bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiologic concentrations of arginine vasopressin activate human platelets in vitro
Authors: Wun Ted , Paglieroni Teresa , Lachant Neil A.
Published: November 2003

Arginine vasopressin (AVP) is a neurohypophyseal peptide hormone with protean effects. Previous reports had shown that AVP stimulates platelets, but only at concentrations 3–6 logs higher than the normal plasma concentrations in humans. In this study we tested the hypothesis that AVP, at physiologic concentrations, stimulated the expression of an activation‐dependent platelet antigen. Platelets obtained from normal volunteers were incubated with increasing concentrations of AVP and the expression of the activation‐dependent platelet antigen P‐selectin (CD62) was determined by monoclonal antibodies and flow cytometry. There was a concentration‐dependent increase in CD62 expression with increasing AVP concentration; at 1 pm AVP, 24.5% (1.3–88.5%) [median (range)] of platelets expressed CD62. The selective vasopressin V1 receptor antagonist d(CH2)5‐Tyr(me)AVP (TM‐AVP) completely abolished AVP‐stimulated CD62 expression. We conclude that AVP can activate platelets at concentrations found in normal humans, at least in vitro, and that this response is mediated by the platelet V1 receptor. AVP may be a physiologic platelet agonist.
Elevated serum interleukin‐6 (IL‐6) is derived from neoplastic lymphoid cells in patients with B‐cell non‐Hodgkin's lymphoma: correlation with extent of IL‐6 expression and serum concentration
Authors: Kato Hirokazu , Kinoshita Tomohiro , Suzuki Sadao , Nagasaka Tetsuro , Murate Takashi , Saito Hidehiko , Hotta Tomomitsu
Published: November 2003

We investigated the expression of interleukin‐6 (IL‐6) mRNA in tumours from 21 patients with B‐cell non‐Hodgkin's lymphoma (NHL). Total RNA, extracted from diagnostic tissue specimens, was subjected to semi‐quantitative analysis for IL‐6 mRNA by the reverse transcription‐polymerase chain reaction (RT‐PCR). The amount of IL‐6 mRNA was semi‐quantified against that in pokeweed mitogen (PWM) stimulated peripheral blood mononuclear cells. The expression of IL‐6 in neoplastic B cells was confirmed by immunohistochemistry. We then looked for correlations between the amount of IL‐6 mRNA produced by neoplastic B cells and serum IL‐6 concentrations. The amount of IL‐6 mRNA correlated with the circulating IL‐6 concentration, suggesting that the malignant cells were the source of IL‐6 in these patients with B‐cell NHL. Our results suggest that the circulating IL‐6 in patients with B‐cell NHL is derived from the neoplastic lymphoid cells, and that neoplastic cells may act as modulators of the general status of patients with B‐cell NHL.
The Fas antigen is detected on immature B cells and the representative cell lines show Fas‐mediated apoptosis
Authors: Nishiuchi Ritsuo , Yoshino Tadashi , Matsuo Yoshinobu , Sakuma Isao , Cao Liu , Seino Yoshiki , Takahashi Kiyoshi , Akagi Tadaatsu
Published: November 2003

The expression and functions of Fas antigen, a major regulator of apoptosis, in T‐cell selection have been intensively investigated, but little is known about its expression in immature B cells which are also selected in the bone marrow, and plasma cells which are at the terminal stage of B‐cell differentiation and are designed to die. We examined bone marrow cells and found Fas antigen on these cells at low levels. Next, we analysed Fas expression and susceptibility to anti‐Fas antibody‐mediated apoptosis on B‐cell lines representing various stages of differentiation. We also examined the expression of Bcl‐2 and Bax on these lines, which were intimately related to apoptosis. Fas antigen was almost negative on pre‐pre‐B cell lines and was detected on pre‐B‐cell lines at low levels. All plasma cell lines expressed Fas at a low to moderate level. Some cell lines with peripheral B‐cell phenotype expressed Fas antigen. Except for the Burkitt cell lines and one plasma cell line, susceptibility to Fas‐mediated apoptosis depended on Fas expression. Bcl‐2 protein was detected on all but one cell line and Bax was detected on 15/23 lines, but neither were related to cellular differentiation or Fas expression.
β‐Thalassaemia in south‐western Spain: high frequency of G → A (IVS I‐1) mutation
Authors: B ENITO ANA , VILLEG AS ANA , PERE Z‐CANO RAMON , BE RNAL RICARDO
Published: November 2003

Huelva province is situated in southwestern Spain; its historic and geographic characteristics contribute to the existence of erythrocyte genetic anomalies, such as β thalassaemia. We have carried out a prenatal study of microcytosis, with a preliminary β‐gene analysis. Our findings show a β‐thalassaemia trait prevalence of 0.81%. In the molecular research there was a high frequency of G → A (IVS I‐1) injury: 55%. By comparing the rates of this mutation in neighbouring Cáceres province in western Spain (47%) and Algarve in south Portugal (32%), it demonstrates that this β‐gene defect has a high frequency in the southwest of the Iberian Peninsula.
Soluble interleukin‐6 receptor as a prognostic factor in multiple myeloma
Authors: Pulkki Kari , Pelliniemi Tarja‐Terttu , Rajamäki Allan , Tienhaara Anri , Laakso Markku , Lahtinen Reino
Published: November 2003

