bluuebunny/crossref_metadata_2025 huggingface dataset:
Pruritus in polycythaemia vera: prevalence, laboratory correlates and management
Authors: Diehn Felix , Tefferi Ayalew
Published: March 2003

In a retrospective cohort, 192 (48%) out of 397 patients with polycythaemia vera had a documented history of pruritus. At diagnosis, the presence of pruritus was significantly associated with a lower mean corpuscular volume and a higher leucocyte count. Among 66 patients with documentation of treatment for pruritus, 389 ‘patient visits’ were reviewed, revealing a significant correlation between active pruritus and low mean corpuscular volume, but not platelet, leucocyte or basophil count. Paroxetine and hydroxyzine were rated by patients to be the most effective drugs in controlling pruritus. These observations suggest a pathogenetic role for both iron deficiency and biogenic amines in polycythaemia vera‐associated pruritus.
Identification of P‐selectin glycoprotein ligand‐1 as a useful marker in acute myeloid leukaemias
Authors: Kappelmayer János , Kiss Attila , Karászi Éva , Veszprémi Anikó , Jakó János , Kiss Csongor
Published: March 2003

Immunophenotyping is considered to be less valuable in the diagnosis of acute myeloid leukaemias (AML) compared with acute lymphoid leukaemias. Here, we present data on the use of quantitative flow cytometry (QFC) of P‐selectin glycoprotein ligand 1 (PSGL‐1, CD162) and three‐colour immunophenotyping including CD162 staining in the identification of myeloid precursors in AML. Analysis of normal peripheral blood (n = 20) and normal bone marrow (n = 5) samples and on 20 samples from de novo M1, M2, M4 and M5 AML patients demonstrated that PSGL‐1 is differentially expressed on various mature and immature leucocyte subsets. It was found by QFC that neutrophils expressed 26500 ± 4500 and monocytes 47200 ± 9900 copies of PSGL‐1 on their surface, whereas AML blasts from M1 and M2 AML patients expressed significantly less PSGL‐1 (12 000 ± 5300) than mature neutrophils (P < 0·001). In M4 and M5 leukaemias, however, the amount of PSGL‐1 on monocytic precursors is displayed in a fairly broad range that is not significantly different from that of mature monocytes (P = 0·084). Using three‐colour immunophenotyping PSGL‐1‐dim staining was co‐expressed with CD7 and C34 positivity and PSGL‐1 staining intensity on immature myeloid cells paralleled with CD45 expression. This would imply a differential expression of PSGL‐1 during myeloid haematopoietic development and suggests that quantification of surface PSGL‐1 may aid in differentiating myeloblasts from monoblasts by immunophenotyping in different AML subsets.
Enhanced T‐lineage acute lymphoblastic leukaemia cell survival on bone marrow stroma requires involvement of LFA‐1 and ICAM‐1
Authors: Winter Stuart S. , Sweatman Jill J. , Lawrence Michael B. , Rhoades Torre H. , Hart Anita L. , Larson Richard S.
Published: March 2003

The bone marrow (BM) microenvironment supports leukaemia cell survival and proliferation. The roles played by adhesive receptor interactions in the survival of T‐lineage acute lymphoblastic leukaemia (T‐ALL) cells on BM stromal cells are not well understood. Recently, we have developed an assay that partially recapitulates the BM microenvironment using HS‐5 BM stromal cells. In this assay, the magnitude of ex vivo T‐ALL lymphoblast survival predicts patient outcome. We examined the molecular basis for cell–cell adhesive events leading to T‐ALL lymphoblast survival on HS‐5 and on donor‐derived BM stroma. Lympho cyte function‐associated antigen‐1 (LFA‐1) on T‐ALL cell lines bound intercellular adhesion molecule‐1 (ICAM‐1) on HS‐5 monolayers, and survival was inhibited 85–98% with monoclonal antibodies directed against LFA‐1 or ICAM‐1. We compared these results with patient‐derived T‐ALL lymphoblasts co‐cultured on either HS‐5 BM or normal BM monolayers and found that LFA‐1 and ICAM‐1 were required, but not alone sufficient for ex vivo leukaemic cell survival. On normal BM stroma, but not HS‐5 monolayers, two additional adhesion molecules, vascular cell adhesion molecule‐1 (VCAM‐1) and E‐selectin, were highly expressed and contributed to T‐ALL cell survival. This is the first report to demonstrate the importance of LFA‐1/ICAM‐1‐mediated adhesion as a critical event in a cascade of cell surface receptor–ligand interactions that regulate T‐ALL survival in the BM microenvironment.
The clinical and biological effects of thalidomide in patients with myelodysplastic syndromes
Authors: Zorat Francesca , Shetty Vilasini , Dutt Diya , Lisak Laurie , Nascimben Fabiana , Allampallam Krishnan , Dar Saleem , York Aaron , Gezer Sefer , Venugopal Parameswaran , Raza Azra
Published: March 2003

