bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma erythropoietin by high‐detectability immunoradiometric assay in untreated and treated patients with polycythaemia vera and essential thrombocythaemia
Authors: Carneskog Jan , Kutti Jack , Wadenvik Hans , Lundberg Per‐Arne , Lindstedt Göran
Published: July 2010

Abstract: By using an immunoradiometric method with a stated detection limit of ≤1 IU/l (stated normal reference limit in adults 3.7–16 IU/l) we determined EDTA‐plasma erythropoietin (EPO) in 58 patients with polycythaemia vera (PV) and 49 patients with essential thrombocythaemia (ET). At the time of blood sampling, 20 of the PV patients were newly diagnosed and untreated, 23 were treated by phlebotomy only, and 30 also received myelosuppressive treatment (with 32P, hydroxyurea or alpha‐interferon). Of the ET patients 24 were untreated and 28 received myelosuppressive therapy. For comparison plasma EPO was also determined in 10 patients with pseudopolycythaemia (PP). In this latter group the results for plasma EPO agreed well with the cited normal reference limits. The majority of untreated PV patients (12/20) had undetectable plasma EPO concentration, and the remainder all had values below the lower normal reference limit. Plasma EPO in PV was not significantly influenced by phlebotomy therapy. Twelve of the 24 untreated ET patients (50%) had plasma EPO values below the reference interval (undetectable in 2 patients). The mean EPO concentration was significantly lower in PV patients receiving phlebotomy therapy than in patients with untreated ET. In the total material of PV and ET treated with myelosuppressive agents the PV patients showed significantly lower values for EPO concentration than did patients with ET. The present results support the view that EPO measurements by high‐detectability methods are diagnostically useful and should be included in the panel of new criteria for the diagnosis of PV.
Antibodies against platelet glycoproteins and antiphospholipid antibodies in autoimmune thrombocytopenia
Authors: Lipp E. , von Felten A. , Sax H. , Müller D. , Berchtold P.
Published: July 2010

Abstract: Autoantibodies against platelet glycoproteins (anti‐GP) are found in the majority of patients with autoimmune thrombocytopenia (AITP) as well as in thrombocytopenia associated with systemic lupus erythematosus (SLE). Some of these patients may have anti‐phospholipid antibodies (anti‐PL). To evaluate the pathogenetic significance of anti‐PL and anti‐GP antibodies in AITP and SLE patients, we investigated anti‐cardiolipin (anti‐CL), anti‐phosphatidylserine (anti‐PS) and anti‐GP antibodies (anti‐GPIIb‐IIIa and anti‐GPIb‐IX) in 71 patients with AITP and 3 thrombocytopenic patients with SLE. Anti‐GP antibodies were detected in 52 (70%) patients. Fifty‐six (73%) patients showed anti‐PL antibodies. Seven patients (6 AITP, 1 SLE) with both anti‐GPIIb‐IIIa and IgG anti‐PL antibodies were followed during treatment with corticosteroids. Antibodies were measured before treatment and at the time of platelet‐peak. Anti‐GPIIb‐IIIa antibodies decreased in all or became undetectable in five. In contrast, IgG anti‐PS and IgG anti‐CL antibodies decreased only moderately or remained positive. Adsorption experiments, using gelfiltered platelets, erythrocyte (Ec)‐inside‐out‐vesicles and purified GPIIb‐IIIa, showed that anti‐GP and anti‐PL antibodies have distinct specificities and do not crossreact. We conclude that anti‐PL and anti‐GP antibodies may be present simultaneously in some patients with immune mediated thrombocytopenia. Although anti‐PS as well as anti‐CL antibodies may be responsible for thrombocytopenia, we speculate that anti‐GPIIb‐IIIa antibodies are more related to the severity of thrombocytopenia.
Serum soluble c‐kit receptor and expression of c‐kit protein and mRNA in acute myeloid leukemia
Authors: Tajima Fumihito , Kawatani Toshio , Ishiga Kiyomi , Nanba Eiji , Kawasaki Hironaka
Published: July 2010

