bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross‐validation of prognostic scores in myelodysplastic syndromes on 386 patients from a single institution confirms importance of cytogenetics
Authors: Pfeilstöcker Michael , Reisner Regina , Nösslinger Thomas , Grüner Helga , Nowotny Hadwiga , Tüchler Heinz , Schlögl Ernst , Pittermann Elisabeth , Heinz Renate
Published: March 2003

In myelodysplastic syndromes (MDS) different prognostic risk analysis systems based on clinical and morphological data are used for predicting survival. Data on diagnostic and prognostic relevance of karyotype aberrations have prompted the development of scores including cytogenetics. The aim of this study was to assess and compare the explanatory power of different scoring systems and to assess the additional explanatory power of cytogenetics by evaluating the clinical and laboratory data of MDS patients from a single institution. Data of 386 MDS patients was available, with cytogenetic analysis at time of diagnosis in 256. Clinical/morphological scores: Bournemouth, modified Bournemouth and Düsseldorf; and scores including cytogenetics: Lausanne–Bournemouth, Lille and the International Prognostic Scoring System (IPSS), were calculated and their predictive power was compared for both overall survival and preleukaemic duration. Each of the scores had significant correlation on both endpoints. Calculating the prognostic value of different cytogenetic aberrations we found that differentiating between evidence for no aberration, single aberrations excluding chromosomes 7 and 8, aberrations on chromosomes 5, 7 or 8 and complex aberrations was important. These data were incorporated in a ‘prognostic index cytogenetics’ (pi score). Cytogenetic scores significantly improved the prognostic value of the best clinical/morphological score in regard to both overall survival and preleukaemic duration. In conclusion, our data further stress the importance of cytogenetics for predicting prognosis in MDS.
CD130 rather than CD126 expression is associated with disease activity in multiple myeloma
Authors: Barillé Sophie , Thabard Wilfrid , Robillard Nelly , Moreau Philippe , Pineau Danielle , Harousseau Jean‐Luc , Bataille Régis , Amiot Martine
Published: March 2003

We analysed the expression of both components of IL‐6R, CD126 the ligand binding protein and CD130 the signal transducing protein, on plasma cells from MGUS and multiple myeloma (MM) cases using flow cytometry. CD126 was detectable in 50% of either MGUS or MM patients without any change of expression during disease progression. In contrast, CD130 expression was up‐regulated during tumoural expansion (43% of MM patients at diagnosis versus 88% at relapse). Finally, combining CD126 and CD130 expression we found a significant increase of the percentage of CD126+ CD130+ patients at relapse, underlying the crucial role of IL‐6 response in the late stage of MM.
Complete remission in acute myeloid leukaemia with t(8;21) following treatment with G‐CSF: flow cytometric analysis of in vivo and in vitro effects on cell maturation
Authors: Ferrara Felicetto , Di Noto Rosa , Viola Assunta , Russo Consiglia , Boccuni Piernicola , Costantini Silvia , Dello Russo Annamaria , Lo Pardo Catia , Del Vecchio Luigi
Published: March 2003

A 75‐year‐old patient diagnosed as having acute myeloid leukaemia with t(8;21) received G‐CSF alone as induction therapy. Complete remission was achieved following 2 weeks of treatment. Flow cytometric analysis, performed by CD45 technique modified by the introduction of preliminary gating with LDS‐751, confirmed the disappearance of blast cells along with myeloid maturation. Finally, in vitro studies demonstrated that G‐CSF, as compared to other differentiation inducers, was able to induce a striking effect toward neutrophilic differentiation.
Early progenitor cells from human mobilized peripheral blood express low levels of the flt3 receptor, but exhibit various biological responses to flt3‐L
Authors: Aurran‐Schleinitz Thérèse , Imbert Anne‐Marie , Humeau Laurent , Bardin Florence , Charbord Pierre , Chabannon Christian
Published: March 2003

The biological effects of flt3‐L, and the expression of its tyrosine kinase receptor (flt3, CD135) were investigated on the immature subsets of human circulating peripheral blood progenitors obtained from cancer patients or normal volunteer donors, after mobilization with rhG‐CSF or chemotherapy. flt3 was expressed at low levels, and its expression increased concomitantly with expression of CD38 within the CD34+ cell population. Despite this low‐level expression, flt3‐L exerted synergistic effects with a combination of c‐kit ligand, IL‐3, IL‐6, GM‐CSF and G‐CSF, mainly to induce proliferation of CD34+/CD38− cells. In addition, flt3‐L increased the detection of HPP‐CFC, both immediately after cell selection, and after 7 and 14 d of cultures. We conclude that flt3‐L is active on circulating early mobilized haemopoietic progenitors, despite the low‐ level expression of its receptor.
Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function
Authors: Celie Patrick H. N. , Van Stempvoort Gunny , Jorieux Sylvie , Mazurier Claudine , Van Mourik Jan A. , Mertens Koen
Published: March 2003

The functional defect caused by substitution of Arg527 (→ Trp) and Arg531 (→ Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII‐R527W and FVIII‐R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII‐R527W, the amount of FVIII‐R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII‐R531G revealed that the activated FVIII‐R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII‐R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII‐derived synthetic peptide Tyr511‐Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three‐dimensional FVIII model residues Val517‐Arg527 are located near the FIXa binding site Ser558‐Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558‐Gln565 and Val517‐Arg527.
Bone marrow effects of anagrelide therapy in patients with myelofibrosis with myeloid metaplasia
Authors: Yoon Soo‐Young , Li Chin‐Yang , Mesa Ruben A. , Tefferi Ayalew
Published: March 2003