Interleukin‐6 (IL‐6) is a major growth factor for the clonal malignant plasma cells in multiple myeloma (MM). The effect of IL‐6 may be enhanced by soluble IL‐6 receptor (sIL‐6R). As there is a clinical need for improved stratification of MM patients at diagnosis, we have studied the role of sIL‐6R as a prognostic marker in 207 newly diagnosed MM patients. Serum sIL‐6R concentration was above the upper reference limit in 47% of the patients at diagnosis. The concentrations of sIL‐6R and two other prognostic factors, IL‐6 and β‐2 microglobulin (β2M), were all significantly higher in the patients who died within 3 years compared with those who survived. However, serum sIL‐6R did not show linear correlation with IL‐6 or β2M levels. In univariate logistic regression analysis sIL‐6R was a significant predictor of 3‐year mortality. Kaplan‐Meier analysis showed that raised levels of sIL‐6R were associated with shorter survival. When the patients were stratified into four groups according to their serum IL‐6 and sIL‐6R levels, the patients with normal serum levels of both parameters had clear survival benefit. As β2M was the most powerful prognostic factor in the multivariate analysis, the patients were also stratified according to their serum β2M and sIL‐6R levels. The patients with raised levels of both β2M and sIL‐6R had shorter survival than the patients in the other three groups. Thus, measurement of these parameters at diagnosis would help to stratify MM patients.
Haemolysis of Plasmodium falciparum trophozoite‐infected erythrocytes after artemisinin exposure
Authors: ORJIH AUGUSTINE U.
Published: November 2003

This study has examined in vitro, how exposure to the antimalarial drug artemisinin affects Plasmodium falciparum and its host erythrocytes. Factors examined include: cell morphology, intracellular haemoglobin levels, and haemoglobin catabolism (haemozoin production). To avoid uninfected erythrocytes complicating the study, P. falciparum ring‐infected erythrocytes were concentrated to 99% parasitaemia, by saponin haemolysis, before the parasites were grown with or without artemisinin. Without artemisinin, the parasites completed their life cycle in the normal time (40 h), during which a mean of 980 pmol of ferriprotoporphyrin IX from haemoglobin was incorporated into haemozoin per 106 parasitized erythrocytes, and intracellular haemoglobin level decreased by 90%. Exposure of ring‐infected erythrocytes to artemisinin (250 ng per ml of culture medium) inhibited parasite growth completely, haemozoin production by 95%, and decreased the intraerythrocytic haemoglobin level by 90%; the infected erythrocytes remained intact during the 64 h of study. Haemozoin production was also inhibited when the drug was administered at the trophozoite stage of parasite growth, but the infected erythrocytes haemolysed. These findings may contribute to understanding of antimalarial actions of artemisinin that promote parasite clearance.
Clinical significance of CD45RO expression on peripheral blood mononuclear cells in HTLV‐I‐infected individuals
Authors: Suzuki Muneou , Uno Hisamitsu , Yamashita Kiyoshi , Toyama Takanori , Kubuki Youko , Maeda Kouichi , Matsuoka Hitoshi , Ohtaki Sachiya , Tsubouchi Hirohito
Published: November 2003

The phenotype of peripheral blood mononuclear cells (PBMC) was examined in 13 healthy volunteers, 26 HTLV‐I carriers, and 58 ATL patients (22 smouldering, five chronic, 24 acute, and seven lymphoma type). The percentage of CD4+, CD25+, CD28+ and CD45RO+ cells in the PBMC of the chronic and acute type patients was significantly higher than that of the volunteers, whereas the percentage of CD8+ and CD45RA+ cells in these patients was significantly low. The histogram for CD45RO fluorescence intensity (FI) revealed two patterns: pattern A consisted of CD45RO+ cells with high FI (CD45ROhigh) and intermediate FI (CD45ROint). Pattern B consisted exclusively of CD45ROhigh. Pattern A was evident in all volunteers. The percentage of subjects showing pattern B was increased in an order that reflected disease progression. In the patients with pattern A, the CD45ROint cells were CD4+ and CD8−, and the FI of CD2, CD3, and Fas within the CD45ROint cells appeared to be lower than that within the CD45ROhigh cells. The acute type patients with pattern A had a significantly longer survival curve than that of these patients with pattern B. These results suggest that the presence of CD45ROint cells may be related to protection against disease progression in HTLV‐I‐infected individuals.
Presence of the seven transmembrane thrombin receptor on human tumour cells: effect of activation on tumour adhesion to platelets and tumour tyrosine phosphorylation
Authors: Nierodzik Mary Lynn R. , Bain R. Morgan , Liu Lin‐Xing , Shivj i Mehboob , Takesh ita Kenichi , Karpatki n Simon
Published: November 2003

Thrombin‐treated tumour cells enhance their adhesion to platelets, fibronectin and von Willebrand factor in vitro, and enhance their pulmonary metastasis in mice in vivo. A unique seven transmembrane spanning thrombin receptor has recently been cloned which is activated following thrombin proteolysis of the N‐terminal end of the receptor with exposure of a tethered ligand. An N‐terminal 14‐mer (SFLLRNPNDKYEPF) or 6‐mer (SFLLRN) of the tethered ligand can serve as a thrombin receptor activation peptide (TRAP) by mimicking the action of thrombin on platelets, endothelial cells and smooth muscle cells. We have examined six human tumour cell lines for their response to TRAP, for the presence of this thrombin receptor mRNA by RT‐PCR, protein by immunoblot and for their in vitro and in vivo response to TRAP. All six cell lines contain the receptor mRNA, and when treated with 100 μm 6‐mer TRAP or 1 u/ml thrombin increase their adhesion to platelets 2–3‐fold. Four of the six cell lines undergo tyrosine phosphorylation within 30 s to 1 min after exposure to 6‐mer TRAP or thrombin. Thus tumour cells respond to thrombin via activation of their seven transmembrane spanning thrombin receptor.
Arg506–Gln mutation in factor V and risk of thrombosis during pregnancy
Authors: Bokarewa Maria , Bremme Katarina , Blombäck Margareta
Published: November 2003

