bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a factor VIII peptide, residues 2315–2330, which neutralizes human factor VIII C2 inhibitor alloantibodies: requirement of Cys2326 and Glu2327 for maximum effect
Authors: Nogami Keiji , Shima Midori , Nakai Hiroaki , Tanaka Ichiro , Suzuki Hiroshi , Morichika Shogo , Shibata Masaru , Saenko Evgueni , Scandella Dorothea , Giddings John , Yoshioka Akira
Published: December 2004

Factor VIII (FVIII) inhibitor alloantibodies react with combinations of the A2, C2 and A3‐C1 domains of the FVIII molecule. Some inhibitors block binding of FVIII to both von Willebrand factor (VWF) and phospholipid, and recognize a C2 domain epitope which overlaps both binding sites. In order to determine the essential binding regions for alloantibodies inhibitory for FVIII activity, we have performed inhibitor neutralization assays and competitive inhibition assays using 10 overlapping synthetic peptides spanning the carboxy‐terminal region of the C2 domain (residues 2288–2332). We found one peptide (2315–2330, L9) which neutralized the anti‐FVIII activity of four out of five different C2 alloantibodies by 50%, 39%, 47% and 57%, respectively. Neutralization of these alloantibodies by recombinant C2 domain (residues 2173–2332) was 68%, 50%, 59%, 86% and >95%, respectively. The inhibitor which was not neutralized by L9 peptide and reacted by immunoblotting with peptide 2218–2307, did not prevent binding of FVIII to VWF and only partially inhibited binding of FVIII to phosphatidylserine. Mutants of the L9 peptide were prepared in which each residue from 2315–2330 was sequentially substituted by glycine. Inhibitor neutralization experiments using these peptides demonstrated that Arg2320 and Cys2326 or Glu2327 are important for the effect of L9 peptide, since their substitution by glycine reduced its neutralizing effect by 60% to >90%, suggesting that they are crucial for formation of the one of the C2 inhibitor epitopes.
Anti‐CD20 monoclonal antibody (Rituximab) for life‐threatening autoimmune haemolytic anaemia in a patient with systemic lupus erythematosus
Authors: Perrotta Silverio , Locatelli Franco , La Manna Angela , Cennamo Lucia , De Stefano Piero , Nobili Bruno
Published: December 2004

Summary. Innovative approaches are needed for patients with systemic lupus erythematosus (SLE) who develop autoimmune haemolytic anaemia (AIHA) that does not respond to conventional treatment. Rituximab, a chimaeric anti‐CD20 monoclonal antibody, has been demonstrated to be highly effective for in vivo B‐cell depletion. We report an 18‐year‐old‐girl with SLE and life‐threatening AIHA that did not respond to steroids, intravenous immunoglobulin and cyclosporin A. Rituximab was given weekly at 375 mg/m2 for two doses. The drug was well tolerated and the patient had no adverse effects. Her haemolytic disorder markedly ameliorated, with a progressive increase of haemoglobin levels, starting a few days after therapy. The patient remains disease‐free 7 months later.
Cytoprotective antioxidant activity of serum albumin and autocrine catalase in chronic lymphocytic leukaemia
Authors: Moran Elizabeth C. , Kamiguti Aura S. , Cawley John C. , Pettitt Andrew R.
Published: December 2004

Summary. Chronic lymphocytic leukaemia (CLL) cells are long lived in vivo but undergo spontaneous apoptosis when cultured in vitro. Intriguingly, CLL cells also appear to have a specific susceptibility to oxidative stress – a potent inducer of apoptosis. Here, we show that serum albumin can function as a cytoprotective antioxidant of potential relevance to circulating CLL cells, and that autocrine catalase – a hydrogen peroxide‐inactivating enzyme that may be released extracellularly – can perform a similar role under the crowded conditions that prevail at sites of tissue involvement. Albumin lowered oxidative stress in cultured CLL cells and inhibited spontaneous and reactive oxidant‐induced apoptosis. Maximal effects were observed at a concentration of 10 mg/ml – fourfold lower than that in plasma and twofold higher than that in standard culture medium containing 10% fetal calf serum. Oxidative stress and spontaneous apoptosis were also decreased by cell crowding and by conditioned medium (CM) from crowded CLL cells, indicating that these processes were subject to autocrine regulation. CLL cells were found to express catalase and release enzyme activity into the culture medium. Exogenous catalase decreased oxidative stress and spontaneous apoptosis, and the anti‐apoptotic effect of CM from crowded CLL cells was abrogated by the specific catalase inhibitor, 3′‐amino‐1,2,4‐triazole. Together, these data strongly implicate autocrine catalase as a cytoprotective antioxidant. Oxidative stress in CLL cells was greatly diminished by ruthenium red – an inhibitor of mitochondrial reactive oxidant production – and by the glutathione (GSH) precursor N‐acetylcysteine, suggesting that the GSH peroxidase antioxidant system may be compromised by lack of available substrate. Our findings highlight the importance of endogenous reactive oxidants in regulating CLL‐cell apoptosis, and help to explain why CLL cells survive for prolonged periods in vivo despite their vulnerability to oxidative stress and spontaneous apoptosis when cultured in vitro.
Rapid recovery of platelet count following administration of liposome‐encapsulated clodronate in a mouse model of immune thrombocytopenia
Authors: Alves‐Rosa Fernanda , Stanganelli Carmen , Cabrera Juana , Cymberknop Dora , Rubel Carolina , Vanzulli Silvia , Van Rooijen Nico , Palermo Marina , Isturiz Martín A.
Published: December 2004