Abstract: To investigate the clinical role of the soluble form of c‐kit receptor (s‐kit) in patients with acute myeloid leukemia (AML), we determined the levels of serum s‐kit and expression of c‐kit antigens and mRNA in leukemic cells. The serum s‐kit level was measured using ELISA assay in 30 AML patients and 20 normal controls. C‐kit antigens of leukemic blasts were stained immunohistologically, and c‐kit mRNA was detected by RT‐PCR. The serum s‐kit level in M1 and M2 were significantly increased (p<0.01) and that in M4 or M5 was significantly decreased (p<0.05) compared to that in the controls. In the comparisons among subtypes of FAB classification, M1 and M2 showed significantly higher levels than M4 or M5 (p<0.05 and p<0.01, respectively). Both expression of c‐kit antigens and mRNA were observed in M0 (1/4), M1 (2/4) and M2 (6/8), but neither was observed in M4 or M5. The serum s‐kit levels were correlated with the absolute number of AML blasts in peripheral blood (r=0.564, p<0.05). These results indicate that the serum s‐kit level is related to the stage of differentiation of AML blasts in accordance with the expression of c‐kit protein and mRNA in AML blasts, and is useful for assessment of leukemic cell burden.
Haematopoietic action of flt3 ligand on cord blood‐derived CD34‐positive cells expressing different levels of flt3 or c‐kit tyrosine kinase receptor: comparison with stem cell factor
Authors: Sakabe Hideaki , Kimura Takafumi , Zeng Zhaozhu , Minamiguchi Hitoshi , Tsuda Shouichiro , Yokota Shouhei , Hodohara Keiko , Abe Tatsuo , Lyman Stewart D. , Sonoda Yoshiaki
Published: July 2010

We compared the effect of human flt3 ligand (FL) and stem cell factor (SCF) on cord blood (CB)‐derived CD34+ cells expressing different levels of flt3 or c‐kit tyrosine kinase (TK) receptor in clonal cell culture. The c‐kit receptor was expressed by 58.5±16.7% of CB CD34+ cells (n = 19), in which c‐kithigh, c‐kitlow and c‐kit‐ cell populations could be identified. In contrast, the flt3 receptor (FR) was weakly expressed on 58.6±8.3% (n = 9) of CB CD34+ cells. FL+erythropoietin (Epo) failed to support erythroid burst (BFU–E) formation by any subpopulation of CD34+ cells. However, SCF+Epo supported BFU–E and erythrocyte‐containing mixed (CFU–mix) colony formation from all subpopulations. Interestingly, FL markedly augmented CFU–mix colony formation supported by interleukin (IL)‐ 3+Epo when CD34+c‐kitlow or CD34+FR+ cells were used as the target. On the other hand, SCF significantly enhanced CFU‐mix colony formation supported by IL‐3+Epo when CD34+c‐kithigh or low and CD34+FR+ cells were used. The replating potential of CFU–mix supported by IL‐3 + Epo + FL was greater when CD34+c‐kitlow or CD34+FR+ cells were used. When the CD34+c‐kitlow cells were used, the number of lineages expressed in secondary cultures of CFU–mix colonies derived from primary cultures containing IL‐3 + Epo+FL or SCF was significantly larger than when the primary cultures contained IL‐3+Epo. Furthermore, the number of long‐term culture‐initiating cells found in CD34+FR+ cells was larger than that in FR‐ cells. CB‐derived CD34+c‐kitlow cells represent a less mature population than c‐kithigh cells, as reported previously. Therefore, these results indicate that both FL and SCF can act on primitive multipotential progenitors. However, it is still uncertain whether CB‐derived CD34+FR+ cells are less mature than CD34+FR‐ cells.
From megakaryocytes to platelets: platelet morphogenesis takes place in the bloodstream
Authors: Behnke Olav , Forer Arthur
Published: July 2010

Abstract:We studied megakaryocyte processes formed in rat bone marrow and spleen, using both the transmission and scanning electron microscopes. Some processes were bulky, others slender and beaded. The bulky megakaryocyte processes developed a specialized arrangement of organelles at the site at which they entered the lumen: filaments present around the outside of the process seemed to support a central cylinder in which organelles flowed along microtubules.