Thirty patients with myelodysplastic syndromes (MDS) were treated with thalidomide at 100 mg/d p.o., increased as tolerated to 400 mg/d for 12 weeks. Levels of apoptosis, macrophage number, microvessel density (MVD), tumour necrosis factor alpha (TNF‐α), transforming growth factor beta (TGF‐β), interleukin 6 (IL‐6), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined in the serum, bone marrow (BM) plasma and BM biopsies before and after therapy. Pretherapy biological characteristics of MDS patients were compared with similar studies performed in 11 normal volunteers. Ten patients demonstrated haematological improvement in the erythroid series, six becoming transfusion independent. Responders had a higher pretherapy platelet count (P < 0·048) and lower BM blasts (P < 0·013). Median time to response was 10 weeks, and four remain in remission beyond a year. Pretherapy MDS BMs showed higher MVD (P < 0·001) and TGF‐β (P < 0·03) and higher serum TNF‐α (P < 0·008) compared with normal control subjects. After therapy, only BM TGF‐β decreased significantly (P < 0·002). Pretherapy haemoglobin was directly related to serum VEGF (P < 0·001) in responders and inversely related in non‐responders (P < 0·05), suggesting the possibility that angiogenesis may be a primary pathology in the former and a consequence of anaemia‐induced hypoxia in the latter. We conclude that thalidomide has important clinical and biological effects in at least a subset of MDS patients, but the precise mechanism of its action remains unknown and requires further study including a larger number of patients.
A sequence of immuno‐chemotherapy with Rituximab, mobilization of in vivo purged stem cells, high‐dose chemotherapy and autotransplant is an effective  and non‐toxic treatment for advanced follicular  and mantle cell lymphoma
Authors: Lazzarino M. , Arcaini L. , Bernasconi P. , Alessandrino E. P. , Gargantini L. , Cairoli R. , Orlandi E. , Astori C. , Brusamolino E. , Pagnucco G. , Colombo A. A. , Calatroni S. , Iacona I. , Regazzi M. B. , Morra E.
Published: March 2003

Summary. Options for relapsed/refractory indolent lymphoma include chemotherapy, immunotherapy and high‐dose therapy with autologous support. The best combination of these approaches, however, is not defined. We treated 10 patients with relapsed/refractory follicular (n = 7) or mantle cell lymphoma (n = 3) using chemotherapy, immunotherapy, high‐dose therapy and autotransplant in a sequence of four phases, each designed to play a specific role in tumour eradication. After the debulking with VACOP‐B (doxorubicin, cyclophosphamide, etoposide, vincristine, prednisone, bleomycin) (phase 1), 9/10 patients responded but none achieved a molecular response. After the immuno‐chemotherapy phase, which combined Rituximab with vincristine and cyclophosphamide, seven patients were in complete response (CR) and three in good partial response (PR), and all those with a molecular marker of disease showed a disappearance of the signal from marrow and blood. Phase 3, which coupled high‐dose cytarabine with Rituximab, was effective in mobilizing an adequate number of progenitor cells that were polymerase chain reaction negative in all informative cases. Phase 4 consisted of high‐dose therapy with autologous support followed by two doses of Rituximab. Autograft was performed in nine patients. The haematopoietic recovery was as expected. This sequence of chemotherapy, immuno‐chemotherapy, stem cell mobilization with in vivo purging and autotransplant, organized in four blocks of treatment, was simple to administer and devoid of toxic effects. It permits rapid attainment of clinical and molecular response and enables the harvest of lymphoma‐free peripheral blood progenitor cells even in heavily pretreated patients with relapsed or refractory disease.
Absence of mutations in the HoxA10, HoxA11 and HoxD11 nucleotide coding sequences in thrombocytopenia with absent radius syndrome
Authors: Fleischman Roger A. , Letestu Rémi , Mi Xiafang , Stevens David , Winters Jeffrey , Debili Najet , Vainchenker William
Published: March 2003

Summary. Recent studies have suggested the HoxA10, HoxA11 and HoxD11 homeobox genes as candidate loci for the thrombocytopenia with absent radius (TAR) syndrome. For example, targeted disruptions of these Hox genes result in abnormal development of the mouse radius, while overexpression of HoxA10 stimulates mouse megakaryocyte (MK) development in vitro. To examine the expression of Hox genes in human MK cells, we utilized reverse transcription polymerase chain reaction with degenerate oligonucleotides to study megakaryocytic cell lines (MEG‐01, DAMI), and primary human MK purified from adult and cord blood. Using this approach, 13 out of 40 clones isolated from cell lines, 10 out of 21 from cord MK, and 11 out of 21 from adult MK were identified as HoxA10, while HoxA11 and HoxD11 sequences were not detected. The normal genomic sequences for the human HoxA10, ‐A11, and ‐D11 genes were then determined and sequenced in 10 unrelated individuals with TAR syndrome. In all patients the derived amino acid sequence for the three Hox genes was identical to normal controls. Southern blotting did not reveal genomic rearrangements or deletions at these loci, and in two patients intact HoxA10 transcripts were detected by amplification in myeloid cells. Although these studies cannot completely exclude the possibility that the TAR syndrome results from non‐coding mutations that affect the level of Hox gene expression in megakaryocytes, mutations in the coding sequence of the Hox genes known to affect radial development are not a common cause of TAR syndrome.
Clonally unrelated Hodgkin's disease following autologous stem cell transplant for B‐cell lymphoma
Authors: Fend Falko , Martinez Antonio , Quintanilla‐Martinez Leticia , Sanz Llucia , Combalia Neus , Raffeld Mark , Jaffe Elaine S , Montserrat Emilio , Campo Elias
Published: March 2003

Summary. Lymphoproliferative disorders after autologous stem cell transplantation (SCT) are rare. We describe two cases of Hodgkin's disease (HD) as a late secondary neoplasia following autologous SCT for mantle cell lymphoma and B‐cell chronic lymphocytic leukaemia respectively. Both HD cases were of mixed cellularity type, showed Epstein–Barr virus (EBV) positivity and followed an aggressive course. Clonal analysis of rearranged immunoglobulin genes from the primary B‐cell neoplasm and the secondary HD provided evidence of separate clonal origins of the two tumours in both patients, thus excluding secondary transformation of the original B‐cell clone through EBV as the causative event for development of HD.
Heterozygosity for two novel null alleles of theKELgene causes the Kell‐null phenotype in a Japanese woman
Authors: Koda Yoshiro , Soejima Mikiko , Tsuneoka Makoto , Yasumoto Kiyoshi , Higashitani Takanori , Sagawa Kimitaka , Kimura Hiroshi
Published: March 2003