In a prospective study investigating the therapeutic role of anagrelide in myelofibrosis with myeloid metaplasia, 20 patients received anagrelide in daily oral doses of 0.5–3 mg. 17 patients were evaluable and received anagrelide for a median of 2 years (range 0.5–4 years). No patient had a clinically appreciable benefit. Bone marrow (BM) examinations at baseline and after 6 and 12 months of treatment were available for 17, 17 and 12 patients, respectively. In all evaluable cases, BM megakaryocyte number increased after 6 months of anagrelide treatment. Also, Ulex europaeus agglutinin‐1 staining of megakaryocytes revealed a left‐shifted maturation pattern in most patients with a platelet response to anagrelide. However, megakaryocyte staining intensity for transforming (TGF‐β) and platelet‐derived (PDGF) growth factors was not affected consistently by treatment. No patient had a 2 grade change in either BM fibrosis or osteosclerosis. These in‐vivo data support our previous in‐vitro observations that anagrelide interferes with megakaryocyte maturation rather than proliferation. Lack of a positive treatment effect is consistent with the finding that anagrelide did not significantly alter megakaryocyte expression of TGF‐β and PDGF.
VH gene sequences from a novel tropical splenic lymphoma reveal a naive B cell as the cell of origin
Authors: Zhu Delin , Thompsett Andrew R. , Bedu‐Addo George , Stevenson Freda K. , Bates Imelda
Published: March 2003

The prevalence of malaria and other infections in tropical Africa provides a setting for the emergence of B‐cell tumours distinct from that in Western countries. Attempts to draw comparisons with Western lymphomas have led to difficulties, with so‐called African chronic lymphocytic leukaemia (CLL) having a different pattern of incidence from Western CLL. Splenomegaly is common in African CLL, and this has posed diagnostic problems in differentiating the tumour from malaria‐associated hyper‐reactive malarial splenomegaly (HMS). One feature of the splenomegalic form of African CLL is that the tumour cells often possess short but fine cytoplasmic projections reminiscent of those observed in Western splenic lymphoma with villous lymphocytes (SLVL). Analysis of Ig VH genes both facilitates discrimination between clonal B‐cell tumours and HMS, and reveals the differentiation status of the cell of origin. This study indicated that VH genes of nine cases of clonal splenic B‐cell tumours with villous lymphocytes from Ghana were relatively unmutated, consistent with an origin from a naive B cell. These features differ from SLVL which arises from a post‐follicular antigen‐selected B cell. One possibility is that these splenic B‐cell tumours derive from a splenic T‐independent B cell, with malaria infection as a potential influence.
High‐sensitive immunophenotyping and DNA ploidy studies for the investigation of minimal residual disease in multiple myeloma
Authors: Almeida Julia , Orfao Alberto , Ocqueteau Mauricio , Mateo Gema , Corral Mercedes , Caballero Ma Delores , Blade Joan , Moro Ma Jesus , Hernandez Jose , San Miguel Jesus F.
Published: March 2003

Sensitive techniques for monitoring minimal residual disease (MRD) in multiple myeloma (MM) are needed to evaluate the effectiveness of new intensive treatment strategies. The aim of the present study was to explore the applicability and sensitivity of flow cytometry immunophenotyping and DNA ploidy studies for the investigation of residual myelomatous plasma cells (PC) in MM patients. Bone marrow (BM) samples from 61 untreated MM patients were immunophenotypically analysed with a panel of 21 monoclonal antibodies, using a high‐sensitive method based on a two‐step acquisition procedure through a SSC/CD38+++‐CD138+‘live‐gate’. Overall, in 87% of MM cases, PC displayed an aberrant phenotype at diagnosis. The most important aberrant criteria were: antigen over‐expression of CD56 (62%), CD28 (16%) and CD33 (6%) and asynchronous expression of CD117 (28%), sIg (21%) and CD20 (10%). DNA aneuploidy was found in 62% of cases. The simultaneous use of these two techniques allowed the detection of aberrant/aneuploid PC in 95% of the cases. Based on dilutional experiments, the detection limit of both techniques ranged from 10−4 to 10−5. In 29 stem cells harvests and 19 BM samples obtained 3 months after autologous transplantation, we have investigated the presence of residual myelomatous PC; they were detected in 44% of the stem cell collections and in 61% of the BM samples obtained after transplant. The percentage of pathological PC did not significantly change during the days of harvest. In summary, the present study shows that the combined use of immunophenotyping and DNA ploidy studies is a suitable approach for MRD investigation in MM patients based on their applicability (95% of cases) and sensitivity (up to 10−5).
Metabolism and functional effects of sphingolipids in blood cells
Authors: Yang Libo , Yatomi Yutaka , Miura Yoshie , Satoh Kaneo , Ozaki Yukio
Published: March 2003

