bluuebunny/crossref_metadata_2025 huggingface dataset:
A prospective study of protein‐specific assays used to investigate idiopathic thrombocytopenic purpura
Authors: WARNER M. N. , MOORE J. C. , WARKENTIN T. E. , SANTOS A. V. , KELTON J. G.
Published: March 2003

Idiopathic thrombocytopenic purpura (ITP) is a disorder in which platelets, sensitized by autoantibodies, are destroyed by the reticuloendothelial system. The diagnosis of ITP has been a clinical one because assays measuring platelet‐associated IgG (PAIgG) have low specificity. The recently introduced assays that measure antibodies against specific platelet glycoproteins (GP) offer the possibility of improved specificity. In this report we describe two prospective studies. In the first study we compared two protein‐ specific assays (AC and MAIPA) looking for the presence of autoantibodies against GP IIb/IIIa in 81 patient samples. These results were compared with an immunoradiometric assay for PAIgG. The second study investigated the enhanced sensitivity of measuring anti‐GP Ib/IX autoantibodies in 76 patient samples. The protein‐specific assays were able to differentiate immune from non‐immune thrombocytopenia (specificity 91%, sensitivity 39%), whereas the PAIgG assay could not (specificity 19%, sensitivity 78%). The addition of the Ib/IX AC assay maintained a specificity of 92% while increasing the diagnostic sensitivity to 66%. In contrast to the PAIgG assay, there was no correlation between the platelet count and the likelihood or degree of positivity within the control samples using the glycoprotein assays. These studies confirm that glycoprotein assays can be used as diagnostic tests for ITP.
Levels of soluble FasL and FasL gene expression during the development of graft‐versus‐host disease in DLT‐treated patients
Authors: Das Hiranmoy , Imoto Shion , Murayama Tohru , Kajimoto Kazuyoshi , Sugimoto Takeshi , Isobe Takashi , Nakagawa Toshitaro , Nishimura Ryuichiro , Koizumi Tamio
Published: March 2003

Three patients with different clinical symptoms of graft‐versus‐host disease (GVHD) who had received donor lymphocyte transfusion (DLT) for the treatment of relapsed leukaemia after an allogeneic bone marrow transplantation (BMT) from HLA‐matched sibling donors were analysed for the presence of soluble FasL (sFasL) in the sera and for the expression of the Fas ligand (FasL) gene in the peripheral blood mononuclear cells (PBMNC). Two patients who demonstrated liver damage with increased levels of serum bilirubin showed significantly increased levels of serum sFasL. The increase in the sFasL level was observed prior to the increase in the bilirubin during the clinical courses of both patients. The high dose of methyl predonisolone administered to one of these patients greatly reduced the levels of sFasL in the serum. The bilirubin levels were also reduced thereafter. The third patient (without liver damage) did not show any increase in the serum sFasL level.

The expression of the FasL gene in the PBMNC of these three patients was examined. All three patients showed increased levels of the FasL gene expression during their clinical courses. However, only one patient showed a parallel alteration of FasL gene expression with sFasL in the serum. These cases provide evidence that the Fas/FasL system is closely associated with human GVHD, especially in the development of liver GVHD.
Molecular characterization of deletion at 11q22.1‐23.3 in mantle cell lymphoma
Authors: Monni Outi , Zhu Ying , Franssila Kaarle , Oinonen Riikka , Höglund Pia , Elonen Erkki , Joensuu Heikki , Knuutila Sakari
Published: March 2003

Chromosomal deletions at 11q21‐23 have recently been reported to be common aberrations in mantle cell lymphoma (MCL). To characterize the structure of the deletion, we studied 41 cases of MCL by fluorescence in situ hybridization using a YAC contig, which spans the region at 11q22.1‐23.3. 17 MCLs were studied using a set of 20 yeast artificial chromosomes (YACs) in a contig, and nine of these cases showed deletion of 11q22‐23. The deletion spanned several megabases in all but one case, where only YAC 755b11 at 11q23.1, covering approximately a 1.6 Mb of DNA, was deleted. Analysis of additional 24 MCLs with YAC 755b11 revealed the deletion in 49% of all cases (20/41). The deleted region at 11q22.1‐23.3 was discontinuous in five lymphomas and in the majority of the cases the distal breakpoint occurred between YACs 785e12 and 911f2 at 11q23.3. We conclude that the deletion of 11q22‐23 and particularly the deletion of YAC 755b11 are very common in MCL and may be important in the genesis or progression of the disease.
A prospective study of risk‐adapted therapy for large cell non‐Hodgkin's lymphoma with VACOP‐B followed by high‐dose CBV and autologous progenitor cell transplantation for high‐risk patients in remission
Authors: Stahel Rolf A. , Jost Lorenz M. , Kroner Thomas , Dommann‐Scherrer Corina , Maurer Robert , Glanzmann Christoph , Jacky Emanuel , Pichert Gabriella , Pestalozzi Bernhard , Marincek Borut , Sauter Christian , Honegger Hanspeter
Published: March 2003