Summary. Immune thrombocytopenic purpura (ITP) is a haematological disorder characterized by increased platelet consumption. The destruction of platelets is mediated by the reticulo‐endothelial system (RES), particularly by splenic and hepatic macrophages. Previously, we demonstrated in a mouse model of thrombocytopenia that the depletion of these cells by liposome‐encapsulated clodronate (LIP‐CLOD) induces the recovery of the platelet count. We now report that LIP‐CLOD is capable of reversing the thrombocytopenia with minimal effects on both, functional RES integrity and platelet functionality. Our data indicate that thrombocytopenic mice treated with low doses of LIP‐CLOD/body weight increase the platelet count to haemostatically safe values within 18 h of treatment. The predictable bleeding time was significantly decreased in these mice, suggesting that the circulating platelets have enhanced haemostatic capacity. Platelet functionality measured through the ADP‐induced fibrinogen‐binding assay showed normal platelet activation after treatment. Regarding immunological competence, mice treated with LIP‐CLOD showed similar antibody titres against sheep red blood cells. However, antibody‐dependent cell‐mediated cytotoxicity carried out by splenocytes was reduced. All these data demonstrate that LIP‐CLOD deserves consideration as a potential therapeutic approach in thrombocytopenic states in which the rapid increase of platelet count is the primary goal.
Long‐term follow‐up of recipients of allogeneic bone marrow grafts reveals no progressive telomere shortening and provides no evidence for haematopoietic stem cell exhaustion
Authors: De Pauw Elmar S. D. , Otto Sigrid A. , Wijnen Juul T. , Vossen Jaak M. , Van Weel Margreet H. , Tanke Hans J. , Miedema Frank , Willemze Roel , Roelofs Helene , Fibbe Willem E.
Published: December 2004

Summary. Accelerated telomere shortening has been proposed as a possible long‐term risk of allogeneic bone marrow transplantation (allo‐BMT). In this study we monitored telomere length in white blood cells (WBC), granulocytes, and naïve and memory CD4+ T lymphocytes in recipients of allo‐BMT at long‐term follow‐up. Peripheral blood was collected from 10 allo‐BMT recipients and donors at a median interval of 18 years after allo‐BMT. Telomere length was determined using Southern blot analysis. Similar to results previously reported at short‐term follow‐up, a small difference in telomere length (0·1–0·3 kb) between recipients and donors was detected in WBC, granulocytes and naïve CD4+ T cells. Our data therefore provide no evidence for sustained telomere shortening in leucocytes, and render the possibility of long‐term haematopoietic graft failure unlikely. In addition, we observed two phenomena that may be related to involution of the thymus. First, the number of naïve CD4+ T cells in the blood was significantly lower in recipients (0·4 × 109/l) than in donors (0·7 × 109/l) (P < 0·05). Second, telomeres in memory CD4+ T cells from recipients were on average 0·6 kb shorter than those from donors (P = 0·01). The latter may be related to the reported rapid peripheral expansion of memory T cells immediately after transplantation.
Spontaneous clinical regression in chronic lymphocytic leukaemia
Authors: Thomas Ranjit , Ribeiro Isabel , Shepherd Patricia , Johnson Peter , Cook Margaret , Lakhani Anil , Kaczmarski Richard , Carrington Patrick , Catovsky Daniel
Published: December 2004

Summary. Chronic lymphocytic leukaemia (CLL) is a B‐cell disorder, which has a median survival of over 10 years from diagnosis for stage A disease. The natural history of stage A disease is generally indolent or only slowly progressive. It is less well known that CLL may undergo spontaneous regression. We report a series of 10 such cases (eight stage A and two stage B) followed at our institutions.
ETV6 (TEL)‐AML1 pre‐B acute lymphoblastic leukaemia cells are associated with a distinct antigen‐presenting phenotype
Authors: Alessandri Angela J. , Reid Gregor S. D. , Bader Sharon A. , Massing Bonnie G. , Sorensen Poul H. B. , Schultz Kirk R.
Published: December 2004

Summary. The recently recognized translocation t(12;21)(p13;q22), which results in the ETV6‐AML1 fusion product, is the most common genetic rearrangement found in childhood pre‐B acute lymphoblastic leukaemia (ALL). It has been associated with a more favourable prognosis and a distinct immunophenotype in terms of myeloid and B cell‐associated antigen expression. Using flow cytometry, we investigated whether the unique ETV6‐AML1 phenotype extended to molecules associated with antigen presentation by analysing 50 diagnostic bone marrow samples from paediatric pre‐B ALL patients. Reverse transcription polymerase chain reaction for the ETV6‐AML1 fusion transcript was positive in 14 patients. ETV6‐AML1‐positive samples were characterized by a significantly higher expression of the co‐stimulatory molecule CD40 (P < 0·0001), as well as a significantly higher class II HLA‐DR mean channel fluorescence (P = 0·001). In contrast, CD86 expression was significantly lower on fusion‐positive samples (P = 0·010) while there was no difference in expression of CD80 or major histocompatibility complex class I between ETV6‐AML1‐positive and ‐negative samples. This is the first observation in acute leukaemia that the distinct immunophenotype associated with specific translocations includes the expression of molecules associated with antigen presentation. In the case of ETV6‐AML1 pre‐B ALL, this characteristic immunophenotype may have implications for the immunogenicity of the leukaemic cells.
Safety and early efficacy assessment of liposomal daunorubicin (DaunoXome) in adults with refractory or relapsed acute myeloblastic leukaemia: a phase I–II study
Authors: Fassas A. , Buffels R. , Anagnostopoulos A. , Gacos E. , Vadikolia C. , Haloudis P. , Kaloyannidis P.
Published: December 2004