Summary. The Kell‐null (Ko) phenotype is rare and it does not express the Kell antigens on erythrocyte membranes. Recently, several distinct missense and nonsense base substitutions in the coding region and the donor splice site of intron 3 were identified in theKELgene in individuals with the Ko phenotype. We analysed both genomic DNA and cDNA sequences of theKELgene in a Japanese woman with the Ko phenotype. She was found to be heterozygous for two novel nullKELalleles. One allele contained an A to G substitution in intron 5 that changes the 3′‐splice site of intron 5 from AAG to AGG, resulting in a reading frameshift by a single guanine insertion inKELmRNA, and the other allele contained a single G to A substitution in exon 12 (codon 459) creating a termination codon. Neither mutation was found in 114 randomly selected Japanese individuals. The results suggested that the Ko blood group phenotype might be owing to several distinct non‐functional alleles without any prevalent allele.
Selective in vitro expansion and efficient retroviral transduction of human CD34+ CD38– haematopoietic stem cells
Authors: Ng Yuk Yin , Bloem Andries C. , Van Kessel Berris , Lokhorst Henk , Logtenberg Ton , Staal Frank J. T.
Published: March 2003

Summary.  Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34+CD38– cells from purified CD34+ cells upon stimulation with Flt3‐ligand, stem cell factor and thrombopoietin. Over a 100‐fold (range 80 to 128‐fold) expansion of CD34+CD38– cells was observed with bone marrow and cord blood (CB). The expanded CD34+CD38– cells remained negative for lineage‐specific markers and could be induced to differentiate into granulocytes, monocytes, megakaryocytes, erythrocytes, and T and B‐lymphocytes in vitro. Lineage differentiation assays with single CD34+CD38– cells showed no loss of multilineage potential of expanded cells after ex vivo culture. We also demonstrated that the increase in frequency of CD34+CD38– cells was not as a result of the downregulation of CD38 expression during the culture. Quantitative analysis showed that the number of 6 week cobblestone area forming cells (CAFCwk6), a measure of proliferating HSC, in cytokine‐stimulated CD34+ cells were increased by 20‐fold. Expanded CD34+CD38– cells could be transduced efficiently with retroviruses encoding the low affinity nerve growth factor receptor (LNGFR) marker gene (17% to 44%, mean 27%), resulting in long‐lasting expression of retroviral‐encoded genes in progeny HSC and differentiated progenitors. We conclude that the combination Flt3‐ligand (FL), stem cell factor and thrombopoietin (TPO) induced strong ex vivo proliferation of CD34+CD38– cells and that the absolute number of expanded cells with stem cell activity increased substantially in this population.
Co‐expression of CD30 ligand and interleukin 4 (IL‐4) receptors by acute myeloid leukaemia blasts is associated with the expansion of IL‐4‐producing CD30+ normal T cells
Authors: Rossi Francesca Maria , Degan Massimo , Mazzocco Francesca Tassan , Di Francia Raffaele , Aldinucci Donatella , Poletto Dalisa , Vellenga Edo , Pinto Antonio , Gattei Valter
Published: March 2003

Summary. CD30 ligand (CD30L), but not its cognate receptor CD30, is frequently expressed on acute myeloid leukaemia (AML) blasts. In the present study, we found that leukaemic blasts presenting surface CD30L displayed a characteristic cytokine‐receptor pattern that makes them ideal targets for those cytokines usually produced by Th2‐type cell subsets. In particular, even though a broad distribution of Th2 cytokine receptors by AML blasts was shown, we demonstrated the almost exclusive expression of interleukin 4 (IL‐4) receptor (R), in the absence of its cognate cytokine, by CD30L+ AML. Furthermore, a number of Th2‐associated markers, including CD30, IL‐4 and GATA‐3, were expressed by residual T cells derived from CD30L+ AML but not from CD30L– AML, in which the presence of the Th1‐associated marker LAG‐3 was documented in some cases. The production of IL‐4 in the absence of interferon γ (IFN‐γ) was also detected in CD3+/CD30+ T cells from CD30L+ AML. These results, along with the shift toward IL‐4‐producing specific T‐cell clones observed in CD30L+ AML samples by enzyme‐linked Immunospot (ELISpot) assay, were consistent with the hypothesis of a Th2 polarization taking place in T cells from CD30L+ AML. The notion that IL‐4 was able to enhance in vitro proliferation of CD30L+/IL‐4R+ purified leukaemic blasts suggests that the selective interaction of IL‐4‐producing CD30+ T cells with CD30L+ leukaemic progenitors may have a role in the progression of this particular AML subset.
Assignment of functional roles to parasite proteins in malaria‐infected red blood cells by competitive flow‐based adhesion assay
Authors: Cooke Brian M. , Glenister Fiona K. , Mohandas Narla , Coppel Ross L.
Published: March 2003

Summary.  Adhesion of parasitized red blood cells (PRBCs) to endothelial cells and subsequent accumulation in the microvasculature are pivotal events in the pathogenesis of falciparum malaria. During intraerythrocytic development, numerous proteins exported from the parasite associate with the RBC membrane skeleton but the precise function of many of these proteins remain unknown. Their cellular location, however, suggests that some may play a role in adhesion. The adhesive properties of PRBCs are best studied under flow conditions in vitro; however, experimental variation in levels of cytoadherence in currently available assays make subtle alterations in adhesion difficult to quantify. Here, we describe a flow‐based assay that can quantify small differences in adhesion and document the extent to which a number of parasite proteins influence adhesion using parasite lines that no longer express specific proteins. Loss of parasite proteins ring‐infected erythrocyte surface antigen (RESA), knob‐associated histidine‐rich protein (KAHRP) or Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) had a significant effect on the ability of PRBCs to adhere, whereas loss of mature parasite‐infected erythrocyte surface antigen (MESA) had no effect. Our studies indicate that a number of membrane skeleton‐associated parasite proteins, although not exposed on the RBC surface, can collectively affect the adhesive properties of PRBCs and further our understanding of pathophysiologically relevant structure/function relationships in malaria‐infected RBCs.
Fluoro‐deoxyglucose positron emission tomography imaging for the detection of occult disease in multiple myeloma
Authors: Orchard Kim , Barrington Sally , Buscombe John , Hilson Andrew , Prentice Hugh Grant , Mehta Atul
Published: March 2003