We examined the sphingolipid metabolism of peripheral blood cells, i.e. platelets, erythrocytes, neutrophils and mononuclear cells. A distinguishing characteristic of sphingolipid metabolism in these highly differentiated cells was their high sphingosine (Sph) kinase activity. The occurrence of [3H]sphingosine 1‐phosphate (Sph‐1‐P) from [3H]Sph (actively incorporated from the outside) in the blood cells was strong, long‐lasting, and independent of cell activation. Hence, the possibility of Sph‐1‐P playing a second messenger role is remote in these cells. About 40% of platelet Sph‐1‐P could be released extracellularly by 12‐O‐tetradecanoylphorbol 13‐acetate, possibly through mediation by protein kinase C. On the other hand, in erythrocytes, neutrophils and mononuclear cells a significant percentage of Sph‐1‐P formed inside the cell was discharged without stimulation, whereas the stimulation‐dependent release was marginal. In contrast to active [3H]Sph conversion to [3H]Sph‐1‐P, formation of [3H]sphingomyelin was barely detectable in the blood cells; this was especially true for anucleate platelets and erythrocytes. The Sph → Sph‐1‐P pathway may become predominant over the Sph → Cer → sphingomyelin pathway during late‐stage differentiation into platelets or erythrocytes. Sph and its methylated derivative, N,N‐dimethylsphingosine, induced apoptosis not only in neutrophils but also in mononuclear cells, whereas Sph‐1‐P elicited Ca2+ mobilization in platelets. Our results suggest that all blood cells may remove plasma Sph, which is harmful or suppressive to cellular functions, and change it into Sph‐1‐P, acting as the source of plasma Sph‐1‐P, which may play a variety of important roles in blood vessels.
BCR‐ABL influences the antileukaemic efficacy of alkylphosphocholines
Authors: Konstantinov Spiro M. , Eibl Hansjörg , Berger Martin R.
Published: March 2003

We have compared the antileukaemic efficacy of a series of new i.v. injectable alkylphosphocholines (APC) with their clinically used congeners miltefosine and perifosine. The test system consisted of four leukaemic cell lines carrying the bcr‐abl rearrangement (K‐562, LAMA‐84, CML‐T1 and BV‐173) and two other leukaemic cell lines (HL‐60 and SKW‐3) without this genetic alteration. The prototype of i.v. injectable APC, erucylphosphocholine, was more active against BCR‐ABL‐positive cell lines than the two reference APC. It induced programmed cell death in HL‐60 and SKW‐3 cells after exposure for 24 h, and in bcr‐abl expressing cells after a prolonged incubation period (48 h). LAMA‐84 cells responded to i.v. injectable APC with increased conversion to an adherent, fibroblast‐like phenotype. Experiments with a cell‐free system showed that the target structures of APC are localized within the cytoplasmic compartment. Blockade of ceramide synthase by fumonisin B1 was insufficient to prevent oligonucleosomal DNA fragmentation. Using RT‐PCR we confirmed that K‐562 and LAMA‐84 cells carry the b3a2 fusion type, and CML‐T1 and BV‐173 the b2a2 variant. BV‐173 cells had the lowest level of bcr‐abl mRNA which correlated with their increased sensitivity. Transfection of K‐562 cells with antisense oligonucleotides directed against bcr‐abl caused a specific suppression of K‐562 clonogenicity. Our data indicated that i.v. injectable alkylphosphocholines are potent inducers of apoptosis and display increased antileukaemic efficacy against BCR‐ABL‐positive blasts as compared with miltefosine and perifosine. The expression of BCR‐ABL cannot prevent apoptosis but delays erucylphosphocholine‐induced programmed cell death. Transfection with bcr‐abl directed antisense oligonucleotides reduces the clonogenicity of K‐562 cells.
Molecular remission of chronic myeloid leukaemia following a non‐myeloablative allogeneic peripheral blood stem cell transplant: in vivo and in vitro evidence for a graft‐versus‐leukaemia effect
Authors: Childs R. , Epperson D. , Bahceci E. , Clave E. , Barrett J.
Published: March 2003

Two patients with chronic myeloid leukaemia (CML) received a non‐myeloablative preparative regimen of cyclophosphamide and fludarabine, followed by an unmanipulated, G‐CSF‐mobilized, peripheral blood stem cell transplant from an HLA‐identical sibling. Chimaerism, evaluated in myeloid and T‐lymphoid lineages by PCR of minisatellite variable regions, showed day 14 post‐transplant haemopoietic recovery to be 90% autologous in both patients. On day 30 the bone marrow showed only 1/20 and 2/18 donor metaphases. By day 100 post transplant both had 100% donor myeloid and lymphoid lineages as assessed by karyotype and minisatellite chimaerism analysis. They subsequently became RT‐PCR negative for BCR‐ABL. Both survive 7 and 14 months post transplant in molecular remission of CML. In one, donor T cells, stimulated with pre‐transplant CML cells, induced 30–50% inhibition of pre‐transplant leukaemic CFU‐GM, but did not inhibit CFU‐GM in the day 60 marrow (46% Ph‐negative recipient, 54% donor). These results show that a non‐myeloablative allotransplant can induce molecular remissions of CML through a graft‐versus‐leukaemia effect.
Plasma IL‐8 and IL‐6 levels can be used to define a group with low risk of septicaemia among cancer patients with fever and neutropenia
Authors: De Bont Eveline S. J. M. , Vellenga Edo , Swaanenburg Joost C. J. M. , Fidler Vaclav , Brummen Petra J. Visser‐van , Kamps Willem A.
Published: March 2003

