summary. Seven patients with chronic immune thrombocytopenic purpura (ITP) were treated with intramuscular anti‐D (anti‐D IgG) five times, on an alternate‐day basis, or until a platelet count of 100 × 109/1 was achieved, and, subsequently, when necessary to maintain platelet counts above 50 × 109/1. Five patients responded to therapy, two of whom entered long‐term remission. Although signs of haemolysis were present in all patients, anaemia was never a problem. No patient developed haematomas at the site of injection. We suggest that intramuscular anti‐D represents a safe and relatively inexpensive alternative to intravenous gamma globulins (IVGG) for children with severe chronic ITP.
A luminescene method for the quantitative determination of phagocytosis of erythrocytes, of malaria‐parasitized eryithrocytes and of malarial pigment
Authors: Schwarzer Evelin , Turrini Franco , Arese Paolo
Published: March 2008

Summary. A method is described for the quantitative measurement of phagocytosis of human erythrocytes, malaria‐parasitized erythrocytes and isolated malarial pigment by adherent human monocytes. The method utilizes measurement of haem‐elicited luminescence both for the assay of ingested haemoglobin or malarial pigment haem and for the quantification of adherent monocytes. The latter is based on assay of luminescence elicited by cytochrome haem. The method utilizes the same low‐cost reagents and equipment for assay of ingested haem and for quantification of adherent monocytes. The method is fast and extremely sensitive. The lower sensitivity limit is 500 monocytes and 10 RBC. or RBC equivalents in the case of malarial pigment, per assay. A detailed protocol with full calculations of a typical phagocytosis experiment of oxidatively damaged RBC, malarial pigment and control RBS is presented.
The endocytic pathway for H‐ferritin established in live MOLT‐4 cells by laser scanning confocal microscopy
Authors: Moss Dean , Hibbs Alan R. , Stenzel Deborah , Powell Lawroe W. , Halliday June W.
Published: March 2008

Summary. We have established the intracellular destination of the putative immunoregulatory protein, human recombinant H (heavy)‐ferritin, in the transformed T‐cell line MOLT‐4, by laser scanning confocal microscopy of live cells. A series of confocal images was collected over a 60min time course using indirect immunofluorescence of H‐ferritin and transferrin, their respective monoclonal antibodies, and fluorescein isothiocyanate (FITC)‐labelled IgG. A marked drop in FITC fluorescence after 40 min of H‐ferritin internalization, indicative of an acidic environment, and co‐localization with tetramethylrhodamine isothiocyanate‐labelled‐dextran strongly suggests that H‐ferritin is transferred to the lysosome. In contrast, transferrin was observed to return to the cell surface. Electron microscopy confirmed that H‐ferritin was transferred to the lysosome. The receptor‐mediated endocytosis and lysosomal delivery of H‐ferritin may thus potentiate its putative immunoregulatory activity.
Expression of shared idiotypes by paraproteins from patients with monoclonal gammopathy of undetermined significance
Authors: Kampe Carsten E. , Hart Sarah , Miller Richard A. , Lichtenstein Alan , Kyle Robert A. , Berenson James R.
Published: March 2008

We have previously demonstrated the expression of shared idiotypes by the paraproteins from approximately one‐quarter of patients with multiple myeloma (MM). We have now investigated whether similar cross‐reactivity is expressed in the paraproteins of patients with monoclonal gammopathy of undetermined significance (MGUS), using a panel of 32 monoclonal antibodies (MAB) generated against follicular B cell lymphomas. The paraproteins from 76/409 (19%) patients with MGUS reacted with at least one of 23 different anti‐idiotypic antibodies used in this study. 18 MABs demonstrated reactivity with more than one patient's paraprotein. Moreover, 10 MABs reacted frequently (with 5–22 paraproteins). Over half (41/76) of the reactive patients’ paraproteins reacted with more than one MAB from this panel. This frequency of anti‐idiotypic reactivity was similar to that of previously studied patients with myeloma, chronic lymphocytic leukaemia (CLL), and follicular B‐cell lymphomas. There was no correlation between specific anti‐idiotypic reactivity and the propensity to develop serious disease (MM, macroglobulinaemia, amyloidosis, or other lymphoproliferative disorders) in patients with MGUS. These results suggest that MGUS is derived from cells producing antibodies that are similar to those of other B‐cell malignancies and that the pattern of idiotype expression is irrelevant to malignant potential.
Subgroup and cost‐benefit analysis of the Finnish multicentre trial of clodronate in multiple myeloma
Authors: Laakso Markku , Lahtinen Reino , Virkkunen Pekka , Elomaa Inkeri
Published: March 2008