Summary. Positron emission tomography with 2‐deoxy‐2‐[18]fluoro‐d‐glucose (FDG‐PET) imaging has been extensively used to detect occult metastatic malignant lesions in patients with carcinoma. We describe its use in three patients with multiple myeloma, each representing a particular clinical situation in which this imaging modality offered advantages over plain radiography, computerized tomography or magnetic resonance imaging. FDG‐PET provides a whole body image showing sites of occult disease. This is of particular value in patients with non‐secretory myeloma, solitary plasmacytoma or for those that relapse with focal disease following autologous or allogeneic stem cell transplantation.
In vitro and in vivo effectiveness of arsenic trioxide against murine T‐cell prolymphocytic leukaemia
Authors: Récher Christian , Chopin Martine , Raffoux Emmanuel , Pierron Gaëlle , Poupon Joël , Sigaux François , Dombret Hervé , Stern Marc‐Henri
Published: March 2003

Summary. T‐cell prolymphocytic leukaemia (T‐PLL) is a rare form of mature T‐cell leukaemia that is generally resistant to conventional chemotherapy. Mice transgenic for MTCP1 develop leukaemia similar to human T‐PLL, providing a model useful for testing therapeutics. We here evaluated the potential effectiveness of arsenic trioxide (ATO) in murine T‐PLL. In vitro, ATO consistently reduced the viability of murine T‐PLL cells at a clinically achievable concentration (1 µmol/l). The percentage of viable cells after 24 h was 77 ± 4%, 56 ± 6%, 31 ± 7% with 0 µmol/l, 0·5 µmol/l and 1 µmol/l ATO respectively. ATO cytotoxicity was enhanced by ascorbic acid (125 µmol/l). Mice were then treated with ATO (5 µg/g/d intra peritoneally, 5 d per week) or saline for 4 weeks, starting 14 d after tumoral engraftment. The appearance of lymphocytosis and splenomegaly was delayed in the group treated with ATO and survival was significantly prolonged (mean survival in days: 57·6 ± 0·8 for ATO versus 45 ± 0 for saline, P < 10−4). No additional effect was observed in vivo by combining ATO with ascorbic acid (500 µg/g/d, 5 d per week, intra peritoneally). These findings provide support for clinical trials to test therapeutic effects of ATO for human T‐PLL.
Flow cytometry analysis of platelet cyclooxygenase‐2 expression: induction of platelet cyclooxygenase‐2 in patients undergoing coronary artery bypass grafting
Authors: Weber Artur‐Aron , Przytulski Boris , Schumacher Marc , Zimmermann Norbert , Gams Emmeran , Hohlfeld Thomas , Schrör Karsten
Published: March 2003

Summary. There are conflicting reports about the expression of cyclooxygenase (COX)‐2 in human platelets. The present study describes a flow cytometric method for the measurement of platelet COX. Both COX‐1 and COX‐2 were shown to be expressed in platelets from patients undergoing a coronary artery bypass graft. There was a significant increase in COX‐2 expression at day 5 as compared with pre‐surgery values (mean fluorescence 12·31 ± 0·88 versus 9·15 ± 0·88; means ± SEM, n = 7, P < 0·05), whereas COX‐1 levels did not change (13·45 ± 1·11 versus 12·38 ± 1·41; n = 7, P > 0·05).
Hb H hydrops fetalis syndrome associated with the interaction of two common determinants of α thalassaemia (‐‐MED/αTSaudiα)
Authors: Viprakasit Vip , Green Sarah , Height Sue , Ayyub Helena , Higgs Douglas R.
Published: March 2003

Summary.  To date, more than 35 single or oligonucleotide mutations of the α genes that cause α thalassaemia have been described. Their interactions give rise to widely variable clinical manifestations, from a mild hypochromic, microcytic anaemia to a lethal intrauterine anaemia associated with hydrops fetalis. Understanding the molecular genetics enables accurate genotyping, genetic counselling and prenatal testing for the most severe forms of α thalassaemia. Here we show for the first time that the interaction between two relatively common forms of α thalassaemia (‐‐MED/αTSaudiα) may be associated with a clinically severe form of α thalassaemia, Hb H hydrops fetalis.
Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen‐induced platelet aggregation by nitroprusside*
Authors: Jang Elliott K. , Azzam Joseph E. , Dickinson Natalie T. , Davidson Monica M. L. , Haslam Richard J.
Published: March 2003

Summary. In studies on human platelets, nitroprusside (NP) alone at 1–10 µmol/l increased platelet cyclic AMP (cAMP) by 40–70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo‐oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2′,5′‐dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen‐induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP‐dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Iβ isoenzyme. An inhibitor of this enzyme (Rp)‐β‐phenyl‐1,N2‐etheno‐8‐bromoguanosine 3′,5′‐cyclic‐monophosphorothioate, diminished the inhibition of collagen‐induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen‐induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP‐dependent protein kinases had supra‐additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.
In vitro evaluation of the efficacy of idarubicin in human tumour cells from patients with low‐grade non‐Hodgkin's lymphoma
Authors: Åleskog Anna , Jonsson Elin , Larsson Rolf , Nygren Peter , Kristensen Jörgen , Sundström Christer , Höglund Martin
Published: March 2003