Seventy women with thrombosis in pregnancy were investigated for the presence of APC resistance and the associated Arg506–Gln mutation in coagulation factor V. The mutation was found in 46% of the investigated women. Carriers of the mutation were more prone to develop thrombosis in the first pregnancy (OR (odds ratio) = 3.41; P < 0.05) and had a higher probability of recurrence (OR = 3.86; P < 0.05) compared to non‐carriers. The incidence of miscarriage was not related to the mutation but its probability increased in women with thrombosis in a second or subsequent pregnancy (P < 0.025). Negative dynamics of APC response during pregnancy was observed in 20/22 women; eight of them developed APC resistance de novo. The suppression of APC response was independent of the mutation.
Deletion mapping indicates that MTS1 is the target of frequent deletions at chromosome 9p21 in paediatric acute lymphoblastic leukaemias
Authors: Guidal‐Giroux Christine , Gérard Bénédicte , Cavé Hélène , Duval Michel , Rohrlich Pierre , Elion Jacques , Vilmer Etienne , Grandchamp Bernard
Published: November 2003

Recent reports have indicated a high frequency of deletions of MTS1 (CDKN2, p16ink4, CDKI4) in acute lymphoblastic leukaemias (ALLs). This gene is located at chromosome 9p21 and encodes an inhibitor of cyclin D‐dependent kinases. In contrast with the observations in some other malignancies, no inactivation of MTS1 by intragenic mutation was demonstrated in leukaemias. A contribution of MTS1 alterations to leukaemogenesis therefore remains questionable. In order to test for the implication of MTS1 as a tumour suppressor gene in paediatric ALLs we have explored the 9p21 chromosomal region in 46 children with this disease. The copy number of the MTS1 gene in blasts from the patients was determined using a quantitative PCR assay enabling us to precisely detect mono‐ and bi‐allelic deletions. Rearrangements of the gene were sought by Southern blot analysis. The extent of the deletions was studied using microsatellite markers spanning the 9p21 chromosomal region. Point mutations were sought in exon 1 and exon 2 of the MTS1 gene in patients with a mono‐allelic deletion. In addition, exon 2 of MTS1, which contains two‐thirds of the coding region, was sequenced in all patients who had no deletion of the gene. Altogether, our data are consistent with the view that MTS1 is the target of 9p21 deletions. Either one or two alleles of the gene were deleted in 36% of non‐selected children with B‐lineage ALL and both alleles were deleted in all seven patients we studied with T‐lineage ALL. The absence of any point mutation implies that the major mechanism of inactivation of MTS1 in ALLs is deletional.
Relative erythroid hyperplasia in the bone marrow at diagnosis of aplastic anaemia: a predictive marker for a favourable response to cyclosporine therapy
Authors: Nakao Shinji , Yamaguchi Masaki , Takamatsu Hideyuki , Takami Akiyoshi , Chuhjo Tatsuya , Ueda Mikio , Shiobara Shintaro , Matsuda Tamotsu
Published: November 2003

Predicting the treatment response of aplastic anaemia (AA) is essential when considering cyclosporine (CyA) therapy among several treatment options, because it requires at least 2 months to determine whether the therapy is beneficial to a patient with AA. To identify the characteristics of patients with AA who are likely to respond to CyA therapy we retrospectively reviewed the clinical records and bone marrow smears of patients treated with CyA. Among 30 patients who received the therapy for at least 3 months within 1 year after diagnosis of AA, and who had not been exposed to antilymphocyte or antithymocyte globulin, 16 (53%) responded with disease remission. CyA‐responsive patients had a significantly higher ratio of erythroblasts to granulocytes (E/G ratio) in the bone marrow at the time of diagnosis as compared with patients refractory to therapy (P = 0.004). Multivariate analysis revealed that a high E/G ratio (>0.6) was significantly associated with a good response to CyA (P = 0.03): 15 (83%) of the 18 patients with an E/G ratio >0.6 responded, but only one (8%) of the 12 with an E/G ratio ≥0.6 did. Although the presence of subclinical paroxysmal nocturnal haemoglobinuria was suspected from the relative erythroblastosis observed in the bone marrow of these patients, flow cytometric analysis of neutrophils in the peripheral blood failed to reveal neutrophils deficient for glycosyl‐phosphatidylinositol (GPI) anchored membrane proteins in all but one case. Identification of the presence of relative erythroid hyperplasia in the bone marrow when AA is diagnosed may help to predict a favourable response to CyA therapy, and therefore facilitate the selection of optimal therapy for AA.
The thrombin receptor gene is centromeric to the common proximal breakpoint in patients with the 5q− syndrome: identification of a previously unrecognized chromosome 5 inversion
Authors: Demetrick Douglas J. , Dewald Gordon W. , Lanman Joseph , Bahou Wadie F.
Published: November 2003

The human thrombin receptor (TR) gene has previously been localized to band q13 of chromosome 5, a site that is at or contiguous with the common proximal breakpoint site identified in the majority of patients with the 5q− syndrome (dysmegakaryocytopoiesis and refractory anaemia). Since thrombin has putative effects on the growth and differentiation of megakaryocytes, we hypothesized that the phenotypic abnormalities in megakaryocytopoiesis observed in the 5q− syndrome may be partially explained by involvement of the TR gene in the interstitial deletion. We have utilized molecular and fluorescence in situ hybridization (FISH) analysis to study potential cytogenetic abnormalities of the thrombin receptor gene in patients demonstrating this abnormality. Dual‐label FISH with a q12‐specific genomic fragment and the TR gene was completed using interphase and metaphase cells from seven patients with a del(5)(q13q33). Our data demonstrate that the TR gene is located on the centromeric side of the common proximal breakpoint, and is grossly present in all patients with the affected 5q− chromosome. In addition, one of the seven patients demonstrated a small proximal rearrangement, most likely representing a paracentric inversion, which was not apparent by conventional cytogenetic techniques. The significance of these alterations is discussed.
The behaviour of red cells in narrow tubes in vitro as a model of the microcirculation
Authors: KUBOTA KAZUO , TAMURA JUN’ICHI , SHIRAKURA TAKUO , K IMURA MASANORI , Y AMANAKA KAZUNORI , ISOZAKI TATSUO , NISHIO IZUMI
Published: November 2003