Megakaryocyte processes were present in platelet‐rich plasma from both human and rat blood. When followed in living preparations, bulky processes developed pointed tips, elongated, and became slender and beaded. Fusiform proplatelets also were present in the platelet rich plasma, with pointed tips at both ends of what appeared to be single “beads”; we assume that the long, beaded megakaryocyte processes would have fragmented were we to have had proper culture conditions.

The straight, shorter fusiform proplatelets in living preparations underwent characteristic curving and bending motions, eventually forming disk‐shaped cells which sometimes had appendages. This behaviour suggests that the entire process of platelet morphogenesis takes place in plasma: megakaryocyte processes first elongate, then bead and fragment, and then curve and fuse to form disk‐shaped platelets. This interpretation is strengthened by finding in freshly isolated plasma many of the shapes seen in the transformations studied in living cell preparations.

The megakaryocyte processes and the proplatelets seemed to appear in plasma with a periodicity related to light and dark cycles – that is, with a circadian rhythm. In particular, megakaryocyte processes appear in human blood within a few hours after sunrise; we argue that this might be related to similar peak periods for heart attacks.
Comparison of methods for retroviral mediated transfer of glucocerebrosidase gene to CD34+ hematopoietic progenitor cells
Authors: Takiyama Nobuaki , Mohney Trina , Swaney William , Bahnson Alfred B. , Rice Erin , Beeler Margaret , Scheirer‐Fochler Sherrie , Ball Edward D. , Barranger John A.
Published: July 2010

Abstract: Gaucher disease is an excellent candidate for gene therapy by transduction of hematopoitic stem cells. In this study, we compared methods which allow an increase in transfer of the glucocerebrosidase gene to human hematopoietic progenitor cells. Several techniques were employed, including the use of cytokines, bone marrow stroma, fibronectin, centrifugal enhancement and in vitro long‐term culture. The effect of prestimulation with cytokines interleukin‐3 (IL‐3), interleukin‐6 (IL‐6) and stem cell factor (SCF) on transduction of cord blood CD34+ cells was examined. The results suggest that 16‐h prestimulation was sufficient for efficient transduction. We examined the effect of bone marrow stroma and fibronectin, both of which increased transduction efficiency up to 36% and 44%, respectively, as measured by PCR for the integrated GC‐cDNA in clonogenic cells (9% without any support). Transduction efficiency of 83% was obtained using 2‐h centrifugation. Combining centrifugation and in vitro culture in long‐term bone marrow culture media containing cytokines (IL‐3/IL‐6/SCF), CD34+ cells from cord blood and peripheral blood of 3 Gaucher patients were transduced weekly for 21 d. The results of 6 separate experiments consistently demonstrated transduction efficiency of 100% after 7‐d in vitro culture. This transduction protocol combining centrifugation and in vitro long‐term culture is an attractive method and can be applied to clinical trials.
Chronic myeloid leukemia in accelerated phase: treatment results with conventional chemotherapy and allogeneic bone marrow transplantation in 96 patients
Authors: Griesshammer M. , Arnold R. , Bangerter M. , Hafner M. , Heinze B. , Hertenstein B. , Heimpel H. , Bunjes D.
Published: July 2010