Summary. Evaluating the potential benefit of the new anthracycline, idarubicin (Ida), in lymphoma, 58 tumour samples from patients suffering from low‐grade non‐Hodgkin's lymphoma (L‐NHL), were analysed in vitro for their sensitivity to 0·5 µg/ml Ida. This was compared with the sensitivity to other anthracyclines (0·5 µg/ml), using the fluorometric microculture cytotoxicity assay. A total of 132 samples from patients with acute leukaemia and a cell‐line panel representing different resistance mechanisms was included for comparison. The median cell survival of L‐NHL cells did not differ after exposing the cells to Ida or daunorubicin (Dnr), whereas epirubicin, doxorubicin (Dox) and mitoxantrone (Mitox) were significantly less cytotoxic than Ida (P < 0·001). The median cell survival in L‐NHL cells did not differ from that of acute leukaemia cells after exposure to 0·5 µg/ml Ida, Dnr, Dox and Mitox. Cells from previously treated patients with L‐NHL had a higher median survival than cells from untreated patients after exposure to all drugs, except for Ida. In samples from previously untreated patients, Spearman rank correlations were high (Rho = 0·81–0·90) between cell survival after exposure to Ida and the other anthracyclines. The same pattern was observed in the cell‐line panel (Rho = 0·78–0·91) (P < 0·05). In contrast, low correlations (Rho = 0·24–0·42) were observed among samples from previously treated patients. Our results indicate a potential benefit of Ida in previously drug‐treated patients with L‐NHL.
Low concentrations of STI571 in the cerebrospinal fluid: a case report
Authors: Petzer Andreas L. , Gunsilius Eberhard , Hayes Michael , Stockhammer Guenther , Duba Hans CH. , Schneller Folker , Grünewald Kurt , Poewe Werner , Gastl Guenther
Published: March 2003

Summary. We report a 53‐year‐old man with lymphoid blast crisis of Ph+ chronic myeloid leukaemia who was treated with STI571, a selective inhibitor of the enzymatic activity of BCR–ABL. He responded excellently to STI571 (600 mg/d), obtaining a complete cytogenetic remission after 3 months of therapy. Although remission in the bone marrow was sustained, the patient developed an isolated central nervous system relapse. Subsequent analyses of STI571 concentrations in the cerebrospinal fluid (CSF) revealed 2‐log lower CSF levels of STI571 than corresponding plasma levels. These are the first data demonstrating a low penetration of orally administered STI571 into the CSF in humans.
Low‐dose continuous oral melphalan for the treatment of primary systemic (AL) amyloidosis
Authors: Sanchorawala Vaishali , Wright Daniel G. , Seldin David C. , Falk Rodney H. , Berk John L. , Dember Laura M. , Finn Kathleen T. , Skinner Martha
Published: March 2003

Summary. Median survival of patients with AL amyloidosis with clinically significant cardiac involvement is 5 months when treated with cyclic melphalan and prednisone. We investigated a regimen of continuous oral melphalan as a single agent for patients with cardiac amyloidosis who were unable to tolerate prednisone or more aggressive chemotherapy. Thirty patients with amyloid cardiomyopathy were treated with continuous oral melphalan. Seven of 13 patients, evaluable after 3–4 months of treatment, achieved a partial haematological response and three achieved a complete haematological response; six patients have survived for > 1 year. This regimen appeared to be effective in inducing haematological responses in patients who received total doses of melphalan > 300 mg.
Expression of cutaneous lymphocyte antigen is associated with a poor outcome of nasal‐type natural killer‐cell lymphoma
Authors: Yoshino Tadashi , Nakamura Shigeo , Suzumiya Junji , Niitsu Nozomi , Ohshima Koichi , Tsuchiyama Junjiro , Shinagawa Katsuji , Tanimoto Mitsune , Sadahira Yoshito , Harada Mine , Kikuchi Masahiro , Akagi Tadaatsu
Published: March 2003

Summary.Nasal and nasal‐type natural killer (NK) lymphoma is a distinct clinicopathological entity mostly associated with Epstein–Barr virus. Cases that have widespread lesions are resistant to ordinary anti‐cancer therapy and take a highly aggressive course. To date, there are no available data on the relationships between the localization, clinical outcome and expression of adhesion molecules in such cases. We examined the expression of cutaneous lymphocyte antigen (CLA) in 52 cases of NK‐cell lymphoma. CLA was highly expressed in cutaneous cases. Also, the CLA+group (n=29) had a much worse prognosis than the CLA–group (n=23), regardless of the primary site or clinical staging. Univariate analysis identified some significant prognostic factors, and multivariate analysis of these factors showed that the expression of CLA was an independent prognostic indicator. In conclusion, the present findings established that CLA is an independent and important prognostic factor in patients with NK‐cell lymphomas.
The β‐globin C→G mutation at 6 bp 3′ to the termination codon causes β‐thalassaemia by decreasing the mRNA level
Authors: Sgourou Argyro , Papachatzopoulou Adamandia , Psiouri Lambrini , Antoniou Michael , Zoumbos Nicholas , Gibbs Richard , Athanassiadou Aglaia
Published: March 2003