Several centres reported a favourable outcome after high‐dose chemotherapy with autologous progenitor cell transplantation in selected patients with high‐risk large cell non‐Hodkgin's lymphoma in first remission. Based on these observations, we wanted to prospectively determine the outcome of a risk‐adapted therapy for patients with large cell lymphoma. Patients aged 60 years or less received 12 weeks of VACOP‐B chemotherapy. For high‐risk patients in remission this was immediately followed by high‐dose chemotherapy with cyclophosphamide, carmustine and etoposide and autologous progenitor cell transplantation. High‐risk criteria were defined before the establishment of the International Index and included large cell lymphoma stage III or IV or mediastinal large lymphoma with sclerosis stage II or higher, and the presence of bulky tumours and/or an elevated LDH. 89 patients fulfilled the clinical selection criteria and were entered onto this multicentre study. 82 patients were evaluable after confirmation of large cell histology by pathology review. Of these, 51 were considered to be in the low‐risk group and 31 in the high‐risk group. The 3‐year event‐free survival for all patients was 68%. The 3‐year event‐free survival was 76% for the low‐risk and 55% for the high‐risk group (P = 0.061). Only 22/31 high‐risk patients were able to receive the high‐dose chemotherapy in first remission as intended. In conclusion, although our study demonstrated that a risk‐adapted therapy for large cell lymphoma could be safely administered, the potential impact on outcome of the strategy chosen here is likely to be small.
High serum levels of M‐CSF and G‐CSF in Kawasaki disease
Authors: Igarashi Hiroshi , Hatake Kiyohiko , Tomizuka Hiroshi , Yamada Muneo , Gunji Yuji , Momoi Mariko Y.
Published: March 2003

To examine any role of macrophage colony‐stimulating factor (M‐CSF), in the immune responses in Kawasaki disease (KD), we serially assayed M‐CSF and several related cytokines using ELISA. In 10 paediatric patients with KD the level of M‐CSF was significantly higher in the acute phase than in the convalescent phase (1476.1 ± 443.6 v 805.0 ± 184.7 U/ml). Higher levels of serum granulocyte colony‐stimulating factor (G‐CSF) and interleukin‐6 were also found in the acute phase. These results suggest that M‐CSF, G‐CSF and interleukin‐6, derived from monocytes as monokines or derived from vascular endothelial cells, might play an important role in the acute phase of KD.
Cellular visualization of tissue prokallikrein in human neutrophils and myelocytes
Authors: Naidoo Yugen , Snyman Celia , Raidoo Deshandra M. , Bhoola Kanti D. , Kemme Micheal , Müller‐Esterl Werner
Published: March 2003

The vasoactive peptides bradykinin and kallidin (lysyl‐bradykinin) have been implicated in diapedesis, a cellular process by which neutrophils migrate through endothelial cell gap junctions. The kinin peptides are released from their precursor moiety, kininogen, by the specific action of endoproteinases, the kallikreins. Kininogens have been demonstrated on the surface of neutrophils, and the presence of a competent processing enzyme such as tissue prokallikrein in neutrophils has been postulated, but firm evidence for this is still lacking. We have raised antibodies to a synthetic peptide that is a sequence copy of the activation segment of human TK and demonstrated that the anti‐peptide antibodies specifically recognized the zymogen but not the active form of kallikrein. Using these anti‐peptide antibodies, we showed by Western blotting, immunocytochemistry and electron microscopy that the tissue prokallikrein antigen was localized in neutrophils and their precursor cells, the myelocytes. We further demonstrated by in situ hybridization the presence of tissue kallikrein mRNA in the mature neutrophils and myelocytes. Our findings lend credence to the hypothesis that upon release and activation, neutrophil‐borne TK acts on cell‐associated kininogens to trigger the release of kinins, which may open endothelial gates for neutrophil diapedesis.
CD99 (MIC2) expression in paediatric B‐lineage leukaemia/lymphoma reflects maturation‐associated patterns of normal B‐lymphopoiesis
Authors: Dworzak Michael N. , Fritsch Gerhard , Fleischer Christine , Printz Dieter , Fröschl Gertraud , Buchinger Petra , Mann Georg , Gadner Helmut
Published: March 2003

We have recently shown that CD99 (MIC2) is differentially expressed during normal early B‐cell development in the bone marrow (BM). Since immature B‐cell precursors (BCP) are assumed to correspond to some extent to acute lymphoblastic leukaemia (ALL) and non‐Hodgkin's lymphoma (NHL) cells with respect to patterns of phenotypic differentiation, we wondered whether the particular maturation‐associated expression patterns of CD99 in the normal BCP stages were conserved also in malignant cells. Therefore we compared malignant and physiological B cells from paediatric ALL/NHL and normal BM samples with respect to CD99 expression using selective gating and semi‐quantitative flow cytometry. Common‐ALLs (n = 45) were similar to their corresponding, very immature BCPs (stage 1) in expressing very high levels of CD99. Most pre‐B ALLs (n = 16) were also CD99hi and thus differed from the patterns found in normal cytoplasmic μ‐chain+ (cμ+) pre‐B cells (stage 2, CD99lo). In particular, we found that those pre‐B‐ALL cases which were CD34+ also showed higher CD99 expression than the CD34− cases. This prompted us to investigate the levels of CD99 in those rare normal BCPs which also coexpress CD34 and cμ; these cells, which are transitory from stage 1 to stage 2, were found also CD99hi, thus precisely reflecting the patterns of CD34+ pre‐B ALLs. The blasts of Burkitt‐type B‐cell ALL/NHL samples (n = 13) expressed considerably less CD99, similarly to the more differentiated BCP stages 2 (cμ+) and 3 (surface μ‐chain+). In summary, we found that paediatric B‐lineage malignancies display remarkable synchrony regarding the levels of CD99 expression compared to their putative normal counterparts.
Endogenous thrombopoietin serum levels during multicycle chemotherapy
Authors: Engel Christoph , Loeffler Markus , Franke Horst , Schmitz Stephan
Published: March 2003