The standard therapy for patients with fever and chemotherapy‐related neutropenia is hospitalization and infusion of broad‐spectrum antibiotics. Early discharge of a defined group of patients at low risk for septicaemia would be of great advantage for these patients. In this study plasma interleukin‐8 (IL‐8) and interleukin‐6 (IL‐6) levels measured at start of fever (n = 72) could define a low‐risk group of febrile patients with neutropenia due to chemotherapy. For this purpose we collected and analysed data on 72 fever episodes from 53 patients with chemotherapy‐related neutropenia, aged between 1 and 66 years. Of the 72 episodes, 18 were classified as bacteraemia and/or clinical sepsis (sepsis group). The IL‐6 and IL‐8 plasma concentration were significantly increased in patients with chemotherapy‐related neutropenia and fever due to bacteraemia versus fever of non‐bacterial origin (P = 0.043 and P = 0.022 respectively). Logistic regression analysis, with sepsis as the outcome variable, revealed significant effects of age combined with either IL‐6 or IL‐8. Sepsis occurrence was lowest for patients <16 years and highest in patients between 16 and 50 years, and was higher in patients with increased IL‐6 (P = 0.032) or IL‐8 (P = 0.049). No significant effect of leucocyte count, C‐reactive protein, sex or underlying malignancy at presentation was detected. The plasma IL‐6 and IL‐8 levels were fairly strongly correlated (Pearson r = 0.62). Using a cut‐off value with 100% sensitivity, both IL‐8 and IL‐6 could define a low‐risk group of neutropenic patients of 28% (CI 15–40%) at the start of the febrile period. Intervention studies are warranted to confirm this result and to investigate whether an early discharge based on IL‐8 or IL‐6 measurement is safe, increases the quality of life, and results in cost savings.
Multiple autoimmune haemopoietic disorders and insidious clonal proliferation of large granular lymphocytes
Authors: Akashi Koichi , Shibuya Tsunefumi , Taniguchi Shuichi , Hayashi Shin , Iwasaki Hiromi , Teshima Takanori , Takamatsu Yasushi , Gondo Hisashi , Okamura Takashi , Harada Mine , Niho Yoshiyuki
Published: March 2003

We report a patient with clonal proliferation of CD3+8+TCRαβ+ large granular lymphocytes (LGL) presenting multiple episodes of autoimmune cytopenia, including autoimmune neutropenia, idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia, and pure red cell aplasia. Each disorder appeared separately or as a combination during an 11‐year clinical course. The increase of blood CD3+8+TCRαβ+ LGL was detected 6 years after the initial diagnosis of cytopenia, but the absolute number of LGL cells was always < 1.0 × 109/l. LGL cells were of monoclonal origin and had a chromosomal abnormality. LGL cells transiently responded to cyclosporine A therapy, which was also effective on all of these autoimmune cytopenias. Accordingly, an undetectable level of proliferation of a clonal LGL population could cause various autoimmune haemopoietic disorders.
p38 MAPK is activated but not necessary in porcine von Willebrand factor‐dependent platelet activation
Authors: Song Seng , Freedman John , Mody Meera , Lazarus Alan H.
Published: March 2003

We have investigated the role of p38 mitogen‐activated protein kinase (MAPK) in von Willebrand factor (VWF)‐dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF‐dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1 min post‐addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S‐transferase‐MAPK activated protein kinase‐2. To determine if p38 MAPK was necessary for porcine VWF‐induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF‐induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580‐inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.
Reduced TGF‐β1 in patients with aplastic anaemia in vivo and in vitro
Authors: Rizzo S. , Killick S. B. , Patel S. , Ball S. E. , Wadhwa M. , Dilger P. , Gordon‐Smith E. C. , Gibson F. M.
Published: March 2003

Transforming growth factor β (TGF‐β) 1 is a ubiquitous bifunctional cytokine implicated in the regulation of haemopoietic stem cells and bone marrow stromal cells. We analysed sera from 63 patients with aplastic anaemia and describe a significant reduction of TGF‐β1 that was directly related to their treatment status. Untreated patients (n = 35), patients who did not respond (n = 15) and those with a partial response (n = 23) to treatment had significantly lower TGF‐β1 than the normal control group (n = 55), P < 0.0001, P < 0.0001 and P = 0.002 respectively. Patients in complete remission (n = 15) exhibited TGF‐β1 serum levels comparable to the control group. In addition, there was a correlation (r = 0.83, P < 0.0001) between serum TGF‐β1 and platelet count at time of sample. We have demonstrated that the primary source of TGF‐β1 in peripheral blood mononuclear cell (PBMC) cultures was not CD3‐positive cells. These data indicate aplastic anaemia is associated with a decreased TGF‐β1 expression in peripheral blood circulation, which may be a direct consequence of thrombocytopenia.  In vitro stromal layers grown from aplastic patient bone marrow (n = 14) produced significantly lower levels of TGF‐β1 (P = 0.02) when compared to normal stroma (n = 15). In the aplastic anaemia bone marrow compartment we postulate that accessory cells down‐regulate TGF‐β1 expression to allow stem cell cycling to counteract hypoplasia. As TGF‐β1 is important in the regulation of haemopoiesis, dysregulation of this cytokine in combination with previously described abnormal cytokine expression may contribute significantly to the pathophysiology of aplastic anaemia by exacerbating primary stem cell defects.
Osteitis fibrosa cystica generalizata with adult T‐cell leukaemia: a case report
Authors: Yamaguchi Tetsuo , Hirano Toru , Kumagai Kenji , Tsurumoto Toshiyuki , Shindo Hiroyuki , Majima Ryuzo , Arima Nobuyuki
Published: March 2003

We report on a 62‐year‐old female initially suffering from extreme pain in both her lower extremities. Plain radiographs revealed multiple osteolytic lesions. Laboratory analyses indicated high levels of serum calcium and parathyroid hormone related protein (PTHrP) and detected HTLV‐1 antibody. Histological examination of biopsied specimens which were obtained from osteolytic lesions indicated osteitis fibrosa cystica (OFC) without tumour cells. After the initial biopsy, a subcutaneous mass developed in the left thigh. Microsections of the biopsied mass disclosed adult T‐cell leukaemia (ATL) neoplastic cells. Immunohistochemical findings revealed that PTHrP secreted from ATL neoplastic cells induced the OFC.
Bone marrow cell trafficking following intravenous administration
Authors: Cui Jisong , Wahl Richard L. , Shen Tingliang , Fisher Susan J. , Recker Elizabeth , Ginsburg David , Long Michael W.
Published: March 2003