Summary.We have studied the expression of the silent β‐thalassaemiaterm+6 (C→G)mutation, at nucleotide 6 after the stop codon within the human β‐globin 3′ untranslated regions (3′UTR), by stable transfection in murine erythroleukaemia (MEL) cells. Steady state mRNA levels from transfected MEL cells containing theterm+6mutant allele were reduced by 52–60%, compared with those obtained from the normal β‐globin gene, in both total and cytoplasmic RNA fractions, showing that the mutation itself is responsible for the similar data obtained from patients. Upon analysis of nuclear RNA, theterm+6mutation was found to also lower the ratio of cleaved/uncleaved transcripts by 22–30%, thus revealing that it interferes with correct 3′‐end formation of β‐globin mRNA. Theterm+6mutation lies within a polypyrimidine track, similar to that in the β‐intervening sequence II (β‐IVSII), which is known to be an important contributor to the promotion of premRNA 3′‐end formation. We propose that the two polypyrimidine tracks flanking the translated region of exon III of the human β‐globin gene may co‐operate during β‐globin mRNA biogenesis.
Influence of lineage‐specific cytokines on commitment and asymmetric cell division of haematopoietic progenitor cells
Authors: Chen Ling , Zhang Jiachang , Tang Delia C. , Fibach Eitan , Rodgers Griffin P.
Published: March 2003

Summary. We examined the influence of cytokines on erythroid‐ and myeloid‐lineage development of AC133+ cells during primary and secondary cultures. Cells cultured for 14 d in liquid medium containing erythropoietin (EPO) were amplified 831‐fold with 98·2% erythroid cells. A similar culture exposed to granulocyte colony‐stimulating factor (G‐CSF) grew 1350‐fold with 97·4% myeloid cells. To assess whether the cells with EPO inducement could respond at this point to G‐CSF signal, or vice versa, the EPO‐stimulated population was re‐grown with G‐CSF, constituting 95·2% myeloid, of 5075‐fold, cells after 14 d of re‐culture. Conversely, reculture of the G‐CSF‐stimulated population with EPO resulted in a 4083‐fold growth with 81·4% erythroid cells. Semisolid culture containing EPO orG‐CSF showed that some individual colonies had self‐ renewal potential after 14 d culture and could be induced todevelop into a different lineage. Analysis of primitive markers, CD34 and Notch1, or lineage markers, EPO‐R and CD13, by single‐cell reverse transcription polymerase chain reaction showed that individual colonies of 2–16 cells contained at least one CD34‐positive cell with expression ofNotch1 and co‐expression of EPO‐R and CD13 appeared on either CD34‐positive or CD34‐negative cells. In situ hybridization with the same cell surface markers in cell populations confirmed the asymmetric cell division and co‐expression from single cell data. The study provides a useful model for the analysis of multipotential progenitor development, and indicates that progenitor cells co‐express genes from different lineage pathways before commitment and that cytokines influence lineage commitment.
Treatment of life‐threatening post‐haemorrhagic anaemia with cell‐free haemoglobin solution in an adolescent Jehovah's Witness
Authors: Anton Natalie , Hitzler Johann K. , Kavanagh Brian P.
Published: March 2003

Summary. Severe blood loss carries an increased risk of a fatal outcome in patients who do not consent to the use of blood products. For patients who base this decision on a religious belief system, such as the Jehovah's Witnesses, important distinctions may exist between the appropriateness of various blood products. We describe the first successful use of purified human cell‐free haemoglobin solution in a paediatric patient, in this case in the management of life‐threatening anaemia in a 14‐year‐old female Jehovah's Witness with immune thrombocytopenia.
Mobilization of dendritic cells in cancer patients treated with granulocyte colony‐stimulating factor and chemotherapy
Authors: Radcliff Fiona J. , Caruso Denise A. , Koina Craig , Riordan Marcia J. , Roberts Andrew W. , Tang Mimi L. K. , Baum Charles M. , Woulfe Susan L. , Ashley David M.
Published: March 2003

Summary. The number of dendritic cells (DC) circulating in the peripheral blood of cancer patients were monitored at multiple time points during chemotherapy and granulocyte colony‐stimulating factor (G‐CSF) support. DC were identified via the lack of expression of standard lineage markers and high expression of HLA‐DR (LN–/DR+). The expression of DC‐associated markers, including CD83, CD11c, IL‐3Rα (CDw123) and CD86, within this LN–/DR+ population was also monitored. Maximal mobilization occurred during recovery on d 12, with a mean 32‐fold increase in LN–/DR+ numbers. The most striking increase was observed in the LN–/DR+/CD83+ cell population: 12 d after commencement of treatment, the proportion of these cells had increased by approximately 120‐fold when compared with baseline. Peripheral blood mononuclear cell (PBMC) and CD34+ cell numbers also peaked 12 d into the treatment regimen in most patients. These data suggest that it should be possible to acquire substantial numbers of DC from leukapheresis products collected from cancer patients undergoing a standard treatment regimen of chemotherapy and G‐CSF. This strategy may be a feasible, low‐risk means of acquiring cells for DC‐based vaccine studies.
The use of anti‐T‐cell receptor‐Vβ antibodies for the estimation of treatment success and phenotypic characterization of clonal T‐cell populations in cutaneous T‐cell lymphomas
Authors: Schwab Cornelia , Willers Jörg , Niederer Eva , Ludwig Elisabeth , Kündig Thomas , Grob Peter , Burg Günter , Dummer Reinhard
Published: March 2003