Summary. We have conducted a phase I/II trial to determine the maximum tolerated dose, early safety and efficacy of single‐agent liposomal daunorubicin in relapsed or refractory acute myeloid leukaemia (AML). Successive cohorts of six patients received escalated doses of 75, 100, 125 or 150 mg/m2 of DaunoXome for three consecutive days. Responding patients received a further consolidation cycle of DaunoXome at a dose identical to the one inducing complete or partial remission at the various dose levels. Twenty‐eight patients with a median age of 50·5 years were enrolled. A maximum tolerated dose was determined at 150 mg/m2. Twelve patients received the second cycle. DaunoXome was well tolerated at all administered levels; dose‐limiting toxicities included nausea and vomiting, mucositis and two episodes of cardiotoxicity resulting in the death of two patients. The overall response rate was 46% with a median duration of response of 180 d and a median duration of survival of 208 d. Ten patients demonstrated a complete response following cycle 1, and a further four entered partial response with the first cycle (marrow blasts between 5% and 10%). Of these, three attained complete response with the second cycle (total complete response 13/28). Our results indicate that DaunoXome at a dose of 150 mg/m2 displays acceptable toxicity in a 3‐d regimen followed by a 3‐d consolidation course at 100 mg/m2/d. At this dose schedule, interestingly high remission rates were achieved, justifying further evaluation of DaunoXome for the treatment of relapsed or refractory AML patients.
Prevalence of IgG antibodies to human parvovirus B19 in haemophilia children treated with recombinant factor (F)VIII only or with at least one plasma‐derived FVIII or FIX concentrate: results from the French haemophilia cohort
Authors: Gaboulaud Valérie , Parquet Armelle , Tahiri Cedric , Claeyssens Ségolène , Potard Valérie , Faradji Albert , Peynet Jocelyne , Costagliola Dominique ,   for the Suivi Thérapeutique National des Hémophiles Group
Published: December 2004

Summary. Human parvovirus B19 (B19) has been transmitted by some brands of virally attenuated plasma‐derived factor VIII (FVIII) or IX (FIX) concentrates. To quantify the differences of human parvovirus B19 risk transmission between albumin‐stabilized recombinant factor and plasma‐derived factor, we studied the prevalence of IgG antibodies to B19 (anti‐B19) in 193 haemophiliac children between 1 and 6‐years of age who had previously been treated with albumin‐stabilized recombinant FVIII only (n = 104), and in children previously treated with solvent/detergent high‐purity non‐immunopurified and non‐nanofiltered FVIII or IX concentrates (n = 89). Association between the prevalence of anti‐B19 and the treatment group was analysed using multivariate logistic regression. Age, severity and type of haemophilia, number of cumulative days of exposure to factor VIII or IX, previous history of red blood cells or plasma transfusion were considered as potential confounding variables. A higher prevalence of anti‐B19 was found in children previously treated with solvent/detergent high‐purity non‐immunopurified and non‐nanofiltered FVIII or IX concentrates than in children treated with albumin‐ stabilized recombinant FVIII only (OR: 22·3; CI: 7·9–62·8), independently of the other factors studied.
Soluble urokinase‐type plasminogen activator receptor (suPAR) as an independent factor predicting worse prognosis and extra‐bone marrow involvement in multiple myeloma patients
Authors: Rigolin Gian Matteo , Tieghi Alessia , Ciccone Maria , Bragotti Letizia Zenone , Cavazzini Francesco , Porta Matteo Della , Castagnari Barbara , Carroccia Rosanna , Guerra Giovanni , Cuneo Antonio , Castoldi Gianluigi
Published: December 2004

Summary. The urokinase‐type plasminogen activator (uPA) system, which consists of a proteinase (uPA), a receptor (uPAR or CD87) and inhibitors, is involved in proteolysis, cell migration, tissue remodelling, angiogenesis and cell adhesion. Recent findings suggest that malignant plasma cells express uPA and uPAR. The expression of these factors could represent a process by which myeloma plasma cells interact with the bone marrow (BM) environment and influence important biological events such as bone matrix degradation, plasma cell invasion and homing and, possibly, clinical evolution. We evaluated uPAR (CD87) and its soluble form (suPAR) in 49 multiple myeloma (MM) patients and correlated their expression and levels with clinico‐biological characteristics of the disease. Flow cytometric analysis demonstrated that CD87 was expressed in all MM patients. High CD87 expression was associated with higher intensity of expression of CD56 (P = 0·038), CD38 (P = 0·058) and CD138 (P = 0·054) and CD45bright positivity (P = 0·014). suPAR levels correlated positively with soluble serum CD138 (P = 0·001), creatinine (P = 0·001), beta2‐microglobulin (P < 0·001), disease stage (P = 0·017) and extra‐BM involvement (P = 0·002). In the 46 evaluable patients, multivariate analysis showed that high levels of suPAR (P = 0·0214) and disease stage (P = 0·0064) were predictive of extra‐BM involvement. In multivariate Cox analysis, 13q deletion (P = 0·0278), high soluble serum CD138 (P = 0·0201) and high suPAR (P = 0·0229) were the only parameters that independently affected survival. We conclude that CD87 is expressed on myeloma plasma cells and that suPAR, which predicts extra‐BM involvement and poor prognosis, possibly represents a molecule with a relevant role in the biology of MM.
The early molecular response to imatinib predicts cytogenetic and clinical outcome in chronic myeloid leukaemia
Authors: Wang Lihui , Pearson Kevin , Ferguson Julia E. , Clark Richard E.
Published: December 2004

Summary. Real‐time quantitative reverse transcription‐polymerase chain reaction (Q‐RT‐PCR) is increasingly used to monitor responses in chronic myeloid leukaemia (CML). The peripheral blood BCR‐ABL/ABL ratio, as assessed by Q‐RT‐PCR, has been shown to correlate with the contemporary cytogenetic response in patients receiving imatinib (Glivec, Gleevec). We have used Q‐RT‐PCR to monitor the early molecular response to 4 weeks and 3 months of imatinib therapy, in 47 patients with established CML. After 4 weeks of imatinib therapy, patients whose BCR‐ABL/ABL ratio had fallen to less than 50% that of baseline had a significantly higher probability of achieving a major cytogenetic response after 6 months of therapy, when compared with those whose ratio did not fall by this amount (P < 0·001). Similarly, patients whose ratio at 3 months was less than 10% of that at baseline had a significantly higher probability of achieving a major cytogenetic remission at 6 months (P < 0·001). Patients who achieved these falls in their BCR‐ABL/ABL ratio at either 4 weeks or 3 months had a superior progression‐free survival at a median follow‐up of 16·5 months (P = 0·01 and 0·003 respectively). These effects were independent of patient age and disease stage. The occurrence of peripheral blood cytopenias sufficiently severe to interrupt therapy was unrelated to progression‐free survival. In conclusion, the data suggest that the early trend in the BCR‐ABL/ABL ratio may be clinically useful for the early identification of patients destined to fare poorly on imatinib.
Demonstration of aberrant T‐cell and natural killer‐cell antigen expression in all cases of granular lymphocytic leukaemia
Authors: Morice William G. , Kurtin Paul J. , Leibson Paul J. , Tefferi Ayalew , Hanson Curtis A.
Published: December 2004