To address trafficking of transplanted marrow cells immediately after intravenous infusion, we examined the early fate of infused non‐adherent, low‐density donor bone marrow cells in a syngeneic mouse model. The presence of infused donor cells, marked with indium‐111 oxine (111In), with the fluorescent dye PKH26, or by a detectable transgene marker, was evaluated at 3–48 h in a variety of tissues, including peripheral blood. All three cell‐marking methods indicated a rapid (< 4 h) influx of cells into the bone marrow, liver, spleen, muscle and other tissues. Moreover, these tissues remained positive for the 48 h observation period. Interestingly, analysis of PKH26‐positive cells in non‐myeloablated animals demonstrated that approximately 17% of infused donor marrow cells localized to the marrow space within 15 h, whereas a smaller proportion of donor cells (~ 1–2%) localized to the marrow in recipients preconditioned by irradiation. In an effort to enrich for cells that specifically home to the bone marrow, PKH26‐labelled donor marrow cells were recovered from the first host and infused into a secondary recipient. Although this was a phenotypically undefined population of cells, no increase was observed in the relative fraction of PKH26‐labelled cells returning or ‘homing’ to the marrow of the second recipient. Taken together, these data suggest both that marrow engraftment may be mediated by non‐specific ‘seeding’ rather than a specific homing signal, and that efficient targeting of transplanted cells to the marrow is a complex multifaceted process.
Molecular, haematological and clinical studies of the −101 C → T substitution of the β‐globin gene promoter in 25 β‐thalassaemia intermedia patients and 45 heterozygotes
Authors: Maragoudaki E. , Kanavakis E. , Traeger‐Synodinos J. , Vrettou C. , Tzetis M. , Metaxotou‐Mavrommati A. , Kattamis C.
Published: March 2003

We report the clinical, haematological, biosynthetic and molecular data of 25 double heterozygote β‐thalassaemia intermedia patients and 45 β‐thalassaemia heterozygotes with the C → T substitution at nucleotide position −101 from the Cap site, in the distal CACCC box of the β‐globin gene promoter. This mutation is considered the most common amongst the silent β‐thalassaemia mutations in Mediterranean populations. Of the 25 compound heterozygotes for the β−101 C → T and common severe β‐thalassaemia mutations, all but one had mild thalassaemia intermedia preserving haemoglobin levels around 9.5 g/dl and haemoglobin F levels < 25%. The only transfused patient was characterized to have an additional α‐globin gene.

Strict assessment of haematological and biosynthetic findings in the heterozygotes for the β−101 C → T mutation (excluding six cases with an α‐globin gene defect) demonstrated that less than half of them had completely normal (silent) haematology; the remainder had either high haemoglobin A2 values (in the range of 3.7–5.1%) and/or low red cells indices and/or raised haemoglobin F values. The α/non‐α‐globin chain synthesis ratios were generally raised, with mean 1.44 (1.07–2.10). Amongst the parents of the compound heterozygotes, who were not selected for molecular analysis following haematological screening, half of the cases were completely silent.

Interaction with severe β‐thalassaemia mutations always resulted in the clinical phenotype of mild non‐transfusion‐dependent thalassaemia intermedia.
The combined effect of total body irradiation (TBI) and cyclosporin A (CyA) on the risk of relapse in patients with acute myeloid leukaemia undergoing allogeneic bone marrow transplantation
Authors: Bacigalupo  , Vitale  , Corvò  , Barra  , Lamparelli  , Gualandi  , Mordini  , Berisso  , Bregante  , Raiola  , Van Lint  , Frassoni
Published: March 2003

One hundred and fifty acute myeloid leukaemia (AML) patients in first remission received an allogeneic bone marrow transplant (BMT), after conditioning with cyclophosphamide 120 mg/kg and total body irradiation (TBI) 3.3 Gy × 3 (total nominal dose 9.9). The received dose, as recorded by thermoluminescent dosimeters, ranged between 7.83 and 12.25 Gy. Patients who received TBI < 9.9 Gy (n = 34) had a significantly higher relapse rate when compared with patients receiving  9.9 Gy (n = 116) (43% vs. 19%; P = 0.002). Graft versus host disease (GvHD) prophylaxis consisted of cyclosporin A (CyA) with or without methotrexate (MTX). The dose of CyA was either 1 or 5 mg/kg/day i.v. from day −1 to + 20, then 10 mg/kg/day orally until day + 365. Patients receiving 5 mg/kg CyA (n = 40) had a higher risk of relapse (49% vs. 15%; P = 0.0001). Thus, low‐dose TBI (< 9.9Gy) and high‐dose CyA (5 mg/kg) were significant predictors of leukaemia relapse. Patients were then divided into three groups: those who had both negative predictors (< 9.9 Gy TBI and 5 mg/kg CyA; n = 26); those who had only one (either < 9.9 Gy TBI or 5 mg/kg CyA; n = 22); and those who had neither ( 9.9 Gy TBI and 1 mg/kg CyA; n = 102). The three groups were comparable for FAB subtype, interval diagnosis transplant and age. The 5‐year actuarial relapse rate for these three groups of patients was 49%, 41% and 15%, with no difference between the first two and a significant difference when compared with the latter (P < 0.01). These data indicate that acute myeloid leukaemia can be cured with allogeneic bone marrow transplantation given an intensive conditioning regimen and low‐dose immunosuppression post‐graft. Either alone is insufficient to produce long‐term disease‐free survival. These results may be relevant for programmes of reduced intensity conditioning designed for patients with acute leukaemia.
T‐prolymphocytic leukaemia with spontaneous remission
Authors: Shichishima  , Kawaguchi  , Machii  , Matsuoka  , Ogawa  , Maruyama
Published: March 2003