Little is known about the behaviour of endogenous thrombopoietin (TPO) serum levels during rapid sequences of dose‐intensified chemotherapy. To characterize the relationship between TPO levels and platelet counts in this setting we serially measured both parameters over the entire treatment period of patients receiving multicycle polychemotherapy. We found TPO and platelet responses to be generally antagonistic through all cycles. However, a cross‐correlation analysis indicated that TPO responses preceded platelet responses by approximately one day in all patients. The cumulative severity of thrombocytopenia observed over successive cycles was accompanied by an increasing TPO response which tended to grow overproportionally in relation to the degree of peripheral thrombocytopenia. These findings are consistent with a model suggesting that both platelet and megakaryocyte mass contribute to a receptor‐dependent consumption process regulating the endogenous TPO level. In order to develop optimal schedules for exogenous TPO administration it might be important to consider endogenous TPO response characteristics.
A novel ELISA‐based primer extension assay for the detection of the factor V Leiden mutation
Authors: Wilde J. T. , O'Sullivan J. J. , Roper J. L. , Aerts P. , Horowitz M. , Navot N.
Published: March 2003

We describe an enzyme‐linked immunosorbent assay (ELISA) based primer extension method for the detection of the factor V Leiden (FVL) mutation. The wild‐type nucleotide at position 1691 or the mutant nucleotide at the complementary position on the antisense strand were detected by the incorporation of biotinylated complementary bases onto fluorescein isothiocyanate (FITC) labelled mini‐sequence primers with specificity for the sense and antisense gene segments downstream from the bases adjacent to position 1691. The reactions took place in pairs of tubes containing the complementary bases to either the wild‐type or mutant nucleotide respectively. Primer extension products from each reaction tube pair which have incorporated biotinylated bases were then captured in streptavidin‐coated microtitre plate wells and detected colourimetrically using an ELISA procedure. 200 patient samples were tested to validate the assay and there was complete genotypic agreement between the ELISA method and restriction site analysis using Mnl I (137 wild type, 55 FVL heterozygotes and eight homozygotes). The method utilizes non‐radioactive reagents and does not require electrophoretic techniques. It is therefore a safe, simple and rapid assay which lends itself to automation.
Isolated isochromosome 17q: a distinct type of mixed myeloproliferative disorder/myelodysplastic syndrome with an aggressive clinical course
Authors: McClure R. F. , Dewald G. W. , Hoyer J. D. , Hanson C. A.
Published: March 2003

A clinicopathologic study was performed on 15 patients with haematological malignancies in which isochromosome 17q [i(17q)] was the sole structural chromosome abnormality identified in bone marrow. The data indicated that an isolated i(17q) is associated with a distinct type of mixed chronic myeloproliferative/myelodysplastic disorder with an aggressive clinical course. The patients ranged in age from 37 to 83 years (median 60) with a M:F ratio of 3:1. All cases were chronic myeloid disorders with mixed proliferative and dysplastic features, making classification difficult. 11 patients tested for BCR/ABL gene fusion were normal. A low bone marrow blast count (<5%) at presentation was a typical finding. All cases had severe myeloid dysplasia which included non‐segmented neutrophils and an increase in the monocyte/macrophage lineage. Fluorescence in situ hybridization (FISH) analysis of one case showed the i(17q) to involve all myeloid lineages, but not the lymphocytes. For cases with complete follow‐up (n = 11) the median survival was 2.5 years (range 0.83–5.25) and 64% progressed to AML prior to death. The following features were identified which defined the haematological disorder associated with an isolated i(17q): (1) adult patient, (2) chronic myeloid disorder with clonal involvement of all myeloid lineages, (3) mixed chronic myeloproliferative/myelodysplastic features, (4) severe hyposegmentation of neutrophil nuclei, (5) prominence of the monocyte/macrophage lineage, (6) high risk for progression to AML, and (7) median survival of 2.5 years.
A population‐based study of childhood myelodysplastic syndrome in British Columbia, Canada
Authors: Hasle Henrik , Wadsworth Louis D. , Massing Bonnie G. , McBride Mary , Schultz Kirk R.
Published: March 2003

Myelodysplastic syndrome (MDS) is considered to be very rare in children. However, the only two published population‐based studies reported widely divergent incidence figures. To further explore the epidemiology of childhood MDS and to evaluate the accuracy of cancer registry and treatment trial data, we conducted a population‐based study of children aged 0–14 years in British Columbia (BC), Canada, between 1982 and 1996. MDS was diagnosed in 31 cases corresponding to an annual incidence of 3.2 per million children or 6% of all leukaemias, compared with an incidence of 6.0/million for acute myeloid leukaemia (AML), and of 0.5/million for chronic myeloid leukaemia. There was a non‐significant (P = 0.19) trend toward an increase in MDS incidence with time, the increase was partly explained by an increasing number of patients with Down syndrome. Associated abnormalities were found in 48% of the MDS cases with Down syndrome as the most common (seven cases). Only one third of the MDS cases were correctly registered in the Cancer Registry and less than half of the eligible MDS patients were enrolled on a cooperative group study. Data on MDS from treatment‐based studies and cancer registries were inaccurate and seemed to significantly underestimate the incidence of MDS in children.
Real‐time RT‐PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21)‐positive AML patients
Authors: Krauter Jürgen , Wattjes Mike Peter , Nagel Stefan , Heidenreich Olaf , Krug Utz , Kafert Sabine , Bunjes Donald , Bergmann Lothar , Ganser Arnold , Heil Gerhard
Published: March 2003