Summary. The diagnosis of granular lymphocytic leukaemia (GLL) requires the presence of an immunophenotypically distinct T‐cell (T‐GLL) or natural killer‐cell (NK‐GLL) population. Flow cytometric immunophenotyping was performed on 21 T‐GLL patients, 11 NK‐GLL patients and 20 normal control subjects using antibodies to T and NK cell‐associated antigens in order to accurately identify the distinguishing features of T‐GLL and NK‐GLL. The NK antigens evaluated included: CD16, CD57, CD94, CD161, and the killing inhibitory receptors (KIRs) CD158a, CD158b and CD158e (p70). Abnormal T‐antigen expression was present in all T‐GLL patients. CD57 was frequently expressed in T‐GLL, however, one‐third of patients showed partial CD57 expression similar to that seen in T cells from normal control subjects. Ten T‐GLL were KIR positive; all expressed a single KIR isoform. All NK‐GLL showed a distinctive, abnormal immunophenotype. Four NK‐GLL expressed a single KIR isoform; the remaining seven patients lacked all tested KIRs, which is also a distinct, abnormal finding. Immunoperoxidase staining of bone marrow biopsy specimens from NK‐GLL patients with antibodies to CD8, TIA‐1 and granzyme B revealed the disease‐specific distinctive staining patterns previously found in T‐GLL. These studies delineate the unique immunophenotypic features diagnostic of T‐GLL and provide strong evidence that NK‐GLL, like T‐GLL, represents a clonal lymphoproliferative disorder.
Autologous stem cell transplant for relapsed and refractory peripheral T‐cell lymphoma: variable outcome according to pathological subtype
Authors: Song Kevin W. , Mollee Peter , Keating Armand , Crump Michael
Published: December 2004

Summary. The purpose of this study was to evaluate the outcome of high‐dose chemotherapy (HDCT) followed by autologous haematopoietic stem cell transplant (ASCT) for patients with relapsed T‐cell non‐Hodgkin's lymphoma. We reviewed 36 patients with peripheral T‐cell lymphoma (PTL) who underwent ASCT between January 1987 and June 2001. Patients had chemosensitive disease, and received high‐dose melphalan and etoposide with or without total body irradiation supported by unpurged autologous stem cells. Comparisons were made with 97 diffuse large B‐cell lymphoma (DLBL) patients. PTL patients had a median age of 46 years (19–62 years). Twenty‐nine had relapsed and seven had primary refractory disease. DLBL patients were statistically similar in baseline characteristics. Of patients with PTL, six (17%) died of treatment‐related complications and 14 (39%) were in remission with a median follow‐up of 42 months (range 6–116 months). Three‐year overall survival and event‐free survival (EFS) were 48% and 37%, respectively, for PTL, compared with 53% and 42% for DLBL (P = 0·41 and 0·29 respectively). There was no significant prognostic variable found by univariate analysis for the PTL cohort. Major PTL subtypes were analysed for outcomes. The 20 patients with PTL, not otherwise specified (PTL‐NOS), had an inferior EFS compared with DLBL patients (23%, P = 0·028). In contrast, the nine patients with anaplastic large T/null cell lymphoma had a non‐significant trend for improved EFS (67%, P = 0·41). While ASCT in patients with relapsed or primary refractory PTL results in long‐term remission rates comparable to DLBL patients, those with PTL‐NOS do significantly worse.
High levels of hyaluronan production by a malignant lymphoma cell line with primary effusion lymphoma immunophenotype OHK
Authors: Kuwabara Hiroko , Yoneda Masahiko , Nagai Masami , Nishio Hajime , Tasaka Taizo , Suzuki Koichi , Mori Hiroshi
Published: December 2004

Summary. We investigated whether hyaluronan (HA) was produced by a malignant lymphoma cell line with a primary effusion lymphoma immunophenotype, OHK. OHK secreted high levels of HA into the culture supernatant, and reverse transcription polymerase chain reaction showed mRNA for the HA synthases 2 and 3. HA binding protein‐reactive HA was present in the plasma membrane and in the cytoplasm. The inoculation of OHK into severe combined immunodeficiency disease mice produced marked viscid ascites with concentrated HA and a vascular‐rich mesenteric solid tumour. These results show that HA is produced and secreted by the malignant lymphoma cell line OHK.
Long‐term follow‐up of chronic autoimmune thrombocytopenic purpura refractory to splenectomy: a prospective analysis
Authors: Bourgeois Emmanuelle , Caulier Marie T. , Delarozee Catherine , Brouillard Marc , Bauters Francis , Fenaux Pierre
Published: December 2004

Summary. Splenectomy remains the most effective treatment of chronic autoimmune idiopathic thrombocytopenia (ITP) (i.e. of > 6 months duration). Treatment of patients refractory to splenectomy (with absence of response or relapse after initial response) is difficult, and their long‐term outcome is not well known. Over a 10‐year period, 183 patients with chronic ITP were splenectomized including 158 adults and 25 children (≤ 15 years). Forty‐seven of them, who were refractory to this treatment, were prospectively followed up for 5‐‐15 years (median 7·5 years). Twelve of them, with moderate thrombocytopenia, remained untreated, and 35 were treated by a median of two regimens (range 1‐‐6), to which 27 responded. Thirty‐six (77%) of the refractory cases reached platelet counts durably > 100 × 109/l, nine of them without treatment and 27 of them with low‐dose steroids or azathioprine; six (13%) remained moderately thrombocytopenic (35 × 109/l to 100 × 109/l platelets); the last five patients, without response to any treatment (up to six regimens), remained severely thrombocytopenic (platelets < 20 × 109/l), and three of them died from bleeding. Twenty‐seven (57%) of the 47 refractory cases required at least one hospitalization, in the majority of cases for intravenous immunoglobulin (IVIg) infusions. Seven of the refractory cases occurred in children. Six of them subsequently reached platelet counts > 100 × 109/l, but one died from bleeding. Our findings confirm the overall favourable long‐term prognosis of chronic ITP refractory to splenectomy.
Expression of human leucocyte antigens and co‐stimulatory molecules on blasts of patients with acute myeloid leukaemia
Authors: Vollmer Markus , Li Li , Schmitt Anita , Greiner Jochen , Reinhardt Peter , Ringhoffer Mark , Wiesneth Markus , Döhner Hartmut , Schmitt Michael
Published: December 2004