Osteolytic lesions and pathological fractures are the major problems in the clinical management of multiple myeloma. We previously reported the main results of a randomized, controlled multicentre trial in 350 Finnish patients with multiple myeloma. All patients received standard melphalan‐prednisolone treatment and were randomized to receive either clodronate 2.4 g daily or a placebo for 24 months. The proportion of patients with progression of osteolytic bone lesions was twice as high in the placebo group as in the clodronate group (24.0%v 12.0%, P= 0.026). The purpose of the present study was to investigate factors associated with the progression of osteolytic lesions and to identify subgroups of patients who would benefit most from clodronate treatment. In univariate logistic regression analysis, including treatment (placebo, clodronate), sex, age, pain index, serum calcium and creatinine, myeloma stage, number of osteolytic lesions at baseline, and number of vertebral fractures at baseline as independent variables and the progression of osteolytic lesions as a dependent variable, only the treatment with a placebo was associated with the progression of osteolytic bone lesions. Separate analyses with respect to the progression of osteolytic bone lesions were carried out in the following subgroups: male v female, ± 64 v > 64 years, stage I v stage II‐III myeloma, no osteolytic lesions at baseline versus osteolytic lesions at baseline, no vertebral fractures at baseline versus vertebral fractures at baseline, and a 50% treatment response to cytotoxic drugs versus no treatment response to cytotoxic drugs. The treatment with clodronate delayed the progression of osteolytic lesions similarly in these subgroups, with the exception of a subgroup of patients who did not have a 50% treatment response to cytotoxic drugs. The treatment with clodronate did not significantly increase treatment costs. We conclude that the treatment effect of clodronate seems to be independent of sex and age of the patients, the stage of myeloma, and the severity of bone lesions at diagnosis, but not of treatment response to cytotoxic drugs.
Intensive chemotherapy with blood progenitor transplantation for primary resistant multiple myeloma
Authors: Dimopoulos Meletios A. , Hester Jeane , Huh Yung , Champlin Richard , Alexanian Raymond
Published: March 2008

Summary. This study assessed the feasibility and effect of blood progenitors as the only source of haemopoietic support for myeloablative therapy for patients with primary resistant multiple myeloma and markedly infiltrated bone marrow. 17 patients with advanced, primary resistant myeloma received a priming regimen of cyclophosphamide (3 g/m2) and etoposide (900 mg/m2) with GM‐CSF. During haematological recovery, at least 2 × 106 CD34+ mononuclear cells/kg were collected from each patient with 4‐12 leukaphereses. High‐dose chemotherapy was then given which consisted of thiotepa (750 mg/m2), busulfan (10 mg/kg) and cyclophosphamide (120 mg/kg) followed by reinfusion of the blood progenitors. Haemopoietic reconstitution was rapid with recovery of granulocytes to >1.0 × 109/1 after a median of 10 d and of platelets to 50 × 109/1 after a median of 29 d. The myeloma responded in 10/17 patients for a projected median duration of at least 12 months. Survival was prolonged significantly in comparison with the outcome of control patients who did not receive intensive treatment. Blood progenitors, assessed from the number of CD34+ cells, produced early haemopoietic recovery after myeloablative therapy that induced sustained control of advanced and resistant multiple myeloma.
Cellular origin and extent of clonal involvement in multiple myeloma: genetic and phenotypic studies
Authors: Takishita Makoto , Kosaka Masaaki , Goto Tetsuya , Saito Shiro
Published: March 2008