AML1/MTG8 was quantified relative to the expression of the GAPDH housekeeping gene by real‐time RT‐PCR in 22 patients with t(8;21)‐positive acute myeloblastic leukaemia (AML) at initial diagnosis and in seven of these patients also during/after chemotherapy and allogeneic bone marrow transplantation. Real‐time PCR was able to specifically detect and quantify AML1/MTG8 over a 5 log range. The detection limit for t(8;21)‐positive cells was a dilution of 1:105. The AML1/MTG8 expression varied considerably among the 22 AML patients at intial diagnosis with a ratio AML1/MTG8:GAPDH of 0.5135±0.536 (range 0.1–2.14, median 0.318). In six patients with t(8;21)‐positive AML a marked decline of AML1/MTG8 could be induced by chemotherapy. These patients are in ongoing complete haematological remission (CR) with a constant low‐level AML1/MTG8 expression. In another patient a rapid rise of AML1/MTG8 transcripts could be detected in CR after allogeneic bone marrow transplantation and the patient relapsed 10 weeks later. In conclusion, real‐time RT‐PCR is a suitable approach for the quantification of AML1/MTG8 transcripts in the monitoring of AML patients with t(8;21) during/after chemotherapy and can provide data of prognostic relevance.
A simple, robust, validated and highly predictive index for the determination of risk‐directed therapy in acute myeloid leukaemia derived from the MRC AML 10 trial
Authors: Wheatley Keith , Burnett Alan K. , Goldstone Anthony H. , Gray Richard G. , Hann Ian M. , Harrison Christine J. , Rees John K. H. , Stevens Richard F. , Walker Helen
Published: March 2003

Data on 1711 patients, aged up to 55 years, in the MRC AML 10 trial were used to create a prognostic index for use in risk‐directed therapy decision making for younger patients with acute myeloid leukaemia (AML). Two parameters, response after course 1 and cytogenetics, were strongly predictive of outcome. For patients with complete remission, partial remission and resistant disease, 5‐year survival from the start of course 2 was 53%, 44% and 22% and relapse rates were 46%, 48% and 69% respectively, and for patients with favourable, intermediate and adverse karyotypic abnormalities, survival was 72%, 43% and 17% and relapse rates were 34%, 51% and 75% respectively (all P < 0.0001). Patients with FAB type M3 but no cytogenetic t(15;17) also had a low relapse rate (29%). These three factors were combined to give three risk groups: good (favourable karyotype or M3, irrespective of response status or presence of additional abnormalities), standard (neither good nor poor), poor (adverse karyotype or resistant disease, and no good‐risk features). Survival for these three groups was 70%, 48% and 15% respectively and relapse rates were 33%. 50% and 78% (both P < 0.0001). The index is simple (based on just three parameters), robust (derived from 1711 patients), highly discriminatory (55% survival difference between good and poor risk) and validated, so can be applied in the clinical setting to assist with therapeutic decisions as in the current AML 12 trial.
Surface CD14 positivity in B‐cell chronic lymphocytic leukaemia is related to clinical outcome
Authors: Callea Vincenzo , Morabito Fortunato , Oliva Bianca Maria , Stelitano Caterina , Levato Domenico , Dattilo Angela , Gangemi Francesca , Iorfida Antonella , Iacopino Pasquale , Nobile Francesco , Molica Stefano , Brugiatelli Maura
Published: March 2003

The aberrant expression of the myelomonocytic antigen CD14 was investigated in 128 untreated patients diagnosed with B‐cell chronic lymphocytic leukaemia (B‐CLL). A cut‐off value of 5 × 109/l CD14‐positive cells was chosen for statistical analysis because it showed the best discriminating power among patients with different clinical features. 56 cases had a CD14+ cell count >5 × 109/l. A significant correlation was found between Rai and Binet stages and total tumour mass (TTM) score on one hand, and the absolute CD14+ cell cut‐off, on the other. This relationship was more evident in Rai 0–II and Binet A–B stages, where a CD14+ cell count >5 × 109/l was preferentially distributed among patients with a higher tumoral mass. In univariate analysis the survival probability at 5 and 10 years showed a significant correlation with Rai and Binet stages, TTM score, CD14+ absolute cell count and median age. The median overall survival (OS) was 63 months for patients with a CD14+ cell count >5 × 109/l and 136 months for those with a CD14+ cell count < 5 × 109/l. In the multivariate Cox regression model, Rai stage, age and CD14+ cell count were independent significant factors for the prediction of OS. Finally, when the same analysis was restricted to Rai stages 0–II, CD14+ cell count was the only significant independent parameter influencing OS, with a relative death risk of 3.8. In conclusion, these data reveal that CD14+ represents an important marker for predicting OS in B‐CLL patients and, therefore, we suggest that it should be included in the immunological characterization of B‐CLL.
Phosphatidylserine‐related adhesion of human erythrocytes to vascular endothelium
Authors: Closse Christèle , Dachary‐Prigent Jeanne , Boisseau Michel R.
Published: March 2003

In pathological conditions such as sickle cell disease, falciparum malaria and diabetes, an abnormal adherence of erythrocytes to endothelium is concomitant with loss of phospholipid asymmetry resulting in phosphatidylserine (PS) exposure. We have investigated the involvement of PS in this interaction by studying adhesion of human erythrocytes, treated with Ca2+‐ionophore A23187 in combination with N‐ethylmaleimide, to human umbilical vein endothelial cells in a flow‐based assay. Results showed that erythrocytes which exposed PS, massively adhered to HUVEC in a Ca2+‐dependent manner. This adhesion was inhibited by PS liposomes and by annexin V, giving clear evidence of the PS dependence of these interactions.
NG2 expression in MLL rearranged acute myeloid leukaemia is restricted to monoblastic cases
Authors: Mauvieux  , Delabesse  , Bourquelot  , Radford‐Weiss  , Bennaceur  , Flandrin  , Valensi  , Macintyre
Published: March 2003

Expression of NG2 has been reported in the majority of paediatric acute leukaemia (AL) cases with MLL rearrangement. We demonstrated 7.1 positivity in 2/3 paediatric and 4/11 adult MLL rearranged acute myeloid leukaemia (AML) but in 0/28 adult AML without MLL rearrangement, thus extending the 100% specificity to adult cases. Positivity correlated with stage of maturation arrest since it was found in 0/6 immature AML but in 6/8 monoblastic cases. These data demonstrate that, if NG2 expression in AL is the (in)direct result of MLL rearrangement, such activation is restricted to a monoblastic population in AML. They also have practical implications for NG2 diagnostic screening strategies.
Specific analysis of the intron 22 XbaI polymorphism of the human factor VIII gene using long‐distance PCR
Authors: De Brasi Carlos D. , Bowen Derrick J. , Collins Peter W. , Larripa Irene B.
Published: March 2003