To investigate the behaviour of red cells in the microcirculation, we established a new capillary method using narrow fluorinated ethylenepropylene copolymer tubes with internal diameters of 12.5 and 25.0 μm. Red cell flow in the tubes under a given range of pressure was analysed through a video system connected to a microscope. The experimental condition was adjusted so that the velocity of the control normocytes would be compatible with that in corresponding vessels in vivo, 0.5–1.5 mm/s. In the 12.5 μm tube, normocytes obtained from 12 young normal volunteers ran in an axisymmetric edge‐on orientation with a folded shape at higher pressures, but rolled along freely without deformation at lower pressures. Deformation during the passage of the microcytes obtained from four patients with polycythaemia vera complicated with iron‐deficient microcytosis and 10 patients with iron deficiency anaemia was relatively mild, whereas that of the macrocytes obtained from eight patients with refractory anaemia was marked. Even after the screening effect at the tube entrance was taken into consideration, the velocities of both microcytes and macrocytes were found to be significantly lower than the control normocytes. Therefore this method may be a new way to investigate the flow properties of red cells in the microcirculation.
Isolated beta‐globin chains reproduce, in normal red cell membranes, the defective binding of spectrin to alpha‐thalassaemic membranes
Authors: Shalev Oded , Shinar Eilat , Lux Samuel E.
Published: November 2003

Alpha‐thalassaemic erythrocytes develop a specific membrane skeletal defect that is manifest as a loss of normal spectrin‐binding sites on the inner surface of the thalassaemic membranes. To test whether this lesion could be caused by the excess free beta‐globin chains that accumulate in alpha‐thalassaemic red cells, we incubated normal red cell membranes with native, haem‐containing alpha or beta globin chains or with haemoglobin A. Spectrin‐depleted inside‐out membrane vesicles (IOVs) derived from membranes incubated with beta‐globin chains bound only 9 ± 3% as much spectrin as IOVs from control membranes incubated with bovine serum albumin. In contrast, IOVs from membranes incubated with alpha‐globin chains or haemoglobin A were nearly normal (79 ± 3% and 86 ± 5% of controls, respectively). This differential effect of globin chains was not seen when membranes were first transformed into spectrin‐depleted IOVs and then incubated with the isolated globin chains. Under these conditions, both alpha and beta globin chains reduced the spectrin‐binding capacity of the IOVs by approximately 45% (alpha 46 ± 7%, beta 43 ± 6%) whereas haemoglobin A had no effect. Unlike IOVs, spectrin isolated from membranes exposed to alpha or beta globin chains bound normally to IOVs and to actin (in the presence of protein 4.1). These studies show that isolated beta‐globin chains (but not alpha‐globin chains) can produce a spectrin‐binding defect in normal red cell membranes similar to that seen in alpha thalassaemia. The existence of similar defects in the membrane skeletons of red cells from other diseases with unstable beta globins suggests a common pathophysiology for the premature destruction of these cells.
Cryopreserved Human Bone Marrow Stroma is Fully Functional In vitro
Authors: Nicol Andrew , Nieda Mie , Donaldson Craig , Denning‐Kendall Patricia , Truman Carol , Bradley Ben , Hows Jill
Published: November 2003

Human marrow long‐term culture (LTC) enables maintenance of both stromal and haemopoietic elements of normal bone marrow (NBM) in vitro for 4–6 months. Stroma‐based cultures are critical for quantitation of long‐term culture initiating cells (LTC‐IC), the most primitive human haemopoietic cells measurable in vitro. Supply of NBM can be sporadic, and up to 3–4 weeks in culture is required for stromal maturity. Stroma availability for experimental purposes can therefore be limited. Efforts to produce transformed human and transfected murine stromal cell lines comparable to NBM stroma have had some success. As an alternative, we investigated cryopreserved NBM and cryopreserved preformed stroma. Function of cryopreserved and control fresh NBM stroma was similar when evaluated for up to 12 weeks in LTC. We have also demonstrated that stroma derived from cryopreserved NBM or preformed cryopreserved NBM stroma can sustain third‐party haemopoiesis as efficiently as fresh NBM stroma in LTC. Batched cryopreserved stroma is a convenient, rapidly available, source of functional stroma which avoids the logistic difficulties and lack of standardization associated with stroma from fresh NBM. This important advance will enhance the use of stroma‐based LTC in studies of human haemopoiesis.
Sensitivity of human acute myeloid leukaemia to diphtheria toxin‐GM‐CSF fusion protein
Authors: Rozemuller Henk , Rombouts Elwin J. C. , Touw Ivo P. , FitzGerald David J. P. , Kreitman Robert J. , Pastan Ira , Hagenbeek Anton , Martens Anton C. M.
Published: November 2003