The cellular origin and extent of clonal involvement in multiple myeloma (MM) are controversial. The third‐complementarity‐determining region (CDR3) of the immunoglobulin heavy chain gene is the target region of VH replacements and somatic mutations. We analysed the CDR3 sequences of myeloma cells from eight newly diagnosed and three relapsed patients in order to elucidate the target cell of malignant transformation in MM. We also examined the extent of clonal involvement in MM using a CDR3 clone‐specific nucleic acid probe. The peripheral lymphocytes from the five MM patients were separated into fractions such as CD34+, CD20+CD10+, CD20+CD21+, CD20+CD19− and CD2+ cells. Amplified CDR3 DNAs from these subpopulations were hybridized with the probe specific to each patient's tumour cells. We found no evidence of ongoing VH replacements or somatic mutations in CDR3 in MM. However, frequent nucleotide mutations in D and JH segments were observed. Circulating malignant cells were detected in the CD34+ and all of the CD20+ subpopulations, but not in the CD2+ fraction. MM is a neoplasm originating from a B‐lineage cell which has already undergone antigendependent selection. Nevertheless, the tumour cells are composed of heterogeneous subpopulations at various stages of differentiation, similar to normal B‐lineage cells. Conversely, T cells were not involved in MM. These results imply that there is an analogous developmental pathway between the normal B‐lineage cells and the tumour cells of MM.
Transient loss of proteins carrying Kell and Lutheran red cell antigens during consecutive relapses of autoimmune thrombocytopenia
Authors: Williamson L. M. , Poole J. , Redman C. , Clark N. , Liew Y. W. , Russo D. C. , Lee S. , Reid M. E. , Black A. J.
Published: March 2008

A patient is described in whom two consecutive relapses of autoimmune throbocytopenic purpura (AITP) were associated with loss of red cell antigens of the Kell and Lutheran blood group systems respectively. During the second relapse the glycoprotein CD44 and to a lesser extent the LW antigen were also depressed. Both relapses were associated with concomitant production of IgG antibody recognizing high‐frequency determinants on the corresponding antigen‐carrying protein. Blocking of antigen sites by these antibodies was not the cause of reduced antigen expression, because immunoblotting studies showed absence of Kell protein during the first relapse, and Lutheran protein during the second. On both occasions the red cell changes reverted to normal with disappearance of the antibody as the AITP entered remission. There was no evidence of clonal lymphocyte expansion as demonstrated using immunoglobulin JH and T cell receptor β chain probes.
Parvovirus B19 as a cause of acquired chronic pure red cell aplasia
Authors: Frickhofen N. , Chen Z. J. , Young N. S. , Cohen B. J. , Heimpel H. , Abkowitz J. L.
Published: March 2008

Parvovirus B19 infection causes chronic anaemia in immunodeficient individuals by selective suppression of erythropoiesis. The bone marrow morphology is character‐istic of pure red cell aplasia (PRCA). To determine the frequency of B19‐induced PRCA we retrospectively analysed a series of 57 PRCA patients. B19 DNA was present in serum of eight patients (14%) and could be extracted from bone marrow aspirate slides from five of these patients. Recent exposure to the virus was confirmed by the presence of anti‐B19 IgM in sera from four and by the finding of giant pronormoblasts in marrow aspirates from five of the B19 DNA‐positive patients. The sensitivities of anti‐B19 IgM and of giant pronormoblasts were only 50% and 63%, respectively; specificites were 90% and 92%. Unexpectedly, PRCA in two B19 DNA‐positive patients remitted after antilymphocyte globulin or cyclosporin A therapy, suggesting that the clinical course of B19‐induced PRCA may be indistinguishable from other forms of PRCA. As therapy with immunoglobulin is uniformly effective for treatment of B19‐associated anaemia, our data suggest that all patients with acquired PRCA should be evaluated for evidence of B19 infection. B19 DNA analysis is the most reliable method to demonstrate infection.
Unexpected remission of acute myeloid leukaemia after GM‐CSF
Authors: Bassan Renato , Rambaldi Alessandro , Amaru Ricardo , Motta Teresio , Barbui Tiziano
Published: March 2008

Summary. The administration of granulocyte‐monocyte colony‐stimulating factor (GM‐CSF) was associated with complete clinical and haematological response in an adult patient with minimally differentiated acute myeloid leukaemia who presented with pneumonia and moderate neutropenia, but no blast cells in the peripheral blood. The response lasted 9 months. At relapse, a second GM‐CSF course resulted in a very good partial remission lasting 5 months, although differences in the kinetics of haemoglobin, neutrophil and platelet recovery were noted. Subsequent recurrences were managed with chemotherapy, a complete remission being obtained twice more and lastly consolidated with myeloablative chemo‐radiotherapy supported by a peripheral blood stem cell autograft. This report suggests that GM‐CSF should be further investigated as a therapeutic agent in selected cases of AML.
GM‐CSF in the treatment of Fanconi's anaemia
Authors: Kemahli Abri , Canatan Duran , Uysal Zümrüt , Akar Nejat , Cin Sükrü , Arcasoy Ayten
Published: March 2008