A rapid, non‐radioactive, PCR‐based method to genotype the XbaI restriction fragment length polymorphism of the human factor VIII gene is described. The method uses long‐distance PCR followed by XbaI restriction digestion and agarose gel electrophoresis. The 6.6 kb amplification product includes a constant XbaI site, which provides a digestion control. The specificity of the method was challenged by a blind experiment with 16 genomic DNA samples previously genotyped by Southern blot analysis: a perfect correlation was obtained between genotypes determined using Southern blot and PCR.
The genetic variability of the VH genes in follicular lymphoma: the impact of the hypermutation mechanism
Authors: Noppe Sofie M. , Heirman Carlo , Bakkus Marleen H. C. , Brissinck Jan , Schots Rik , Thielemans Kris
Published: March 2003

Follicular lymphoma (FL) cells have inherited an activated hypermutation mechanism from their origin of germinal centre B cells. Based on today's knowledge of the intrinsic properties related to this mechanism and the VH base composition, reconsideration of previous reports should be made on a broader range of samples. The present study examined the mutation pattern of the VH genes expressed by 55 cases of FL. FL VH genes showed evidence of antigenic selection in 30% of cases with 88% carrying a functional sIg and 78.2% showing intraclonal variation. VH family and gene segment utilization was found to be roughly similar to that of normal B lymphocytes. FL VH genes revealed extensive variations. 17% of the VH exons harboured a total of five deletions, three duplications and two insertions as compared to the most homologous germline counterpart. The VH genes of one tumour displayed three populations with varying CDR3 length at diagnosis. At relapse, emergence of a differently mutated gene, additional mutations reminiscent of ongoing mutations or no variation was prominent. From this study the heterogeneity of FLs is well established and ongoing mutations are seen in the scope of the activated status of the hypermutation mechanism rather than antigen‐stimulated tumour growth.
A chronic hypercoagulable state in patients with β‐thalassaemia major is already present in childhood
Authors: Eldor A. , Durst R. , Hy‐Am E. , Goldfarb A. , Gillis S. , Rachmilewitz E. A. , Abramov A. , Maclouf J. , Godefray Y. C. , De Raucourt E. , Guillin M. C.
Published: March 2003

A higher than normal incidence of thromboembolic events has been observed in adult patients with β‐thalassaemia major (TM) and certain haemostatic anomalies found in these patients suggest the existence of a chronic hypercoagulable state. Thalassaemic red blood cells (RBC) were demonstrated to facilitate thrombin formation due to altered asymmetry of the membrane phospholipids with enhanced exposure of phosphatidylserine. Since RBC anomalies exist in thalassaemia from the first months of life, we studied markers of hypercoagulability and thrombophilia in 36 adult patients (range 19–38 years) and 26 children (range 2–18 years) with β‐TM. All the patients were in steady state and none had experienced clinical signs or symptoms of thrombosis. Highly elevated urinary levels of 11‐dehydro‐thromboxane B2 and significantly elevated plasma levels of thrombin–antithrombin III (TAT) complexes were observed to the same extent in TM children and adults. The levels of factor II were decreased while factors V, VII + X and plasminogen were within the normal range. The natural coagulation inhibitors, protein C (PC) and protein S (PS) were significantly decreased in all TM patients investigated, regardless of age, but the PS binding protein (C4bBP) and antithrombin III levels were normal. The frequency of other thrombophilic mutations was not increased. Thus, a chronic hypercoagulable state already exists in thalassaemia in childhood and may contribute to the cardiac and pulmonary anomalies and the thrombotic events which occur later.
Immune reconstitution after transplantation of autologous peripheral CD34+ cells: analysis of predictive factors and comparison with unselected progenitor transplants
Authors: Rutella Sergio , Rumi Carlo , Laurenti Luca , Pierelli Luca , Sora' Federica , Sica Simona , Leone Giuseppe
Published: March 2003

The recovery of lymphocyte count, CD4+ and CD8+ T‐cell subsets, natural killer (NK) cells and CD19+ B‐cells was evaluated in a cohort of 15 patients receiving autologous CD34+ peripheral blood progenitor cells (PBPCs; group A) for haematological malignancies and in 20 patients transplanted with autologous unselected PBPCs (group B). Lymphocyte count recovered in both patient cohorts, being significantly lower in group A than in group B 1 (P = 0·008) and 2 months (P = 0·0035) after progenitor cell infusion. The repopulation of CD3+ T‐cells occurred more rapidly in group B than in group A (P = 0·034 on week 4); CD19+ B‐lymphocytes did not return to reference ranges in either group of patients. The count of CD4+ T‐lymphocytes remained < 200/μl during the study period in patients transplanted with CD34+ PBPCs, significantly lower than group B levels (P = 0·034 and P = 0·021 on weeks 4 and 8 respectively). CD8+ T‐cells increased rapidly in both groups; thus, the CD4 to CD8 ratio was severely reduced. CD4+ and CD8+ T‐cells displayed an activated phenotype in both groups of patients, co‐expressing the HLA‐DR antigen throughout the study period. NK cells followed a similar repopulation kinetics in both study groups, although their expansion was greater in group B than in group A (P = 0·014 on week 4). In the CD34+ group, post‐transplant administration of granulocyte colony‐stimulating factor predicted a faster lymphocyte recovery in multivariate analysis (P = 0·025); interestingly, the amount of passively transferred lymphocytes correlated inversely with time to achieve a lymphocyte count > 0·5 × 109/l (r = –0·63, P = 0·01). Further investigations are necessary to characterize T‐cell competence after transplantation of CD34+ PBPCs.
Therapy with thalidomide in refractory multiple myeloma patients — the revival of an old drug
Authors: Kneller  , Raanani  , Hardan  , Avigdor  , Levi  , Berkowicz  , Ben‐Bassat
Published: March 2003