The potential to selectively eliminate acute myeloid leukaemia (AML) cells with the GM‐CSF‐diphtheria toxin fusion protein (DT‐GM‐CSF) was studied under conditions of autonomous proliferation in vitro with no growth factors (GFs) added and after growth stimulation with a mixture of human (hu)G‐CSF, huIL‐3 and huSCF. DNA synthesis was maximally inhibited after 48 h exposure to DT‐GM‐CSF. Cell viability and AML colony forming ability in vitro were reduced. 18/22 samples were found to be sensitive to DT‐GM‐CSF, with 50% inhibition of DNA synthesis (ID50) at concentrations ranging from 0.1 to 16 ng/ml, and four samples were minimally or not sensitive to DT‐GM‐CSF (ID50  99 ng/ml). From the 15 samples which showed autonomous proliferation, 13 were sensitive to inhibition of proliferation by DT‐GM‐CSF. The level of GM‐CSF receptor (GM‐CSFR) expression was determined by flow cytometry after labelling with specific antibodies for the alpha and beta subunits. Although the toxicity to DT‐GM‐CSF was specifically mediated by the GM‐CSFR, no correlation was found between the level of expression of the GM‐CSFR alpha or beta subunit and the sensitivity for DT‐GM‐CSF. These in vitro studies show that the DT‐GM‐CSF fusion protein can be used for specifically targeting and eliminating leukaemic cells in the majority of AML cases.
Alpha interferon gene deletions in post‐transplant lymphoma
Authors: Wood Angela , Angus Brian , Kestevan Patrick , Dark John , Notarianni Giancarlo , Miller Sue , Howard Martin , Proctor Steve , Middleton Pete
Published: November 2003

Post‐transplant lymphoproliferative disorder (PTLD) is a well‐recognized complication of organ transplant and has been associated with high mortality using conventional chemotherapy. We have investigated 11 cases of PTLD for alterations to the interferon alpha (IFNA) and p16 genes on chromosome 9p using archival material. 4/9 (44%) cases had deletions of the IFNA genes, in contrast to 1/59 (1.7%) cases of intermediate/high‐grade de novo NHL drawn from the same geographical region. PTLD may therefore represent a distinct NHL subgroup exhibiting distinct gene pathology.
Ultraviolet irradiation induces multiple DNA double‐strand breaks and apoptosis in normal granulocytes and chronic myeloid leukaemia blasts
Authors: Bogdanov Konstantin V. , Chukhlovin Alexei B. , Zaritskey Andrey Yu , Frolova Olga I. , Afanasiev Boris V.
Published: November 2003

Major leucocyte subpopulations were isolated from peripheral blood of healthy donors, and patients with chronic myeloid leukaemia (CML) and chronic lymphoid leukaemia (CLL). In vitro UV irradiation was performed at the wavelength of 257 nm (UVC band). DNA double‐stranded breaks (DNAdsbs) were detected immediately after UV‐irradiation, by means of agarose gel electrophoresis. Cell viability was estimated after 18 h in culture, as relative numbers of residual non‐apoptotic cells. Evaluation of the dose–response curves revealed that normal CLL lymphoid cells showed only moderate damage after UV‐irradiation, as assessed by DNAdsbs and cell viability criteria. However, normal granulocytes and myeloid blasts from CML patients expressed a sharp increase in DNAdsbs, even at lower doses of UV‐radiation. UV‐induced amplification of endogenous oxidative systems (e.g. NADPH‐dependent oxidase) is suggested as a probable reason for enhanced DNA breakage and apoptosis in cells of the granulocytic lineage.
Long‐term follow‐up of de novo myelodysplastic syndromes treated with intensive chemotherapy: incidence of long‐term survivors and outcome of partial responders
Authors: Wattel Eric , De Botton Stéphane , Laï Jean Luc , Preudhomme Claude , Lepelley Pascale , Bauters Francis , Fenaux Pierre
Published: November 2003

The percentage of long‐term survivors after intensive chemotherapy and the outcome of MDS patients who achieve partial remission (PR) with intensive chemotherapy (IC) are not known.

Between 1981 and 1996 we treated 99 patients with de novo MDS who had high‐risk MDS or progression to AML, with IC. 41 (41%) achieved CR, 16 (16%) achieved partial remission (PR), 26 (26%) had failure, and 16 (16%) died in aplasia. Eight of the patients who achieved CR were autografted, three were allografted and the remaining cases received moderate consolidation chemotherapy.

After IC, the 16 PR patients fulfilled the criteria for RA in 15 cases and CMML in one case. Median PR duration was 17 months, and three PR were > 3 years (39, 50+, 82+ months). Median actuarial survival of patients who achieved PR and CR was 18 months and 20 months from the onset of IC, respectively (difference not significant).

Of the 71 patients treated before 1993, with sufficient follow‐up, 10 (14%) had survived > 4 years (long‐term survivors). Four of them were alive in first CR after 49+ to 110+ months and probably cured, two were alive in PR after 50+ and 82+ months and four had died after 49–78 months. Long‐term survivors were characterized by a significantly higher incidence of RAEB‐T at diagnosis, and with normal or favourable cytogenetic findings. In patients with RAEB‐T at diagnosis included before 1993, 8/23 (35%) cases who had no unfavourable karyotype had survived > 4 years.

Our findings suggest that MDS patients who achieve PR with IC, and not only those who achieve CR, can benefit from this type of treatment. The percentage of long‐term survivors remains low, however, and is almost restricted to patients with RAEB‐T at diagnosis and no unfavourable karyotype.
Engraftment of normal stem cells in W/Wv mice assessed by a novel quantitative PCR analysis
Authors: Tanosaki R. , Migliaccio A. R.
Published: November 2003