We have used recombinant human (rh) GM‐CSF in two 12‐year‐old Fanconi's aplastic anaemia patients. They had not received any previous therapy except blood transfusions. Each patient was given three 21 d courses of rh‐GM‐CSF, the first two at a dose of 3.5μg/kg/d and the third at 7 μg/kg/d s.c. There were significant increases in WBC and absolute neutrophil counts after the first week of rh‐GM‐CSF which lasted as long as the treatment was continued. Following the cessation of treatment, WBC and ANC dropped rapidly. We conclude that rh‐GM‐CSF can be used in FAA, especially in severely neutropenic cases.
Familial thrombophilia and activated protein C resistance: thrombotic risk in pregnancy?
Authors: Cook G. , Walker Isobel D. , McCall Frances , Conkie J. A. , Greer I. A.
Published: March 2008

An abnormal anticoagulant response in vitro to activated protein C (aPC) has been proposed as an aetiological factor in familial thrombophilia. It is postulated that this phenomenon is due to an inherited molecular defect of factor V resulting in poor inactivation by aPC. We conducted a family study when the proband presented in her second pregnancy with superficial phlebitis, a history of deep venous thrombosis and a family history of venous thromboembolic disease. No abnormality of antithrombin activity, protein C activity or deficiency of protein S were demonstrated in the family members tested. The proband had aPC ratios below the laboratory range on three consecutive occasions. In addition, her mother, who had a history of recurrent DVTs and a pulmonary embolus, and also an asymptomatic nulliparous sister, both had aPC resistance ratios below the laboratory range on consecutive samples. Further information about the combined risk of aPC resistance and pregnancy is needed before guidance on the management of affected women can be formulated.
The plasma concentration of soluble Fc‐gamma RIII is related to production of neutrophils
Authors: Huizinga T. W. J. , Haas M. de , Oers M. H. J. van , Kleijer M. , Viléa H. , Wouw P. A. van der , Moulijn A. , Weezel H. van , Roos D. , Borne A. E. G. Kr.von dem
Published: March 2008

Summary. FcγRIII (the CD16‐antigen), a low‐affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes and macrophages. A soluble form of FcγRIII has been identified in human plasma. This soluble form of FcγRIII (sFcγRIII) originates from release by neutrophils. In the present study we show by transfusions of plasma that contains sFcγRIII of one allotype (NA1‐FcγRIII) in recipients homozygous for the other allotype (NA2‐FcγRIII) that the clearance of sFcγRIII is about 0.7 ml/min. Because the concentration of sFcγRIII was found to be constant in a small cohort of donors followed for about 1‐5 years, the half‐life of NA1‐sFcγRIII is about 1.8 d, assuming a one‐compartment model.

The plasma concentration of sFcγRIII depended mainly on the production of neutrophils in the bone marrow, and was not influenced by shifts of neutrophils from one pool to another (storage, marginating or circulating pool). Because FcγRIII is only expressed on mature neutrophils, this implies that the concentration of sFcγRIII depends on production of mature neutrophils.
Studies of neutrophil and monocyte oxidative responses in polycythaemia vera and related myeloproliferative disorders
Authors: Samuelsson Jan , Forslid Jan , Hed Jan , Palmblad Jan
Published: March 2008