Summary. Recently, leukaemia‐associated antigens (LAA) recognized by T lymphocytes, such as Wilm's tumour‐1 (WT‐1) or pathogenesis‐related protein‐1 (PR‐1), have been identified. For immunotherapies that employ antigen peptides, either alone or pulsed on dendritic cells (DC), the expression of human leucocyte antigen (HLA) molecules on the targeted leukaemic blasts (LB) is crucial. The co‐stimulatory molecules CD80 and CD86 give the secondary signal to T lymphocytes that is necessary for the lysis of leukaemia cells, and CD40 enhances the efficacy of antigen presentation. Here, the expression of HLA‐ABC, HLA‐A2, HLA‐DR, CD40, CD80 and CD86 was flow cytometrically examined in blood samples from 24 healthy volunteers (HV), 24 patients with newly diagnosed acute myeloid leukaemia (AML) and five patients with relapsed AML. The expression of HLA‐ABC, CD40, CD80 and CD86 was significantly reduced on LB in comparison with monocytes of HV. HLA‐A2 and HLA‐DR expression was similar on LB and on monocytes of HV. In AML patients, the expression of HLA and CD86 molecules was significantly higher on LB than on CD33/CD34‐negative monocytes. CD40 and CD80 molecules were deficient on AML blasts. The preservation of HLA molecules and CD86 on LB of the majority of AML patients at the time of diagnosis and even at relapse of the disease are prerequisites for LAA‐targeted immunotherapies in these patients.
Prognostic significance of CD38 and CD20 expression as assessed by quantitative flow cytometry in chronic lymphocytic leukaemia
Authors: Hsi Eric D. , Kopecky Kenneth J. , Appelbaum Frederick R. , Boldt David , Frey Tom , Loftus Margaret , Hussein Mohamad A.
Published: December 2004

Summary.CD38 expression on chronic lymphocytic leukaemia (CLL) cells is a poor prognostic factor, however, methods for measuring this vary. The QuantiBRITETMflow cytometry (FC) system yields an absolute antigen expression value (antibodies bound/cell, ABC) and may be useful in standardizing CD38 expression analysis. We evaluated cryopreserved pretreatment CLL cells for CD20 ABC, CD38 ABC, and percentage of CD38+B cells from 131 patients requiring therapy. The 92 patients (70%) with ≥ 100 CD38 ABC had worse overall survival (OS; 34% at 5 years) compared with those with < 100 CD38 ABC (70% at 5 years, mortality hazard ratio 2·30, 95% confidence interval 1·28–4·12; two‐tailedP = 0·003). Among the 64 patients with < 30% CD38+cells, OS of the 25 with ≥ 100 ABC was worse than that of the 39 with < 100 ABC (P = 0·018). OS of patients with < 30% CD38+cells and ≥ 100 ABC was actually similar to that of patients with ≥ 30% CD38+cells.BrightCD20 expression (≥ 20 000 ABC) was not associated with a worse OS (P = 0·10). The presence of ≥ 100 CD38 ABC in CLL patients requiring therapy is an unfavourable prognostic factor for OS and quantitative FC may be superior to percentage CD38+cell assessment. Prospective trials are required to determine more precisely the prognostic significance of absolute expression levels in fresh CLL cells.
Over‐expression of short isoforms of Helios in patients with adult T‐cell leukaemia/lymphoma
Authors: Fujii Keiko , Ishimaru Fumihiko , Nakase Koichi , Tabayashi Takayuki , Kozuka Teruhiko , Naoki Kyoko , Miyahara Masayuki , Toki Hironobu , Kitajima Koichi , Harada Mine , Tanimoto Mitsune
Published: December 2004

Summary. In previous studies, we demonstrated an over‐expression of the dominant‐negative isoform of the transcription factor Ikaros in patients with blast crisis of both chronic myelogenous leukaemia and B‐cell acute lymphoblastic leukaemia (ALL). Recently, we reported an over‐expression of the short isoforms of Helios, which is one of the members of the Ikaros gene family, in a patient with T‐cell ALL. In the present study, we found over‐expression of short isoforms of Helios in human T lymphotropic virus‐I (HTLV1)‐infected patients who had developed chronic and acute forms of adult T‐cell leukaemia/lymphoma. In contrast, we could not detect any over‐expression of short isoforms of Helios in healthy HTLV1 carriers. By Southern blotting, we detected a small deletion in the Helios gene locus of adult T‐cell leukaemia/lymphoma patients. The present results suggest that Helios gene abnormalities might be one of the important mechanisms in the disease progression of HTLV1 infection.
A pooled analysis of karyotypic patterns, breakpoints and imbalances in 783 cytogenetically abnormal multiple myelomas reveals frequently involved chromosome segments as well as significant age‐ and sex‐related differences
Authors: Nilsson Thérèse , Höglund Mattias , Lenhoff Stig , Rylander Lars , Turesson Ingemar , Westin Jan , Mitelman Felix , Johansson Bertil
Published: December 2004