We describe a quantitative PCR based on the unique Nsi 1 restriction site generated by the Wv mutation (C to T substitution at position 2007 of c‐kit) to quantify the percentage of engraftment of wild‐type stem cells in W/Wv mice. Primers flanking this mutation specifically amplify c‐kit sequences from either genomic DNA or cDNA. After Nsi 1 digestion, wild‐type products were identified by electrophoresis as a single 104 bp (genomic DNA) or 118 bp (cDNA) band whereas W/Wv products migrated as two bands: the 104–118 bp band (corresponding to the W allele) and the 85 bp band (corresponding to the Wv allele). The relationship between the ratio/Wv/total c‐kit product versus the ratio W/Wv cells/total cells obtained in PCR amplification of artificial cell mixtures was expressed by a statistically significant linear regression. This indicated that the Wv/total c‐kit ratio depends mainly upon the ratio between the two cell types in the preparation to be analysed. Therefore it is possible to determine the percentage of donor cells in tissues of W/Wv transplanted with normal stem cells by comparing the amplification ratio obtained with DNA extracted from the tissues with the ratio obtained in standard calibration curves. As applications of the technique, we determined the percentage of donor‐derived cells in mononuclear blood fractions, in preparations of splenic B, T and myelo‐monocytic cells and in GM colonies cultured from the marrow of W/Wv mice transplanted with wild‐type stem cells.
Granulocyte‐macrophage colony stimulating factor receptor α and β chain complexes can form both high and intermediate affinity functional receptors
Authors: WHEADON HELEN , DEVEREUX STEPHEN , KHWAJA ASIM , LINCH DAVID C.
Published: November 2003

Scatchard analysis of primary human haemopoietic cells using iodinated GM‐CSF suggests that there are low, intermediate and high affinity classes of the GM‐CSF receptor. To investigate the molecular basis of this, we generated a clone of transfected NIH3T3 cells that constitutively expressed the human granulocyte‐macrophage colony stimulating factor receptor (GM‐CSF R) β chain and inducibly expressed the human GM‐CSF R α chain. In the cells fully induced to express the α chain the overall level of expression of the α and β chains at the cell surface was comparable with that found in primary haemopoietic cells and cell lines. When cells were partially induced to express the α chain, the α:β ratio determined by antibody binding was approximately 1:1 and Scatchard analysis revealed a single class of intermediate affinity receptors (Kd = 614 ± 88 pm). In cells with fully induced α chain expression, the α:β ratio was approximately 3:1 and there was a switch to a dual high and low affinity receptor with Kds of 67 ± 32 pm and 1.7 ± 0.56 nm respectively. The change from intermediate to high affinity was not associated with changes in αβ stoichiometry as detected by cross‐linking with radiolabelled GM‐CSF and gel electrophoresis. Both the high and intermediate affinity receptors were able to activate the STAT 5 and the MAP kinase pathways, although there was a difference in the ligand dose–response curves which was compatible with the different affinities of the receptors. It is proposed that the switch from an intermediate to high affinity receptor was due to the availability of free α chains presenting ligand to the αβ chain complexes at the surface of the cell membrane.
Allogeneic bone marrow transplantation for agnogenic myeloid metaplasia
Authors: Guardiola Philippe , Esperou Helene , Cazals‐Hatem Dominique , Ifrah Norbert , Jouet Jean‐Pierre , Buzyn Agnes , Sutton Laurent , Gratecos Nicole , Tilly Herve , Lioure Bruno , Gluckman Eliane
Published: November 2003

Agnogenic myeloid metaplasia is a rare indication for allogeneic bone marrow transplantation (BMT). We have retrospectively studied 12 patients allografted for this disease within the French BMT group. Prior to BMT, the mean age was 40 years (range 14–49). Diagnosis was based on the Polycythaemia Vera Study Group criteria. Before BMT, 10 patients had been splenectomized, eight required transfusions, and four had received at least two lines of chemotherapy. Cyclophosphamide and total body irradiation was the main conditioning regimen used (n=8). The donor was an HLA‐identical sibling except in one case where there was one HLA‐DR mismatch. Acute graft‐versus‐host disease (GVHD) prophylaxis consisted of methotrexate and cyclosporine A. 11 patients engrafted with median times to achieve absolute neutrophil count > 0.5 × 109/l and platelet count > 50 × 109/l of 17 (range 12–44) and 29 (range 12–196) days respectively. One primary graft failure occurred. 10 patients developed grade II–IV acute GVHD, four developed extensive chronic GVHD. One patient relapsed 16 months post‐BMT and was untreated and well 14 months later. Three patients died from the BMT procedure. In May 1996 the median follow‐up was 25 months and the 4‐year overall and event‐free survivals were 71% and 59%, respectively. Thus, we conclude that extensive myelofibrosis is not associated with delayed engraftment, and that HLA‐identical sibling allogeneic BMT can be considered in a small proportion of patients with agnogenic myeloid metaplasia.
Clonal stability in late‐relapsing childhood lymphoblastic leukaemia
Authors: Frost Lindsay , Goodeve Anne , Wilson Gillian , Peake Ian , Barker Helen , Vora Ajay
Published: November 2003

We report stability of a clonal immunoglobulin heavy chain (IgH) gene rearrangement in a case of childhood acute lymphoblastic leukaemia (ALL) relapsing 17 years after completion of first‐line therapy. Clonal stability was shown by polymerase chain reaction amplification of the hypervariable CDRIII region of IgH gene. Identically sized products from the original diagnostic and the second presentation samples were obtained and direct sequencing confirmed complete sequence homology. Absence of clonal evolution together with recent reports of persistent minimal residual disease in patients in long‐term complete remission, suggests that ‘cure’ of childhood ALL may be critically dependent on effective immune surveillance to keep such disease below clinically significant levels.
Dendritic cells generated from the blood of patients with multiple myeloma are phenotypically and functionally identical to those similarly produced from healthy donors
Authors: Pfeiffer S. , Gooding R.P. , Apperley J.F. , Goldschmidt H. , Samson D.
Published: November 2003