T‐prolymphocytic leukaemia (T‐PLL) is a rare dis‐order with a poor prognosis. A 69‐year‐old man was diagnosed as having a small‐cell variant of T‐PLL according to the French–American–British classification by haematological, immunological and ultrastructural studies, although the cells had a CD7− phenotype and no chromosomal abnormality. He had no symptoms or organomegaly. The number of his lymphocytes, 53.7 × 109/l at the time of diagnosis, gradually decreased without therapy, and he was in complete remission 39 months later. A rearranged band in the T‐cell antigen receptor‐β gene, which was detected at the time of diagnosis, decreased or disappeared. This is the first report of a T‐PLL case with spontaneous complete remission.
Haemophagocytic lymphohistiocytosis in X‐linked severe combined immunodeficiency
Authors: Grunebaum E. , Zhang J. , Dadi H. , Roifman C. M.
Published: March 2003

Haemophagocytic lymphohistiocytosis (HLH) is characterized by destruction of haematopoietic elements, and is associated with a variety of manifestations including immune abnormalities. We describe an infant with HLH who had no evidence of infection or malignancy. He had markedly reduced natural killer (NK) and T‐cell numbers and mitogen responses, consistent with severe combined immune deficiency. Western blot and flow cytometry analyses revealed an absence of interleukin (IL)‐2 receptor γ (γ common) chain expression and a transition (C → T) at nucleotide 684 in the γ common gene. This novel case highlights the need for a thorough evaluation of immunological phenotype and genotype in patients with HLH.
Reduction of intra‐ and interlaboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training
Authors: Barnett D. , Granger V. , Kraan J. , Whitby L. , Reilly J. T. , Papa S. , Gratama J. W. ,   for and on behalf of the CD34 Task Force of the European Working Group of Clinical Cell Analysis (EWGCCA)
Published: March 2003

The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single‐platform flow cytometric protocol for the enumeration of CD34+ stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34+ stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34+ Stem Cell Quantification that analysed the same specimens using non‐standardized methods. Two bead‐counting systems, Flow‐Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow‐Count, the intralaboratory coefficient of variation (CV) was ≤ 5% in 39% of the laboratories (trial 1), increasing to 65% by trial 3. Interlaboratory variation was reduced from 23.3% (trial 1) to 10.8% in trial 3. In trial 2, 70% of laboratories achieved an intralaboratory CV ≤ 5% using TruCount, increasing to 74% for trial 3; the interlaboratory CV was reduced from 23.4% to 9.5%. Comparative analysis of the EWGCCA and the UK NEQAS cohorts revealed that EWGCCA laboratories, using the standardized approach, had lower interlaboratory variation. Thus, the use of a common standardized protocol and targeted training significantly reduced intra‐ and interlaboratory CD34+ cell count variation.
Dynamics of telomere shortening in neutrophils and T lymphocytes during ageing and the relationship to skewed X chromosome inactivation patterns
Authors: Robertson Jane D. , Gale Rosemary E. , Wynn Robert F. , Dougal Mark , Linch David C. , Testa Nydia G. , Chopra Rajesh
Published: March 2003

Human haemopoiesis undergoes profound changes throughout life, resulting in compromised regenerative capacity of haemopoietic stem cells. It has been suggested that telomere shortening results in senescence of haemopoietic stem cell subsets and may influence the balance between stem cell renewal and proliferation. Telomere length and telomerase activity was measured in whole blood leucocytes, neutrophils and T cells from cord blood and individuals aged from 1 year to 96 years. Rapid telomere shortening [700 base pairs (bp)] was demonstrated in the first year of life, followed by a gradual decline of 31 bp/year. T cells were shown to have longer telomeres than neutrophils (mean difference 372 bp, P = < 0·001) but demonstrated similar rates of shortening (20 ± 0·3 bp/year vs. 22 ± 0·3 bp/year). Telomerase was detectable in T cells but not in neutrophils, suggesting that telomerase is not the rate‐limiting step for regulation of telomere length in haemopoietic cells. Stem cell utilization as measured by X chromosome inactivation patterns was found to be independent of telomere length. This supports the concept that age‐dependent skewed haemopoiesis is the result of random stem cell loss or X‐allelic exclusion rather than telomeric senescence. These studies provide insight into the ageing process and a reference point for evaluating replicative stress in individuals of different age groups.
Neutropenia and anaemia due to carbimazole‐dependent antibodies
Authors: Bux J. , Ernst‐Schlegel M. , Rothe B. , Panzer C.
Published: March 2003

Carbimazole‐dependent antibodies to erythrocytes were detected in the sera of three anaemic patients who had been treated with carbimazole for hyperthyroidism. By the use of Rhnull‐typed erythrocytes, we could show that some of these were directed against the proteins of the Rh complex. Carbimazole‐dependent antibodies eluted from erythrocytes showed no binding to other blood cells. One patient also presented with neutropenia and mild thrombocytopenia. Additional carbimazole‐dependent antibodies against the neutrophil‐specific Fcγ receptor IIIb (FcγRIIIb, CD16b) and the broadly expressed platelet endothelial cell adhesion molecule 1 (PECAM‐1; CD31) were detected in this patient's serum. Surprisingly, the PECAM‐1‐reactive drug‐dependent antibodies were also detectable in the sera of the other two patients with normal leucocyte and platelet counts. We assume that carbimazole can induce cell lineage‐specific drug‐dependent antibodies that cause cytopenia and also drug‐dependent antibodies against the broadly expressed PECAM‐1 molecule that may cause mild but not severe cytopenia.
Human bone marrow stroma‐dependent cell line MOLP‐5 derived from a patient in leukaemic phase of multiple myeloma
Authors: Matsuo Yoshinobu , Drexler Hans G. , Nishizaki Chiharu , Harashima Akira , Fukuda Shunichi , Kozuka Teruhiko , Sezaki Tatsuo , Orita Kunzo
Published: March 2003