We have treated 17 refractory or relapsed multiple myeloma patients resistant to chemotherapy with thalidomide at a dose of 200–800 mg/day. Eleven patients responded, five of whom had a very good partial response (> 75% decline in M protein) and another five exhibited a partial response (> 50% decline in M protein). Except for one patient, treatment was well tolerated with only mild side‐effects. Thalidomide should be included in the therapeutic options for refractory myeloma.
New genotypes in Fy(a− b−) individuals: nonsense mutations (Trp to stop) in the coding sequence of either FY A or FY B
Authors: Rios  , Chaudhuri  , Mallinson  , Sausais  , Gomensoro‐Garcia  , Hannon  , Rosenberger  , Poole  , Burgess  , Pogo  , Reid
Published: March 2003

Duffy blood group antigens are carried on a glycoprotein that is predicted to pass through the erythrocyte membrane seven times and is a promiscuous chemokine receptor. The Fy(a− b−) phenotype is present in two‐thirds of African‐American Blacks but is rare in Caucasians. In Blacks, the phenotype is due to a non‐functional GATA‐1 motif in the FY B, which silences the gene in erythrocytes but not in other tissues, and these patients do not generally make anti‐Fyb or anti‐Fy3. We describe here the molecular analysis of FY in three unrelated Caucasians who were studied because they had strong anti‐Fy3 in their serum. Each was found to have a point mutation that was predicted to change a tryptophan to a premature stop codon in the coding sequence. In one patient (patient 1), the nonsense mutation was at nucleotide 287 of the major transcript in FY A; in another (patient 2), it was at nucleotide 407 in the major transcript of FY B; and in a third (patient 3), it was at nucleotide 408 of the major transcript of FY A.
Flow cytometric assessment of CD15+CD117+ cells for the detection of minimal residual disease in adult acute myeloid leukaemia
Authors: Nakamura Kyoko , Ogata Kiyoyuki , An Emi , Dan Kazuo
Published: March 2003

There is little information available regarding immunophenotypic monitoring of minimal residual disease (MRD) in acute myeloid leukaemia (AML). We investigated leukaemic cells co‐expressing CD15 and CD117 (CD15+CD117+) in 72 adult AML cases at diagnosis. In 22 cases (31%) with various AML subtypes, more than 5% of leukaemic cells showed the CD15+CD117+ phenotype (range 5.22–55.48%). These 22 cases were younger and had a higher complete remission (CR) rate than the other AML cases, but the CD15+CD117+ cell percentage at diagnosis showed no correlation with the CR duration among the 72 cases. The CD15+CD117+ cell percentage showed a range of 0.00–0.08% in bone marrow cells from 10 haematologically normal subjects. We also investigated CD15+CD117+ cells in sequential bone marrow samples from 17 AML patients who achieved CR and who had had more than 5% CD15+CD117+ leukaemic cells at diagnosis. Because the CD15+CD117+ cell percentage varied among these AML cases, we calculated the percentage of MRD {MRD% = [CD15+CD117+ cells (%) in each sequential marrow sample] ÷ [CD15+CD117+ cells (%) at diagnosis of the corresponding case] × 100}. A high MRD% after 10 months of CR was significantly associated with a short CR duration (P = 0.0004), whereas continuation of a well‐reduced MRD% was associated with a long CR duration. The leukaemic cells conserved the CD15+CD117+ phenotype in all of the eight cases who relapsed. Flow cytometric monitoring of CD15+CD117+ cells is simple and can be applied to a substantial fraction of AML cases. This monitoring may be useful for predicting relapse of adult AML.
In vivo biotinylation studies: specificity of labelling of reticulated platelets by thiazole orange and mepacrine
Authors: Robinson Monique , Machin Samuel , Mackie Ian , Harrison Paul
Published: March 2003

Animal in vivo biotinylation studies have demonstrated that thiazole orange (TO) labels the youngest cells in the circulation. TO has since been widely used for the measurement of reticulated platelets. As recent findings suggest that at high concentrations TO also labels platelet dense granules non‐specifically, the value of previous work is unclear. Mepacrine also labels platelet dense granules and can detect storage pool defects. In this study, a mouse in vivo biotinylation model was used to determine the specificity of TO and mepacrine staining on platelets recently released into the circulation. The mean life span of biotin/TO (low), biotin/TO (high) and mepacrine/TO dual‐positive platelets was 1.4 d (SD 0.5), 2.2 d (SD 0.2) and 2.3 d (SD 0.3) respectively (n = 6) compared with a life span for biotin‐positive platelets of 4.9 d (SD 1.6). TO (low), TO (high) and mepacrine labelled 8.0% (SD 3.1), 43.9% (SD 8.3) and 40.0% (SD 9.9) of the total platelet population respectively (results of 30 samples from six mice), which decreased to 6.8% (SD 3.9), 26.6% (SD 6.9) and 25.7% (SD 10.6) after thrombin degranulation. The shorter life span and lack of thrombin sensitivity of TO (low)‐positive platelets, suggests that TO (low) measures reticulated platelets specifically. The comparative life spans and thrombin sensitivity of TO (high) and mepacrine‐positive platelets suggest that TO (high) labels platelet dense granules. These data also suggest that dense granules are lost during platelet ageing.
Autosomal dominant type 1 von Willebrand disease due to G3639T mutation (C1130F) in exon 26 of von Willebrand factor gene: description of five Italian families and evidence for a founder effect
Authors: Castaman Giancarlo , Eikenboom Jeroen C. J. , Missiaglia Edoardo , Rodeghiero Francesco
Published: March 2003