Using a combination of GM‐CSF, SCF, flk‐2/flt‐3 ligand, and IL‐4, dendritic cells (DC) have been generated in vitro from the adherent fraction of mononuclear cells isolated from the blood of patients with MM. Analysis of cell yield showed no significant difference in DC yield (numbers or percentage of leucocytes) or total number of leucocytes generated in myeloma cultures compared to similar cultures prepared using mononuclear cells from the blood of healthy donors. The mean number of DC produced after 10 d of culture were 8.19×105 and 9.87×105 cells (41% and 51% of all leucocytes) for the myeloma and normal cultures respectively. Flow cytometry investigation of phenotypic markers including CD1a, HLA‐DR, CD80 (BB1/B7.1) and CD86 (B70/B7.2), and functional status (stimulatory potential in allogeneic mixed leucocyte reactions (MLR)) confirmed the generation of cells phenotypically identified as cultured DC. In addition, these cells were more effective than PBMC at stimulating allogeneic PBMC proliferation. These data demonstrate no difference between DC generated from patients with MM and healthy donors. This study was considered a prerequisite for future investigations directed towards developing effective immunotherapies for myeloma.
Expression of several members of the TNF‐ligand and receptor family on tonsillar lymphoid B cells
Authors: Dürkop Horst , Anagnostopoulos Ioannis , Bulfone‐Paus Silvia , Stein Harald
Published: November 2003

Although the expression patterns of the members of the tumour necrosis factor receptor and ligand families have extensively been studied by flow‐cytometry on stimulated peripheral blood mononuclear cells (PBMNC), little or no flow‐cytometric or immunohistological data exist about their expression in lymphoid tissue. According to the data obtained from stimulated PBMNC, several members of these molecule families (e.g. CD40 ligand [CD40L], CD30, CD27, hOX40) have been considered to be either T‐cell restricted or strongly T‐cell associated. The present study on samples from palatine tonsils revealed that most of these molecules are also expressed by tonsillar B cells. The additional analysis of the co‐expression of these molecules also disclosed the existence of CD40+/CD40L+ and CD27+/CD70+ (CD27L+) lymphoid cells in tonsillar tissue.
Two cytotoxic pathways of natural killer cells in human cord blood: implications in cord blood transplantation
Authors: Umemoto Masakazu , Azuma Eiichi , Hirayama Masahiro , Nagai Masataka , Hiratake Shinnya , Qi Jiang , Kumamoto Tadashi , Komada Yoshihiro , Sakurai Minoru
Published: November 2003

Using K562 cells as a target we investigated cord blood (CB)–natural killer (NK) cytolytic pathways. The cytotoxicity of fresh CB‐NK cells was significantly lower than that of peripheral blood mononuclear cells (PB MNCs). When CB was incubated with IL‐2, the level of CB‐NK cytotoxicity was increased and boosted to the level observed in PB‐NK cells. Fresh CB‐NK cells induced apoptosis in target cells. Activated CB cells induced apoptosis and necrosis in target cells, at the same level as PB MNCs. CB stem cell transplantation may also induce graft‐versus‐host disease (GVHD)/graft‐versus‐leukaemia (GVL), similar to bone marrow transplantation.
Identification of del(6)(q21q25) as a recurring chromosomal abnormality in putative NK cell lymphoma/leukaemia
Authors: Wong K. F. , Chan J. K. C. , Kwong Y. L.
Published: November 2003

The putative natural killer (NK) cell lymphomas/leukaemias are a group of recently characterized haematolymphoid malignancies sharing an immunophenotype of CD3/Leu4− CD3ɛ+ CD56+, and a genotype of germline T‐cell receptor genes. They frequently present in extranodal sites and exhibit a highly aggressive clinical course. Information on the cytogenetic or molecular events leading to the tumourigenesis in this group of tumours is very scarce. In this study we analysed the cytogenetic findings of seven patients with CD56‐positive putative NK cell lymphoma/leukaemia. Three cases, including one nasal, one extranasal and one leukaemic form, showed a common region of deletion at 6q21‐q25, suggesting that this may be a non‐random chromosomal aberration.
Megakaryocytes derived from CD34‐positive cord blood cells produce interleukin‐8
Authors: Higuchi Tsukasa , Koike Kenichi , Sawai Nobukuni , Hemed Mwamtemi Hadija , Takeuchi Kouichi , Shiohara Masaaki , Kikuchi Toshimi , Yasui Kozo , Ito Susumu , Yamagami Osamu , Sasaki Yumiko , Okumura Nobuo , Kato Takashi , Miyazaki Hiroshi , Ikeda Masayuki , Yamada Muneo , Komiyama Atsushi
Published: November 2003

In a serum‐free liquid culture, thrombopoietin (TPO) selectively stimulated the growth of megakaryocytic cells from CD34‐positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Day 10 megakaryocytes (2 × 105) secreted > 1000 pg/ml of interleukin (IL)‐8, in contrast to small amounts of IL‐1β and IL‐6. A time‐course study showed that the IL‐8 production of megakaryocytes occurred at the late phase of the culture period. The megakaryocyte‐conditioned medium had the chemotactic potential of polymorphonuclear leucocytes, which was abrogated by the addition of anti‐IL‐8 antibody, suggesting the secretion of biologically active IL‐8. The combination of TPO and IL‐1α was required for a significant augmentation of the IL‐8 secretion. Direct evidence for IL‐8 synthesis in megakaryocytes was provided by reverse transcription–polymerase chain reaction on purified CD41b+ cells and by the detection of intracellular IL‐8 in CD41b+ cells. These results suggest that TPO stimulates not only the proliferation and differentiation of the progenitors capable of megakaryocytic lineage expression but also IL‐8 release by the megakaryocytic cells with the aid of IL‐1.
Addition of granulocyte colony‐stimulating factor to chemotherapy in patients with AIDS‐related lymphoma: effects on neutrophil Fcγ receptor expression and soluble FcγRIII plasma levels
Authors: Kersten M. J. , De Jong S. , Evers L. M. , Koene H. R. , Van Oers M. H. J.
Published: November 2003