Summary. Sézary syndrome and Mycosis fungoides are the most common forms of cutaneous T‐cell lymphomas. To assess the response to different therapies especially in Sézary syndrome, it is helpful to monitor the percentage of circulating tumour cells in the blood. The use of T‐cell receptor (TCR)‐Vβ specific monoclonal antibodies provides a suitable tool for detecting Sézary cells. In this study, we analysed the levels of clonal CD4+Vβ+ cells of seven patients with various treatment modalities using flow cytometry and investigated the immunophenotype of the clonal cells by double staining with a panel of antibodies recognizing lymphatic surface markers. Additionally, a polymerase chain reaction‐denaturing gradient gel electrophoresis assay was performed on clonal CD4+Vβ2+ cells, showing that these cells carry a Vγ10/11, JγP1/2 TCR rearrangement. Follow‐up studies revealed close association of the Vβ+ clone developmentwith the clinical response to different therapiesinsixpatients. Intwo cases, the CD4+Vβ+ cells decreased accompanied by partial regression or even complete remission. In four cases, a stable or increasing clonal CD4+Vβ+ population reflected well a stable or progressing course of the disease. Double staining of Vβ+ cells revealed the following pattern, CD3+, CD5+, CD7+, CD28+, CD80–, CD86+ and human leucocyte antigen (HLA) class I+. In contrast, HLA‐DR was heterogeneously expressed. We conclude that identification and monitoring of CD4+Vβ+ clonal T cells by fluorescence‐activated cell sorting with double staining is a suitable method to assess clinical responses to different therapies.
Human monocytes synthesize hyaluronidase
Authors: Girard Nicole , Maingonnat Catherine , Bertrand Philippe , Tilly Hervé , Vannier Jean‐Pierre , Delpech Bertrand
Published: March 2003

Summary. The involvement of hyaluronic acid (HA) oligosaccharides and blood‐derived mononuclear cells in inflammatory processes prompted us to determine whether peripheral blood mononuclear cells (PBMC) possess hyaluronidase activity. PBMC were incubated with macromolecular‐tritiated HA at pH 3·8 and supernatants were analysed by size exclusion chromatography to reveal digestion of HA. This digestion was due to the CD14‐positive (CD14+), adherent, non‐specific esterase‐positive, subpopulation of PBMC. Hyaluronidase activity (72 kDa) was found in aqueous and non‐ionic detergent PBMC extracts but not in the medium in which the cells had been cultured. These results indicate that hyaluronidase is, at least in part, linked to the membrane rather than excreted. Hence, monocytes have one or more hyaluronidases that can generate a pool of active HA fragments within tissues. Hyaluronidase activity was also found in 3/3 myelomonocytic lineage leukaemias but not in 3/3 lymphoblastic leukaemias.
Minimal residual disease quantification in patients with acute myeloid leukaemia and inv(16)/CBFB‐MYH11 gene fusion
Authors: Van Der Reijden Bert A. , Simons Annet , Luiten Erna , Van Der Poel Sonja C. , Hogenbirk Pauline E. , Tönnissen Evelyn , Valk Peter J. M. , Löwenberg Bob , De Greef Georgine E. , Breuning Martijn H. , Jansen Joop H.
Published: March 2003

Summary. We have designed a real‐time CBFB‐MYH11 reverse transcription polymerase chain reaction (RT‐PCR) assay to quantify minimal residual disease (MRD) in patients with inv(16)‐positive acute myeloid leukaemia (AML). Six patients were followed for a median of 17·5 months after diagnosis during which 120 evaluable samples were analysed. The CBFB‐MYH11 expression at diagnosis varied only fourfold between the six patients and was virtually identical to that observed in the CBFB‐MYH11‐positive cell line ME‐1. For two cases, a patient‐specific real‐time PCR for CBFB‐MYH11 quantification at genomic DNA level was designed. Similar disease levels were found at the RNA and genomic DNA level during and after treatment, indicating that CBFB‐MYH11 gene expression was unaltered during treatment and that the percentage of malignant cells can be accurately quantified at the RNA level. Following successive courses of chemotherapy, the reduction of malignant cells was found to be significantly more pronounced (80–250‐fold greater) in peripheral blood compared with bone marrow in five out of six cases tested. Treatment with gemtuzumab ozogamicin as sole agent at relapse did not result in a selective decrease of tumour cells in three cases analysed. We conclude that real‐time PCR is a powerful method of monitoring MRD levels and quantifying the antileukaemic effect of separate (experimental) courses of chemotherapy.
Persistent aplasia after chemotherapy for acute myeloid leukaemia treated with stem cell transplantation from a matched unrelated donor after dose‐reduced conditioning
Authors: Hambach Lothar , Heil Gerhard , Hertenstein Bernd , Ganser Arnold
Published: March 2003

Summary. Persistent aplasia is a rare complication with poor prognosis after intensive chemotherapy for acute leukaemia. A 59‐year‐old man with acute myeloid leukaemia (AML), was treated after 186 d of chemotherapy‐induced persistent aplasia with an allogeneic peripheral blood stem cell transplantation (PBSCT) from a matched unrelated donor (MUD) after dose‐reduced conditioning. The patient remains in complete haematological remission more than 8·5 months after haematological recovery. We believe that this is the first reported case of treatment for chemotherapy‐induced persistent aplasia with MUD‐PBSCT after dose‐reduced conditioning, a procedure that can be successfully performed even in elderly patients.
Unusual phenotypic observations associated with a rare HbH disease genotype (‐ ‐Med/αTSaudiα): implications for clinical management
Authors: Traeger‐Synodinos Joanne , Papassotiriou Ioannis , Karagiorga Markissia , Premetis Evangelos , Kanavakis Emmanuel , Stamoulakatou Alexandra
Published: March 2003

Summary.  A single patient with a rare Haemoglobin H (HbH) disease genotype (‐ ‐Med/αTSaudiα) was observed to have exceptionally high levels of HbH (> 60%) and paradoxically high total haemoglobin levels. Studies of haematological parameters, blood biochemistry and oxygen transport properties revealed a severe functional anaemia, associated with marked erythropoietic stimulation and a markedly raised cardiac output. This rare case illustrates the complexity of interactions that may be associated with the clinical course of HbH disease, highlighting that haematological parameters alone may lead to spurious evaluation of clinical status. Issues related to the therapeutic management of unusual cases are raised.
Influence of different radioprotective compounds on radiotolerance and cell cycle distribution of human progenitor cells of granulocytopoiesis in vitro
Authors: Klingler Werner , Kreja Ludwika , Nothdurft Wilhelm , Selig Christoph
Published: March 2003