The novel multiple myeloma (MM) cell line MOLP‐5 and its homologous sister cell line B407, a lymphoblastoid cell line (LCL), were established from the peripheral blood of a 71‐year‐old Japanese patient with Bence‐Jones κ‐type multiple myeloma (stage IIIB with hyperammonaemia and hypercalcaemia). The growth of MOLP‐5 cells is constitutively dependent on bone marrow stroma (BST) cells; none of the cytokines tested nor the culture supernatant of the bone marrow stroma cells could support the growth of MOLP‐5. Wright–Giemsa‐stained MOLP‐5 cells showed typical plasma cell morphology with abundant cytoplasm and one to three nuclei. The immunoprofile of MOLP‐5 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) κ light chain, CD28, CD29, CD38, CD40, CD44, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA‐1; the cells were negative for surface Ig and various other B‐cell, T‐cell and myelomonocyte‐associated immunomarkers. Interleukin 6 (IL‐6) receptor mRNA was found in the reverse transcriptase polymerase chain reaction (RT‐PCR) analysis. IL‐6 and IL‐10 could induce cellular proliferation in short‐term induction experiments. IL‐6 or IL‐10 production was not detected by specific enzyme‐linked immunoabsorbent assay (ELISA). MOLP‐5 cells expressed parathyroid hormone‐related protein (PTHrP) at the mRNA level. Cytogenetic analysis showed the typical t(11; 14) chromosome abnormality. The novel MOLP‐5 cell line together with the B407 B‐LCL sister line will be useful model systems in the investigation of the biology of MM.
Chronic overexpression of membrane‐bound flt3 ligand by T lymphocytes in severe aplastic anaemia
Authors: Pfister Otmar , Chklovskaia Elena , Jansen Wendy , Mészáros Kinga , Nissen Catherine , Rahner Christoph , Hurwitz Nina , Bogatcheva Natalia , Lyman Stewart D. , Wodnar‐Filipowicz Aleksandra
Published: March 2003

Aplastic anaemia (AA) is an immune‐mediated bone marrow failure associated with high serum levels of flt3 ligand (FL). We examined expression of the membrane‐bound isoform of FL in peripheral blood and bone marrow cells from AA patients at diagnosis (n = 16) and after immunosuppressive (IS) treatment (n = 36). Flow cytometry demonstrated strongly increased FL levels on the cell surface of T lymphocytes in AA relative to normal controls (P < 0·0001). T‐cell‐specific expression of membrane‐bound FL was confirmed by confocal microscopy. FL mRNA and total cellular FL protein levels were increased about threefold. Overexpression of FL in AA was observed for up to 20 years after IS treatment. FL levels correlated inversely with CD34+ cell numbers and the colony‐forming ability of AA bone marrow (R = −0·68 and −0·85 respectively). Histological examination of spleen specimens and bone marrow biopsies gave no evidence of degeneration or fibrosis due to prolonged exposure to high FL. Levels of membrane‐bound FL were not increased in autoimmune diseases (n = 23), including rheumatoid arthritis and lupus erythematosus, nor in graft‐versus‐host disease (n = 8). Chronic overexpression of FL on the surface of T lymphocytes in AA, but not in other T‐cell‐mediated disorders, suggests that membrane‐bound FL plays a role in cell–cell interactions in bone marrow failure and may be important for long‐term haemopoietic recovery.
Fatal cold anti‐i autoimmune haemolytic anaemia complicating hairy cell leukaemia
Authors: Mainwaring C. J. , Walewska R. , Snowden J. , Winfield D.A. , Ng J. P. , Chan–lam D. , Nolan B. , Booker D. J. , Stamps R. , Sokol R. J.
Published: March 2003

Hairy cell leukaemia (HCL) is a rare lymphoproliferative disorder associated with pancytopenia, splenomegaly and the presence of typical hairy B lymphocytes in the bone marrow and/or peripheral blood. The most significant complication relates to opportunistic infections that arise as a consequence of neutropenia and monocytopenia. HCL is occasionally associated with systemic autoimmune disorders including polyarteritis nodosa and rheumatoid disease. Secondary autoimmune haemolytic anaemia (AIHA) appears to be rare. We report on two cases of HCL complicated by fatal cold anti‐i AIHA. Fulminant haemolysis causing death is rare in cold AIHA and only a few individual cases have been reported, none having anti‐i specificity.
In vitro and in vivo studies with human carrier erythrocytes loaded with polyethylene glycol‐conjugated and native adenosine deaminase
Authors: Bax B. E. , Bain M. D. , Fairbanks L. D. , Webster A. D. B. , Chalmers R. A.
Published: March 2003