Summary. We have previously reported that polymorphonuclear granulocyte (PMN) chemiluminescence (CL) and superoxide anion production are abnormally low in patients with polycythaemia vera (PV) after simulation with n‐formylmethionyl‐leucyl‐phenylalanine (fMLP). but normal when elicited by phorbol myristate acetate (PMA). This study documents that both fMLP and PMA induced CL was normal in PMN from patients with chronic myelogenous leukaemia (CML) and essential thrombocythaemia (ET). Furthermore, we monitored intracellular hydrogen peroxide (H2O2) production in PMN and monocytes from patients with PV, CML and ET by flow cytometry. H2O2) production in resting and PMA‐stimulated cells was normal in all diseases. So also was fMLP induced H2O2 generation in ET PMN and monocytes. In contrast, fMLP‐induced H2O2 production was significantly lower both in PV PMN (1.8 ± 0.7 mean fluorescence intensity units in PV compared to 8.4 ± 3.4 in healthy controls; P < 0.02), and in PV monocytes (0.3 ± 0.5 compared to 2.5 ± 0.7 in controls; P < 0.02). A less pronounced reduction of fMLP stimulated H2O2 production was noted in CML PMN (3.8 ± 3.1 compared to 8.4 ± 3.4 in controls; P < 0.05), and monocytes (1.3 ± 0.6 compared to 2.5 ± 0.7 in controls: P < 0.05). The reduction of H2O2 generation in PV and CML PMN was not attributed to subpopulations of less responsive cells. However, one ET and one CML patient showed a subpopulation of less responsive PMN. Thus intracellular H2O2 (as well as extracellular release of superoxide ions) is reduced in fMLP‐stimulated PV PMN and monocytes but normal after PMA stimulation, a phenomenon that is not consistently found in other myeloproliferative disorders.
Minor myeloid component in Ph chromosome‐positive acute lymphoblastic leukaemia: correlation with cytogenetic pattern and implication for poor response to therapy
Authors: Cuneo Antonio , Demuynck Hilde , Ferrant Augustin , Louwagie Andries , Doyen Chantal , Stul Michel , Cassiman Jean‐Jacques , Cin Paola Dal , Negrini Massimo , Carli Maria Gretel , Boogaerts Marc , Michaux Jean‐Louis , Castoldi Gianluigi , Berghe Herman van den
Published: March 2008

Morphological, immunological and cytogenetic features were studied in 27 adults presenting with Ph chromosome‐positive acute lymphoblastic leukaemia (ALL), in correlation with clinical outcome.

Twenty patients (group 1) were diagnosed as having typical ALL according to the FAB criteria supported by immunological findings. Less than 1% blast cells with azurophilic granules were detected in all cases. Myeloid cytochemistry, i.e. peroxidase and Sudan black‐B stain, was negative in all cases. A minor phenotype deviation consisting of the expression of the CD13 myeloid‐associated marker was detected in two patients.

In seven patients (group 2) a diagnosis of ALL with a minor myeloid component was made because of the presence of a majority of lymphoid blasts and of 5‐15% blast cells with morphological cytochemical and immunological features of the myeloid lineage.

Abnormal metaphases were found in 6/20 (30%) patients in group 1, compared with 7/7 (100%) patients in group 2.

All patients were treated by antilymphoid regimens: however, complete remission was achieved in 17/20 (85%) patients in group 1 versus 1/7 (14.3%) patients in group 2. Median survival was 16 months, range <1‐120+ in group 1 and 9 months, range <1‐15 in group 2.

It is concluded that morphological, immunological and cytogenetic studies allow for the recognition of two cytological subsets of Ph+ ALL. The presence of a minor myeloid component in otherwise typical Ph chromosome‐positive ALL may be associated with a distinct cytogenetic pattern and poor responses to antilymphoid therapy.
Trisomy 12 is uncommon in typical chronic lymphocytic leukaemias
Authors: Criel A. , Wlodarska I. , Meeus P. , Stul M. , Louwagie A. , Hoof A. Van , Hidajat M. , Mecucci C. , Berghe H. Van den
Published: March 2008

The incidence of trisomy 12 was studied by conventional chromosome analysis in 111 patients referred as B‐cell chronic lymphocytic leukaemia (B‐CLL). Fluorescent in situ hybridization (FISH) was also applied in 34 of those patients with either a normal karyotype or no analysable mitoses. By karyotyping, trisomy 12 was present in 11.7% (13/111), whereas additional FISH increased the incidence to 14.4% (16/111). When subdividing our cases in either typical CLL (n= 90), fulfilling the FAB classification criteria, or atypical CLL (n= 21), with one or more variations from those criteria, the incidence of +12 by metaphase analysis was 3% and 48%, respectively. Additional FISH increased the incidence to 4% and 57%. The most common aberration in atypical CLL was FMC7 positivity (n= 11), followed by CD5 negativity (n= 8), strong surface immunoglobulin staining (n= 7) and atypical morphology (n = 6). Trisomy 12 could only be demonstrated in a small proportion of neoplastic cells in all positive cases. By FISH and/or karyotyping, all available samples at diagnosis of the disease were positive.