Summary. The cytogenetic features (ploidy, complexity, breakpoints, imbalances) were ascertained in 783 abnormal multiple myeloma (MM) cases to identify frequently involved chromosomal regions as well as a possible impact of age/sex. The series included MM patients from the Mitelman Database of Chromosome Aberrations in Cancer and from our own laboratory. Hyperdiploidy was most common, followed by hypodiploidy, pseudodiploidy and tri‐/tetraploidy. Most cases were complex, with a median of eight changes per patient. The distribution of modal numbers differed between younger and older patients, but was not related to sex. No sex‐ or age‐related differences regarding the number of anomalies were found. The most frequent genomic breakpoints were 14q32, 11q13, 1q10, 8q24, 1p11, 1q21, 22q11, 1p13, 1q11, 19q13, 1p22, 6q21 and 17p11. Breaks in 1p13, 6q21 and 11q13 were more common in the younger age group. The most frequent imbalances were + 9, – 13, + 15, + 19, + 11 and – Y. Trisomy 11 and monosomy 16 were more common among men, while –X was more frequent among women. Loss of Y as the sole change and + 5 were more common in elderly patients, and – 14 was more frequent in the younger age group. The present findings strongly suggest that some karyotypic features of MM are influenced by endogenous and/or exogenous factors.
Carboxy‐terminal fragment of osteogenic growth peptide in vitro increases bone marrow cell density in idiopathic myelofibrosis
Authors: Fazzi Rita , Pacini Simone , Testi Rossana , Azzarà Antonio , Galimberti Sara , Testi Cesarina , Trombi Luisa , Metelli Maria Rita , Petrini Mario
Published: December 2004

Summary. Idiopathic myelofibrosis (IMF) is a clonal stem cell disorder characterized by reactive fibrosis of bone marrow sustained by a complex cytokine network. At present, no efficacious therapy for this disease exists. Synthetic carboxy‐terminal pentapeptide of osteogenic growth factor (sOGP10‐14) can increase bone marrow cellularity and the number of haematopoietic colonies; this study evaluated the activity of sOGP10‐14 in IMF. Fragments of bone marrow biopsies from patients affected by IMF were cultured with or without the addition of sOGP10‐14. Cellular density was evaluated by image analysis, and transforming growth factor‐β1 (TGF‐β1) concentration was immunologically assayed in the supernatant of cultured bone marrow biopsies. The proliferation rate of the megakaryoblastic M07‐e cell line, cultured in the presence of either granulocyte–macrophage colony stimulating factor or thrombopoietin (TPO), and with or without sOGP10‐14, was evaluated. Megakaryocyte colony forming unit (CFU‐Mk) assay was performed on bone marrow samples of IMF patients with or without sOGP10‐14. After 14 d, bone marrow cellularity was significantly increased in samples cultured with the pentapeptide. Moreover, sOGP10‐14 induced a significant increase of TGF‐β in culture supernatants. TPO‐primed proliferation of M07‐e was reduced by sOGP10‐14, and the pentapeptide significantly reduced CFU‐Mk on IMF bone‐marrow‐derived cells. sOGP10‐14 increased ex vivo bone marrow cellularity in IMF. This action could be related to the megakaryocyte inhibition induced by the interference of this pentapeptide with growth factor activities. These findings suggest that a deficiency of osteoblast‐related factors may play a role in bone marrow failure in IMF.
Thromboembolic events in patients with myelodysplastic syndrome receiving thalidomide in combination with darbepoietin‐alpha
Authors: Steurer Michael , Sudmeier Irene , Stauder Reinhard , Gastl Günther
Published: December 2004

Summary. A phase II trial was conducted to explore the efficacy and tolerability of combining thalidomide (100 mg/d p.o.) with an erythropoietic growth factor (darbepoietin‐alpha 2·25 µg/kg/d s.c.) in patients with low‐to‐intermediate‐risk myelodysplastic syndromes (MDS). However, the trial had to be discontinued early because of an unexpectedly high incidence of thromboembolic events. Of the first seven patients enrolled, two developed deep‐vein thrombosis and one died of massive pulmonary embolism. We concluded that thalidomide might significantly increase the thromboembolic risk of erythropoietic proteins in MDS patients. Careful clinical surveillance and thrombosis prophylaxis (heparin or oral anticoagulation) should be considered for MDS patients undergoing combined treatment with thalidomide and erythropoietic growth factors.
Expression and functional analysis of granulocyte colony‐stimulating factor receptors on CD34++ cells in patients with myelodysplastic syndrome (MDS) and MDS‐acute myeloid leukaemia
Authors: Sultana Tanvira Afroze , Harada Hironori , Ito Kinro , Tanaka Hideo , Kyo Taiichi , Kimura Akiro
Published: December 2004

Summary. CD34++ cells from 45 patients with myelodysplastic syndrome (MDS) and MDS‐acute myeloid leukaemia (MDS‐AML) were observed by flow cytometry for the expression of granulocyte colony‐stimulating factor receptor (G‐CSFR). Ten patients had a significantly reduced expression of G‐CSFR. Late stages of disease showed a higher proportion of either high or low G‐CSFR expression than earlier stages. In MDS refractory anaemia (RA), G‐CSFR was inversely related to CD33 expression. Most patients (9/10) with low G‐CSFR expression had neutropenia of the peripheral blood. Neutropenia was less common in the normal group, but also occurred in the high expression group. No neutrophil response was observed following G‐CSF administration to MDS‐AML patients (6/6) with low G‐CSFR expression. In the high expression group, patients (3/3) showed a response to G‐CSF while, in the normal group (1/2), the response was minor. In the normal‐ or high‐receptor‐expressing groups, the receptors were functionally active in terms of apoptosis but not proliferation and clonogenic growth, although no clear correlation to receptor expression was observed. The G‐CSFR signal transduction pathway in the normal and high group was not deficient of messenger RNA for either janus kinases (Jaks) or signal transducers and activators of transcription (Stats). These findings suggest that the lowered expression of G‐CSFR may cause neutropenia in MDS and MDS‐AML patients and, therefore, may partially explain the neutropenia in myelodysplastic patients.
Over‐expression of the dominant‐negative isoform of Ikaros confers resistance to dexamethasone‐induced and anti‐IgM‐induced apoptosis
Authors: Sezaki Nobuo , Ishimaru Fumihiko , Takata Minoru , Tabayashi Takayuki , Nakase Koichi , Kozuka Teruhiko , Fujii Keiko , Nakayama Hiroyuki , Teshima Takanori , Harada Mine , Tanimoto Mitsune
Published: December 2004