Twenty‐four apparently unrelated Italian patients with autosomal dominant type 1 von Willebrand disease (VWD) and a clear autosomal pattern of inheritance of bleeding symptoms were screened for the C1149R and C1130F mutations. None of the patients had the C1149R mutation; three patients and four affected relatives were heterozygous for the C1130F mutation. The mutation appeared to be linked to a single haplotype, defined by five genetic markers [variable number tandem repeat (VNTR) I and II in intron 40, RsaI in exons 13 and 18 and HphI in exon 28], suggesting a founder effect. The patients responded well to desmopressin infusion. The C1130F mutation might have a dominant negative effect on the secretion of the normal protein that desmopressin would appear to overcome.
Antithrombotic effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, on platelet plug formation in in vivo experiments
Authors: Sheu J. R. , Hung W. C. , Wu C. H. , Lee Y. M. , Yen M. H.
Published: March 2003

In this study, platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. Rutaecarpine (200 µg/g) significantly prolonged the latent period of inducing platelet plug formation in mesenteric venules when it was intravenously injected. Rutaecarpine (200 µg/g) prolonged occlusion time by approximately 1·5‐fold (control 127 ± 29 vs. taecarpine 188 ± 23 s). Furthermore, aspirin (250 µg/g) also showed a similar prolongation of the occlusion time in this experiment. On a molar basis, rutaecarpine was approximately twofold more potent than aspirin at prolonging the occlusion time. Furthermore, rutaecarpine was also effective in reducing the mortality of ADP‐induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 25 and 50 µg/g. Intravenous injection of rutaecarpine (50 µg/g) significantly prolonged the bleeding time by approximately 1·5‐fold compared with normal saline in the severed mesenteric arteries of rats. Continuous infusion of rutaecarpine (5 µg/g/min) also significantly increased the bleeding time 1·5‐fold, and the bleeding time returned to baseline within 60 min after cessation of rutaecarpine infusion. These results suggest that rutaecarpine has an effective anti‐platelet effect in vivo and that it may be a potential therapeutic agent for arterial thrombosis, but it must be assessed further for toxicity.
P‐glycoprotein (PGP), lung resistance‐related protein (LRP) and multidrug resistance‐associated protein (MRP) expression in acute promyelocytic leukaemia
Authors: Michieli Mariagrazia , Damiani Daniela , Ermacora Anna , Geromin Antonella , Michelutti Angela , Masolini Paola , Baccarani Michele
Published: March 2003

We analysed the expression of three drug transporter proteins [p‐glycoprotein (PGP), lung resistance‐related protein (LRP) and multidrug resistance‐associated protein (MRP1)] involved in anthracycline resistance that are frequently overexpressed in poor‐risk adult acute non‐lymphocytic leukaemia (ANLL), in 23 acute promyelocytic leukaemia (APL) patients at onset managed at a single institution. Cellular daunorubicin accumulation was also evaluated. At onset, no case had PGP or MRP1 expression that exceeded that of non‐multidrug‐resistant (MDR) cell lines. Only one case showed LRP overexpression. No peculiar MDR features distinguished the seven patients who relapsed from those who maintained complete remission. In the onset vs. first relapse, only one patient showed an increased (threefold) PGP expression at relapse. At second relapse, three out of four patients showed a PGP expression two‐ to threefold higher than baseline values. These results are consistent with the view that low PGP, LRP and MRP1 expression and the absence of defects in intracellular drug accumulation may account for the peculiarly high sensitivity of APLs to anthracycline. It does not support the screening of MDR markers in APL patients at onset as predicting factors of early relapse. The results suggest that no significant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse. In contrast, PGP expression is likely to increase later in the patient history as a result of additional chemotherapy courses.
Cell‐surface expression of transrearranged Vγ–Cβ T‐cell receptor chains in healthy donors and in ataxia telangiectasia patients
Authors: Hinz Thomas , Allam Atef , Wesch Daniela , Schindler Detlev , Kabelitz Dieter
Published: March 2003

Transrearrangements between the T‐cell receptor (TCR) VγI family and JβCβ loci occur at increased frequencies in patients with ataxia telangiectasia (AT). We have used an optimized reverse transcriptase polymerase chain reaction (RT‐PCR) approach to investigate the occurrence of TCRVγ–JβCβ transrearrangements involving the dominantly used Vγ element in peripheral blood γδ T cells, i.e. Vγ9. We detected in frame transcripts of Vγ9‐JβCβ transrearrangements in 4/16 AT patients and in 3/13 healthy control donors. A panel of monoclonal antibodies (mAb) against all expressed TCRVγ elements was used to monitor cell‐surface expression of transrearranged TCR. A very low proportion (< 1%) of peripheral blood TCRαβ cells expressed Vγ instead of Vβ elements. For the first time, we have isolated and molecularly characterized αβ T cells with a Vγ9‐JβCβ transrearrangement from two AT patients and we have shown that such TCR are functional. We conclude that functional TCR transrearrangements can also involve Vγ9, the dominant Vγ element in peripheral blood γδ T cells.
Mesenchymal progenitor cells in human umbilical cord blood
Authors: Erices Alejandro , Conget Paulette , Minguell José J.
Published: March 2003