Polyethylene glycol‐conjugated adenosine deaminase (pegademase) is used for enzyme replacement therapy for patients with severe combined immunodeficiency caused by adenosine deaminase deficiency. The entrapment of pegademase within human energy‐replete carrier erythrocytes using a hypo‐osmotic dialysis procedure was investigated with the objective of prolonging the in vivo circulatory half‐life of the enzyme and maintaining therapeutic blood levels. Native unmodified adenosine deaminase (ADA) was similarly studied. The efficiency of pegademase entrapment was low (9%) whereas the entrapment of native unmodified ADA was substantial (50%), suggesting that the polyethylene glycol side‐chains were impeding intracellular entrapment. The biochemical characteristics and the osmotic fragility of these carrier erythrocytes were not adversely affected by the entrapment of either pegademase or native ADA. In vivo survival studies of pegademase‐loaded 51Cr‐labelled carrier erythrocytes in an ADA‐deficient adult patient showed a mean cell half‐life of 16 d. Carrier erythrocyte‐entrapped pegademase and native ADA had in vivo half‐lives of 20 and 12·5 d, respectively, demonstrating that entrapment prolongs the half‐life over that of plasma pegademase, which has a circulating half‐life of 3–6 d. These results provide the basis for a more extensive clinical evaluation of carrier erythrocyte‐entrapped native adenosine deaminase therapy.
Deletion of the 5′‐ABL region: a recurrent anomaly detected by fluorescence in situ hybridization in about 10% of Philadelphia‐positive chronic myeloid leukaemia patients
Authors: Herens C. , Tassin F. , Lemaire V. , Beguin Y. , Collard E. , Lampertz S. , Croisiau C. , Lecomte M. , De Prijk B. , Longrée L. , Koulischer L.
Published: March 2003

Inclusion of the BCR–ABL ES probe in routine cytogenetics led to the identification of a subgroup of Philadelphia positive (Ph+) chronic myeloid leukaemia patients characterized by a 5′‐ABL deletion. This anomaly was observed in 5/51 cases (9·8%). Cytological and clinical data suggest that the 5′‐ABL deletion may be associated with dysplastic features of polymorphonuclear cells and metamyelocytes and a short chronic phase duration.
Erythropoietin‐induced rheological changes of rat erythrocytes
Authors: Bor‐Kucukatay Melek , Yalcin Ozlem , Meiselman Herbert J. , Baskurt Oguz K.
Published: March 2003

The effects of recombinant human erythropoietin (rhEPO) on red blood cell (RBC) rheological properties were investigated in rats. Rats received intramuscular injections of 150 U/kg/d rhEPO for 5 d, following which blood samples were obtained 1, 5 or 10 d later. RBC deformability was assessed by determining cell transit times through 5‐µm micropores (CTA) and RBC shape recovery time constants via photometry, aggregation in plasma and dextran was measured by photometry and RBC electrophoretic mobility was determined in a cylindrical electrophoresis system. RBC aggregation was found to be significantly decreased on day 5 after rhEPO treatment (P < 0·05), yet was unchanged from control on days 1 and 10. Mean RBC micropore transit times remained unchanged, but the distributions of transit times were altered; compared with control, the 5th percentiles on both days 1 and 5 were decreased and the 95th percentile on day 1 was elevated. Electrophoretic mobility of RBCs in phosphate‐buffered saline was significantly increased on day 5 after rhEPO treatment (P < 0·05), with mobility measurements in dextran 500 (MW = 500 kDa) solutions suggesting that the cells' surface properties related to the formation of a ‘depletion layer’ may be altered on day 1. These results indicate that the rheological behaviour of RBC as a consequence of rhEPO treatment are temporal and are affected by the presence of reticulocytes as well as by the average age of the circulating cells.
Dose–response relationship of megakaryocyte progenitors from the bone marrow of thrombocytopenic and non‐thrombocytopenic neonates to recombinant thrombopoietin
Authors: Sola Martha C. , Du Yan , Hutson Alan D. , Christensen Robert D.
Published: March 2003

Megakaryocyte (MK) progenitors from the marrow of adults undergo dose‐dependent clonogenic proliferation in response to recombinant thrombopoietin (rTpo). It is unknown whether progenitors from the marrow of thrombocytopenic neonates display rTpo dose‐dependent proliferation and whether they are more or less sensitive to rTpo than progenitors from non‐thrombocytopenic neonates or adults. To assess this, we cultured marrow from four thrombocytopenic and four non‐thrombocytopenic neonates, and from six healthy adults, in a serum‐free system in the presence of increasing concentrations of rTpo (0–100 ng/ml). Marrow from the thrombocytopenic and non‐thrombocytopenic neonates generated three times more MK colonies/105 light density cells (129 ± 39 and 167 ± 30 respectively) than marrow from adults (54 ± 30, P < 0·0001) at a rTpo concentration of 50 ng/ml. Neonatal and adult samples had a rTpo dose‐dependent increase in MK colonies. However, neonates reached a maximal number of colonies at a rTpo concentration of 10 ng/ml, compared with 50 ng/ml in adults, resulting in a larger area under the rTpo dose–response curve for neonatal progenitors (P = 0·0047). Neonates also generated more large MK colonies than the adults (24% vs. 2% at 100 ng/ml).
Severe pancytopenia triggered by recombinant hepatitis B vaccine
Authors: Viallard Jean‐François , Boiron Jean‐Michel , Parrens Marie , Moreau Jean‐François , Ranchin Valérie , Reiffers Josy , Leng Bernard , Pellegrin Jean‐Luc
Published: March 2003

We describe the case of a teenager who developed fever, arthritis, cutaneous vasculitis and severe pancytopenia 3 weeks after the third vaccination boost with a recombinant hepatitis B vaccine. Bone marrow examination showed paucity of late myeloid elements and, subsequently, maturation arrest. Interferon‐γ (IFN‐γ) production by peripheral blood mononuclear cells from the patient was dramatically increased. An underlying immune predisposition (HLA‐DR3) may have indirectly enabled the vaccine to trigger a hepatitis B virus‐specific cytotoxic T‐lymphocyte response. It is therefore possible that the pancytopenia was induced by a dysregulation of the CD8+ T‐cell compartment via increased IFN‐γ production.