Summary. In previous studies, we demonstrated an over‐expression of the dominant‐negative isoform of the transcription factor Ikaros, Ik‐6, in patients with B‐cell malignancies, including blast crisis of chronic myelogenous leukaemia and acute lymphoblastic leukaemia. To investigate the consequence of over‐expression of Ik‐6 in B cells, we constructed Ik‐6 transfectants of the FDH‐1 and Ramos cell lines. FDH‐1, which was established from a patient with early pre‐B acute lymphoblastic leukaemia, undergoes apoptosis with dexamethasone treatment, whereas Ramos undergoes apoptosis following anti‐IgM antibody treatment. Compared with the wild type, the over‐expression of Ik‐6 rendered the FDH‐1 and Ramos transfectants resistant to dexamethasone‐induced and anti‐IgM‐induced apoptosis respectively. An immunoblotting study demonstrated bcl‐2 upregulation in anti‐IgM‐induced Ramos Ik‐6 transfectants, but not in FDH‐1 Ik‐6 transfectants. Further investigations of the mechanism of leukaemogenesis associated with the over‐expression of Ik‐6 are warranted.
Cytokine imbalance in hyper‐IgE syndrome: reduced expression of transforming growth factor β and interferon γ genes in circulating activated T cells
Authors: Ohga Shouichi , Nomura Akihiko , Ihara Kenji , Takahata Yasushi , Suga Naohiro , Akeda Hideki , Shibata Rumiko , Okamura Jun , Kinukawa Naoko , Hara Toshiro
Published: December 2004

Summary. Hyper‐IgE syndrome (HIES) is a primary immunodeficiency disease characterized by recurrent infections and marked immunoglobulin (Ig)E elevation. To assess the proper T‐cell defects of HIES, the cytokine profile of naturally activated T cells was compared between HIES, atopic dermatitis and chronic granulomatous disease (CGD). Intracellular flow cytometric analysis after in vitro stimulation showed no difference in the proportion of interferon (IFN)γ‐ or interleukin 4 (IL‐4)‐producing T cells among these diseases. Quantitative polymerase chain reaction (PCR) for the cytokine genes was performed using circulating highly fractionated HLA‐DR+ and HLA‐DR– T cells. The IFNγ/IL‐4 or IFNγ/IL‐10 ratios were lower in HLA‐DR+ T cells of HIES than in CGD (P = 0·0106, 0·0445), but did not differ between HIES and atopy. The transforming growth factor‐β (TGFβ)/IL‐4 ratio in HLA‐DR+ T cells of HIES was lower than that of atopy (0·0106) or CGD (0·0062). The TGFβ/IL‐4 ratio in HLA‐DR– T cells of HIES was also lower than that of atopy (0·0285). Stepwise logistic regression analysis identified TGFβ/IL‐4 ratios in HLA‐DR+ (0·0001) or HLA‐DR– (0·0086) T cells as the most powerful parameters to distinguish HIES from atopy and/or CGD. Serum IgE levels negatively correlated with IFNγ/IL‐4 (0·0108), IFNγ/IL‐10 (0·0254), or TGFβ/IL‐4 (0·0163) ratios in HLA‐DR+, but not HLA‐DR–, T cells. These results suggested that the in vivo activated T cells of HIES did not sufficiently express the IFNγ and TGFβ genes, which could affect IL‐4‐dependent IgE production. The reduced TGFβ expression may involve the indigenous T‐cell defects of HIES.
BH3‐domain mimetic compound BH3I‐2′ induces rapid damage to the inner mitochondrial membrane prior to the cytochrome c release from mitochondria
Authors: Feng Wan‐Yu , Liu Feng‐Ting , Patwari Yasmeen , Agrawal Samir G. , Newland Adrian C. , Jia Li
Published: December 2004

Summary. The Bcl‐2 family proteins are major regulators of cell survival and death in human leukaemia. BH3‐containing peptides induce apoptosis by binding to the hydrophobic pocket of the anti‐apoptotic proteins, such as Bcl‐2 or Bcl‐XL. A small cell‐permeable compound, BH3I‐2′ (3‐iodo‐5‐chloro‐N‐[2‐chloro‐5‐((4‐chlorophenyl)sulphonyl)phenyl]‐2‐hydroxybenzamide), has been recently reported to have a function similar to Bak BH3 peptide. BH3I‐2′ induces apoptosis by disrupting interactions mediated by the BH3 domain, between pro‐apoptotic and anti‐apoptotic members of the Bcl‐2 family. This study found that BH3I‐2′ induced cytochrome c release from the mitochondrial outer membrane in a Bax‐dependent manner and that this correlated with the sensitivity of leukaemic cells to apoptosis. Moreover, it also induced rapid damage to the inner mitochondrial membrane, represented by a rapid collapse of mitochondrial membrane potential (ΔΨm), prior to the cytochrome c release. This occurred both in whole cells and isolated mitochondria, and was not associated with the sensitivity of cells to BH3I‐2′‐induced apoptosis. Exogenous Bcl‐2 or Bcl‐XL neutralized BH3I‐2′in vitro and diminished its effect on the inner mitochondrial membrane. Our results indicate that BH3I‐2′ not only induces cytochrome c release from the outer mitochondrial membrane but also damages the inner mitochondrial membrane, probably by interacting with Bcl‐2 family proteins.
Generation of dendritic cells from CD14+ monocytes positively selected by immunomagnetic adsorption for multiple myeloma patients enrolled in a clinical trial of anti‐idiotype vaccination
Authors: Motta Maria R. , Castellani Samantha , Rizzi Simonetta , Curti Antonio , Gubinelli Francesco , Fogli Miriam , Ferri Elisa , Cellini Claudia , Baccarani Michele , Lemoli Roberto M.
Published: December 2004