AIDS‐related neutropenia and neutrophil dysfunction can (partly) be reversed by granulocyte‐colony stimulating factor (G‐CSF). We studied the effect of G‐CSF on neutrophil increment and levels of soluble Fcγ receptor type III in 15 patients with AIDS‐related lymphoma (ARL) undergoing chemotherapy. In six of these patients we performed a detailed kinetic analysis of the membrane expression of the functionally important Fcγ‐receptors type I, II and III. In all these patients G‐CSF induced FcγRI positive neutrophils with a decreased expression of the FcγRIII receptor. These changes were similar to those seen both in healthy volunteers and in non‐HIV‐infected individuals treated with chemotherapy. Interestingly, the mean neutrophil and sFcγRIII increment were significantly lower and more patients had a nadir granulocyte count < 0.5 × 109/l after the first cycle than after the second cycle of chemotherapy. This may be related to a therapy‐associated decrease in HIV‐1 viral load.

The conclusion is that patients treated with chemotherapy for ARL have a qualitatively normal response to G‐CSF.
Tumour cell u‐PA as a cause of fibrinolytic bleeding in metastatic disease
Authors: Bennett Bruce , Croll Alison M. , Robbie Linda A. , Herriot Richard
Published: November 2003

Tumour cells may express urokinase type plasminogen activator (u‐PA). This may influence the invasive properties of the cells but has seldom been implicated in production of a systemic bleeding state. Two patients are described in whom severe bleeding occurred in association with disseminated malignancies. Thrombin generation was little disturbed and platelet numbers were insufficient to account for the bleeding. Florid plasmin generation was evident in the circulation and the fibrinolytic inhibitor tranexamic acid controlled the bleeding well. Free active u‐PA was demonstrated in the circulation and u‐PA antigen on the malignant cells which invaded the marrow of one of the patients. Tumour cell u‐PA may occasionally be responsible for a bleeding state.
Brefeldin A inhibits thrombin receptor regeneration in human endothelial cells
Authors: Storck Josef , Vahland Michael , Breer Thorsten , Zimmermann E. R.
Published: November 2003

Endothelial cells, once stimulated with thrombin, are resistant to subsequent stimulation. After a recovery period of about 60 min the cells are sensitized again for activation by thrombin. The resensitization is independent of the receptor de novo synthesis. Therefore an intracellular pool of thrombin receptors that is possibly co‐localized with the Golgi apparatus has been assumed. Brefeldin A (BFA) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organization of the Golgi apparatus. Accordingly we have examined the effects of BFA on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. The von Willebrand factor (VWF) release from Weibel‐Palade vesicles and the intracellular calcium mobilization were used as physiological parameters of thrombin receptor activation. The addition of BFA (2 μg/ml) to endothelial cells or the reduction of the incubation temperature from 37°C to 16°C blocked the receptor response regeneration almost completely.
Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor‐α concentration
Authors: Peddie Clare M. , Wolf C. Roland , McLellan Lesley I. , Collins Andrew R. , Bowen David T.
Published: November 2003

Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor‐α (TNF‐α) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single‐cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor‐enriched bone marrow CD34+ compartment from MDS patients (P = 0.039), which are absent in both CD34− MDS cells (P = 0.53) and also CD34+ cells from normal subjects (P = 0.55). MDS CD34+ blood cells also showed oxidized pyrimidine nucleotides compared with CD34− cells (P = 0.029). Within normal subjects no differences were seen between CD34+ and CD34− bone marrow cell compartments. CD34+ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF‐α and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF‐α, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.
ALL R‐87 protocol in the treatment of children with acute lymphoblastic leukaemia in early bone marrow relapse
Authors: Giona Fiorina , Testi Anna Maria , Rondelli Roberto , Amadori Sergio , Arcese William , Meloni Giovanna , Moleti Maria Luisa , Ceci Adriana , Pillon Marta , Madon Enrico , Comis Margherita , Pession Andrea , Mandelli Franco
Published: November 2003

Seventy‐three children with acute lymphoblastic leukaemia (ALL) in first bone marrow (BM) relapse, occurring within 30 months from complete remission (CR), were enrolled in an Italian cooperative study (ALL R‐87 protocol). This treatment programme consisted of an induction phase with intermediate‐dose cytarabine (IDARA‐C) plus idarubicin (IDA) and prednisone (PDN), followed by a multidrug consolidation therapy and bone marrow transplant (BMT). 55/73 children achieved CR (75.3%); 15 (20.5%) failed to respond and three (4.2%) died during induction. The response rate was significantly higher for children with a first CR duration 12 months (P = 0.0005) and for those with a white blood cell (WBC) count at relapse <20 × 109/l (P=0.004). The estimated disease‐free survival (DFS ± SE) at 82 months was 0.18 ± 0.05 for all responders, and 0.70 ± 0.14 for allotransplanted patients versus 0.05 ± 0.05 for those autografted (P = 0.001). The estimated probabilities of survival ± SE and event‐free survival (EFS ± SE) at 83 months were 0.16 ± 0.07 and 0.13 ± 0.04, respectively, for all enrolled children. Univariate analysis showed that age <10 years at initial diagnosis and B‐lineage immunophenotype favourably influenced both DFS (P = 0.001) and EFS probabilities (P = 0.0014 and P = 0.012, respectively), whereas a first CR duration 12 months and a WBC count at relapse <20 × 109/l were associated only with a better EFS rate (P = 0.026 and P = 0.004, respectively).

Our results show the efficacy of the IDA plus IDARA‐C schedule used in the ALL R‐87 protocol in high‐risk relapsed ALL children. Allogeneic BMT proved effective for patients with an HLA sibling donor. In a multivariate analysis, age 10 years at initial diagnosis (P = 0.016) and WBC count at relapse 20 × 109/l (P = 0.048) were independently associated with a worse disease outcome.