Summary. Ficoll‐separated mononuclear cells (MNC) of cryopreserved human bone marrow were incubated with isotoxic doses of diltiazem, N‐acetylcysteine (NAC), glycopolysaccharide extract of spirulina platensis (SPE), tempol, thiopental, WR2721 and WR1065. After irradiation with a single dose of 0·73 Gy, survival of granulocyte/macrophage colony‐forming cells (GM‐CFC) was determined at d 10–14, using an agar culture system. Diltiazem, NAC, tempol and WR1065 significantly improved radiotolerance with protection factors (PF) between 1·21 and 1·36 (n = 5, P < 0·05) at 0·73 Gy (PF‐0·73 Gy). The survival curves of diltiazem (D0 = 0·88 Gy, n = 1·00), NAC (D0 = 0·92 Gy, n = 1·10), tempol (D0 = 0·99 Gy, n = 1·10), WR1065 (D0 = 0·89 Gy, n = 1·16) and control (D0 = 0·78 Gy, n = 1·00) over 0·36–2·91 Gy showed a significant radioprotective effect for D0 only for tempol (P = 0·018) and for the extrapolation number ‘n’ only in the case of NAC (P = 0·023). Cell cycle analysis of the CD34+ cell subpopulation (control‐0 h: G1 = 82·7%, S = 13·7%, G2/M = 3·6%) revealed that all compounds with a significant PF‐0·73 Gy also caused a significant increase in CD34+ cells in S phase up to 48 h. Within the first 24 h, only NAC (26·7 ± 4·1%), tempol (14·3 ± 1·0%) and possibly WR1065 (15·5 ± 1·6%) had higher fractions of CD34+ S‐phase cells compared with controls. This observation and the improvement of GM‐CFC cloning efficiency indicated that only NAC was able to recruit progenitor cells in the cell cycle, whereas tempol and WR1065 possibly inhibited cell cycle progression by S and G2/M arrest. Of the radioprotectors tested, NAC, tempol and WR1065 may be suitable to support, alone or combined with cytokine therapy, accelerated haematopoietic recovery after irradiation.
Potassium antimonyl tartrate induces caspase‐ and reactive oxygen species‐dependent apoptosis in lymphoid tumoral cells
Authors: Lecureur Valérie , Le Thiec Aurélien , Le Meur Alexandre , Amiot Laurence , Drenou Bernard , Bernard Marc , Lamy Thierry , Fauchet Renée , Fardel Olivier
Published: March 2003

Summary. The metalloid salt potassium antimonyl tartrate (PAT), previously used as an antiparasitic agent, has recently been shown to exert cytotoxicity towards acute promyelocytic leukaemia cells like arsenical compounds. In this study, we have investigated its effects towards human lymphoid malignant cells and compared them with those of arsenic trioxide (As2O3). Like As2O3, PAT was found to inhibit cell growth of various lymphoid cell lines, deriving from either acute lymphoid leukaemias (Jurkat, Molt‐4 and Nalm‐6) or lymphomas (Daudi, Raji and Rec1). PAT toxicity was linked, at least in part, to induction of apoptosis in both Daudi and Jurkat cells, which was dependent on caspase activity. This apoptotic process was also associated, similarly to that triggered by As2O3, with loss of mitochondrial potential and enhanced cellular production of reactive oxygen‐related species. It was enhanced by co‐treatment with the pro‐oxidant buthionine sulphoximine and abolished in response to the antioxidant N‐acetylcysteine, thus underlining that PAT toxicity, similarly to that of As2O3, is probably modulated by the redox status of the cells. PAT, used at concentrations in the micromolar range that are thought to be clinically achievable, was also demonstrated to markedly decrease the viability of primary cultured tumoral B cells that originated from 18 patients suffering from chronic lymphoid leukaemia whereas normal lymphocytes were less sensitive. These data therefore suggest that PAT may deserve to be evaluated in the treatment of some lymphoid malignancies.
Novel flow‐cytometric analysis based on BCD5+ subpopulations for the evaluation of minimal residual disease in chronic lymphocytic leukaemia
Authors: Maloum Karim , Sutton Laurent , Baudet Sylvie , Laurent Caroline , Bonnemye Patrick , Magnac Christian , Merle‐Béral Hélène
Published: March 2003

Summary. We describe a new flow‐cytometric analysis using quadruple labelling with anti‐CD19, CD20, CD5, CD79b monoclonal antibodies and sequential gating. We determined a novel criteria defined by BCD5+CD79b–/low/total BCD5+ cells ratio (BCD5+R), and compared it with the previous definition of phenotypic remission, based on CD19+CD5+ coexpression, and with complementarity‐determining region 3 polymerase chain reaction (CDR3 PCR) and clonotypic PCR (cPCR). A series of 54 peripheral blood samples from 21 chronic lymphocytic leukaemia (CLL) patients in complete haematological remission and a series of 16 from normal volunteers were analysed. In normal controls, the BCD5+R was always < 0·2. The sensitivity of the BCD5+R was 1 × 10−4vs 5 × 10−2 for CDR3 PCR and 1 × 10−5 for cPCR. Among the 54 CLL samples, 35 had a BCD5+R < 0·2 and showed polyclonal CDR3 PCR, whereas the cPCR was positive in 12 out of 20 tested. In the remaining 19 samples, BCD5+R was > 0·2, CDR3 PCR was monoclonal in 16 out of 19 and cPCR positive in 14 out 14 tested, including one out of three samples with polyclonal CDR3 amplification. Even though cPCR remains the most sensitive method to evaluate MRD, this new, sensitive and specific flow cytometric parameter, the BCD5+R, is more suitable than CDR3 PCR for routine clinical MRD assessment in CLL.