Haemopoiesis is sustained by two main cellular components, the haematopoietic cells (HSCs) and the mesenchymal progenitor cells (MPCs). MPCs are multipotent and are the precursors for marrow stroma, bone, cartilage, muscle and connective tissues. Although the presence of HSCs in umbilical cord blood (UCB) is well known, that of MPCs has been not fully evaluated. In this study, we examined the ability of UCB harvests to generate in culture cells with characteristics of MPCs. Results showed that UCB‐derived mononuclear cells, when set in culture, gave rise to adherent cells, which exhibited either an osteoclast‐ or a mesenchymal‐like phenotype. Cells with the osteoclast phenotype were multinucleated, expressed TRAP activity and antigens CD45 and CD51/CD61. In turn, cells with the mesenchymal phenotype displayed a fibroblast‐like morphology and expressed several MPC‐related antigens (SH2, SH3, SH4, ASMA, MAB 1470, CD13, CD29 and CD49e). Our results suggest that preterm, as compared with term, cord blood is richer in mesenchymal progenitors, similar to haematopoietic progenitors.
Hypermethylation of P16ink4a and P15ink4b genes as a marker of disease in the follow‐up of non‐Hodgkin's lymphomas
Authors: Martinez‐Delgado Beatriz , Richart Alberto , García Maria Jose , Robledo Mercedes , Osorio Ana , Cebrian Arancha , Rivas Carmen , Benitez Javier
Published: March 2003

The hypermethylation of p16ink4a and p15ink4b genes have been described as an inactivating mechanism alternative to deletions and mutations that accounts for a relatively high proportion of cancers, including non‐Hodgkin's lymphomas (NHLs). To investigate whether detection of abnormal methylation could have clinical applications in the management and follow‐up of lymphomas, we have analysed the behaviour and evolution of p16ink4a and p15ink4b methylation in 13 NHL cases undergoing chemotherapy. All cases were also analysed for the presence of monoclonal rearrangements of immunoglobulin or T‐cell receptor genes. Six patients showed methylation in at least one of these genes at diagnosis, whereas in two other cases methylation appeared during the treatment. The other five cases were always unmethylated. Methylation was detected when any histological or molecular evidence of disease was present, suggesting a good correlation between methylation and disease. In some cases, we were able to detect methylation in patients at complete remission and without evidence of monoclonal cell population, indicating a high sensitivity of the PCR to detect methylation. These results suggest that p16ink4a and p15ink4b methylation could be good markers of disease and could be helpful in identifying lymphoma patients at risk of relapse.
Expression of the NUP98/HOXA9 fusion transcript in the blast crisis of Philadelphia chromosome‐positive chronic myelogenous leukaemia with t(7;11)(p15;p15)
Authors: Yamamoto Katsuya , Nakamura Yuichi , Nakamura Yuko , Saito Kenji , Furusawa Shinpei
Published: March 2003

The t(7;11)(p15;p15) translocation is a recurrent aberration observed in acute myeloblastic leukaemia (AML) and chronic myelogenous leukaemia (CML). It has been shown that the NUP98 gene at 11p15 is fused with the HOXA9 gene at 7p15 in AML with t(7;11). We report the first case with CML expressing the NUP98/HOXA9 fusion transcript. A 27‐year‐old Japanese man was initially diagnosed as in the chronic phase of Philadelphia‐positive CML. At the diagnosis of myeloid blast crisis, the karyotype evolved to 46, XY, t(7;11)(p15;p15), t(9;22)(q34;q11). Reverse transcriptase polymerase chain reaction identified the NUP98/HOXA9 transcript, suggesting that the NUP98/HOXA9 fusion protein could play a critical role in the progression to blast crisis.
Increased platelet activation and abnormal membrane glycoprotein content and redistribution in myeloproliferative disorders
Authors: Jensen Morten Krogh , De Nully Brown Peter , Lund Birgit Villadsen , Nielsen Ove Juul , Hasselbalch Hans Carl
Published: March 2003

Chronic myeloproliferative disorders (MPDs) are characterized by a high incidence of thrombohaemorrhagic complications, possibly caused by platelet dysfunction. In an attempt to define platelet functional abnormalities, we assessed the expression of activation‐dependent membrane proteins in unstimulated and agonist [ADP and thrombin receptor‐activating peptide (TRAP)]‐stimulated platelets using quantitative whole blood flow cytometry in samples from 50 MPD patients and 30 controls. The receptor densities of activation markers and glycoproteins (GPs) were quantified using standardized fluorescent beads. Compared with controls, the mean percentage of P‐selectin‐positive (15·3% vs. 7·2%; P < 0·001) and thrombospondin (TSP)‐positive (6·6% vs. 3·7%; P = 0·003) platelets was increased in unstimulated platelets from patients. Patients having experienced a thrombotic event had a higher mean percentage of TSP‐positive non‐stimulated platelets than patients without a history of thrombosis (9·0% vs. 4·6%; P = 0·02) and a higher GPIV molecules of equivalent fluorochrome (MEF) value (33113 vs. 24471 MEF; P = 0·02). Mean MEF values of monoclonal antibodies (mAbs) against GPIb (34055 vs. 38945 MEF; P < 0·001) and GPIIb/IIIa (1416 vs. 1648 MEF; P < 0·001) were significantly reduced among patients, whereas surface expression of GPIV was increased in patients (28273 vs. 16258 MEF; P < 0·001). In TRAP (10 µmol/l) stimulated whole blood, the MEF of P‐selectin (9611 vs. 13293 MEF; P = 0·004) and CD63 (2385 vs. 5177 MEF; P < 0·001) and the ratio of PAC‐1/GPIIb/IIIa MEF (0·98 vs. 2·00; P < 0·001) was reduced in patients, indicating either a reduced granule GP content or an intrinsic cellular defect in receptor‐mediated granule secretion and activation of the GPIIb/IIIa complex. Expressed as the relative change of MEF compared with unstimulated platelets, TRAP induced decrease of GPIb (7·8% vs. 45%; P < 0·001) and increase of GPIIb/IIIa (49·1% vs. 95·7%; P < 0·001) and GPIV expression (17·8% vs. 55·2%; P < 0·001) was attenuated in patients.