Summary. Circulating monocytes from multiple myeloma patients enrolled in a clinical study of anti‐idiotype vaccination were labelled with clinical‐grade anti‐CD14 microbeads and positively selected with the CliniMACS instrument. Cells were then grown, according to good manufacturing practice guidelines, in fetal‐calf‐serum‐free medium in cell culture bags and differentiated to dendritic cells (DC) with granulocyte–macrophage colony stimulating factor plus interleukin 4 (IL‐4), followed by either tumour necrosis factor‐α (TNF‐α) or a cocktail of IL‐1β, IL‐6, TNF‐α and prostaglandin‐E2. The CD14+ cell yield was increased from 17·6 ± 6·5% to 93·8 ± 6·3% (recovery 64·4 ± 15·4%, viability > 97%). After cell culture, phenotypic analysis showed that 86·7 ± 6·8% of the cells were DC: 2·27 ± 0·9 × 108 DC/leukapheresis were obtained, which represented 20·7 ± 4·6% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (28·6 ± 3% of initial CD14+ cells) of DC than TNF‐α alone, with secretion of larger amounts of IL‐12, potent stimulatory activity on allogeneic T cells and efficient presentation of tumour idiotype to autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of preloaded DC. The recovery of thawed, viable DC was 78 ± 10%. Finally, interferon‐α‐2b was at least as efficient as IL‐4 in inducing the differentiation of mature, functional DC from monocytes.
Platelets may inhibit leucotriene biosynthesis by human neutrophils at the integrin level
Authors: Chabannes Bernard , Moliere Patrick , Merhi‐Soussi Faten , Poubelle Patrice E. , Lagarde Michel
Published: December 2004

Summary. Polymorphonuclear leucocytes and blood platelets co‐operate in several pathophysiological processes, and arachidonic acid (AA) metabolites produced in response to the activation of these cells are potent mediators of their functions. We studied the role of platelets in the formation of 5‐lipoxygenase products from AA by autologous neutrophils, especially the chemotactic agent leucotriene (LT) B4. The formation of all products, namely 5‐hydroxy‐eicosatetraenoic acid (5‐HETE), LTB4 and the other LTA4‐derived metabolites, in response to the calcium ionophore A23187 was evaluated by high‐performance liquid chromatography. All the 5‐lipoxygenase products were significantly diminished by physiological concentrations of platelets. This inhibitory effect was lost when platelets were previously degranulated by thrombin in non‐aggregating conditions. Peptides containing the Arg‐Gly‐Asp‐Ser or His‐His‐Leu‐Gly‐Gly‐Ala‐Lys‐Gln‐Ala‐Gly‐Asp‐Val sequence, which prevent the adhesion of platelets to neutrophils via the fibrinogen released from platelet granules and the integrin glycoprotein IIb/IIIa, markedly decreased the inhibitory effect of non‐degranulated platelets. The production of transcellular metabolites of AA such as LTC4, the dual 5‐ and 12‐lipoxygenase product 5,12‐diHETE and lipoxins could not account for the decreased formation of 5‐HETE and LTA4‐derived metabolites. It is concluded that platelets may inhibit the neutrophil 5‐lipoxygenase activity at the integrin level and in turn may play a role in slowing down the production of LTB4 in the course of inflammation.
Quality of life measurement in patients with transfusion‐dependent myelodysplastic syndromes
Authors: Jansen A. J. G. , Essink‐Bot M.‐L. , Beckers E. A. M. , Hop W. C. J. , Schipperus M. R. , Van Rhenen D. J.
Published: December 2004

Summary. The myelodysplastic syndromes (MDS) are clonal disorders characterized by dysplasia in at least two myeloid cell lines. Fatigue is one of the most significant symptoms. MDS patients are treated with blood transfusions to improve their health‐related quality of life (HRQoL). A cross‐sectional pilot study was performed for psychometric evaluation of three internationally established HRQoL measures in MDS patients, and for investigation of the association between the severity of chronic anaemia and HRQoL. Fifty consecutive MDS patients completed the Short Form 36, the Multidimensional Fatigue Inventory and the EuroQoL‐5D Visual Analogue Scale. Hb level was measured during the same visit. Psychometric analysis focused on feasibility, construct validity and reliability. The questionnaires showed a high feasibility, reliability and validity. MDS patients had worse HRQoL scores than the age‐ and sex‐matched general population. We found a positive correlation between haemoglobin (Hb) level and HRQoL. This study provides insights into the suitability of established HRQoL measures for the evaluation of interventions in MDS patients. Hb value and HRQoL are complementary variables for evaluation of the severity of chronic anaemia in patients with MDS.
Impact of graft‐versus‐host disease in reduced‐intensity stem cell transplantation (RIST) for patients with haematological malignancies
Authors: Mineishi Shin , Kanda Yoshinobu , Saito Takeshi , Nakai Kunihisa , Makimoto Atsushi , Kami Masahiro , Tanosaki Ryuji , Wakasugi Hiro , Tobinai Kensei , Takaue Yoichi
Published: December 2004

Summary. To clarify the impact of graft‐versus‐host disease (GVHD) on the outcome of reduced‐intensity stem cell transplantation (RIST), 40 patients who received RIST were compared with those who received conventional stem cell transplantation (CST). RIST regimens consisted of either cladribine (0·11 mg/kg/d × 6, n = 13) or fludarabine (30 mg/m2/d × 6, n = 27) with busulphan (BU, 4 mg/kg/d orally × 2), with or without antithymocyte globulin (ATG). CST regimens were either cyclophosphamide/total body irradiation (CY/TBI, n = 23), BU/CY (n = 19) or others (n = 6). The RIST group contained more patients who were at high risk of transplant‐related mortality, including older patients, while the two groups contained the same percentages of patients at high risk of relapse. There were no differences between these groups in the incidences of acute (grade II–IV, 31·6% RIST vs 33·3% CST, P = 0·6742) and chronic GVHD (56·2%vs 64·1%, P = 0·8512), relapse rate (15·0%vs 18·8%, P = 0·6642), or overall (69·3%vs 65·6%, P = 0·4817) and progression‐free survival (64·7%vs 63·8%, P = 0·6920) at d 500. Multivariate analysis of progression‐free survival identified only grade III–IV acute GVHD and relapse risk dose as adverse risk factors. Although GVHD is a major threat in RIST, appropriate induction of GVHD may be associated with anti‐tumour activity in RIST comparable to that of CST.