Abstract: The treatment results of 96 patients with Philadelphia‐positive chronic myeloid leukemia (CML) in accelerated phase (AP) were reviewed retrospectively. Treatment of AP consisted of allogeneic bone marrow transplantation in 20 (14 related and 6 unrelated donors) or conventional chemotherapy (CC) in 76 patients. Three main treatment strategies were followed in the CC group: continuation (group A) or dose escalation (group B) of chronic phase therapy or change of chronic phase therapy to hydroxyurea (group C). Median survival was 7.0 months in group A (range 1.8–110), in group B 8.3 months (range 0.9–40) and in group C 9.6 months (range 1.5–47.6), p = 0.89. Survival in CC was dependent on response to therapy as the achievement of a second chronic phase was significantly associated (p < 0.001) with a longer median survival (21 months) compared with stable accelerated phase disease (11 months) or treatment failure (5 months). Median survival in the BMT group was 16.7 months (range 5–77), the 5‐yr probability of relapse was 25% and the 5‐yr disease‐free survial was 36%. For patients < 55 yr median survival after BMT was significantly prolonged compared with median survival after CC (n = 45, 8.3 months, p = 0.008). After developing criteria of AP, median survival in our analysis has been less than 1 yr. The results of conventional chemotherapy in the treatment of accelerated phase CML are disappointing. If a suitable donor is available allogeneic BMT should be performed without delay in patients with AP.
Production of phospholipid antibodies in selected thrombophilic women differing in genotype at the 506 site of factor V
Authors: Bokarewa Maria I. , Bremme Katarina
Published: July 2010

Abstract: The composition of antibodies against phospholipids (PLa) was analysed in 54 PLa‐positive thrombophilic women, different in the factor V genotype (Arg506‐Gln mutation carriers, n = 23; and noncarriers, n = 31). The presence of antibodies against prothrombin was also studied. The incidence of cardiolipin antibodies (CLa) and lupus anticoagulant (LA) activity was similar among the carriers and noncarriers, while phosphatidylserine antibodies (PSa) were often the only PLa detected in the carriers of the mutation (7/23 vs. 2/31; χ2 = 5.47, p < 0.05). Antibodies against prothrombin were found in 11 of 54 patients. They segregated with PSa (8/19 vs. 3/35; χ2 = 8.54, p < 0.025), but were equally distributed between carriers and noncarriers. The analysis of nAPC‐ratio in patients with different PLa showed that it was consistently lower in the mutation carriers in whom none of the PLa caused further suppression. The noncarrier group positive for LA in several assays was associated with a reduction of nAPC‐ratio (0.82 ± 0.25 vs. 1.12 ± 0.22, p < 0.05). Our findings indicate that PLa contributing to the development of APC resistance are restricted to those possessing LA activity.
Selective induction of apoptosis of NB4 cells from G2+M phase by sodium arsenite at lower doses
Authors: Ma Dong‐Chu , Sun Ying‐Hui , Chang Kui‐Zhong , Ma Xiao‐Feng , Huang Shi‐Lin , Bai Yue‐Hui , Kang Jian , Liu Ya‐Ge , Chu Jun‐Jie
Published: July 2010

Abstract: Apoptosis of NB4 cells induced by sodium arsenite and arsenate was studied using flow cytometry and DNA gel electrophoresis in order to investigate their effects on cell cycle and determine the relationship between apoptosis and cell cycle. In this study, we found that: 1) at low doses, sodium arsenite selectively induced apoptosis of NB4 cells in G2+M phase of cell cycle after its being arrested in G2 phase. With increment of the cells blocked in G2 phase, dUTP‐specifically labeling cells in G2+M phase increased without concomitant increment of dUTP‐labeling cells in other two phases of cell cycle; 2) at high doses, extensive apoptosis was induced in NB4 cells from all phases of cell cycle without cell cycle preference and cell cycle blockade; 3) sodium arsenite‐induced apoptosis of NB4 cells occurred in the presence of bcl‐2 expression as the unapoptotic cells; 4) sodium arsenite with As3+ induced apoptosis of NB4 cells more strongly than sodium arsenate with As5+ did although both of them affected NB4 cells in the same pattern. These results not only suggested that both arsenite and arsenate induced apoptosis of NB4 cells through 2 different mechanisms ‐ at low doses, arsenical might directly induce apoptosis through regulation of cell cycle checkpoint, while at high doses they might directly induce it, but also indicated that bcl‐2 might not play an important role in arsenite or arsenate‐induced apoptosis of NB4 cells, whereas chemical valence of As in a compound might be related to efficiency in arsenical induction of apoptosis.
Kinetics of circulating selectin levels during bone marrow aplasia
Authors: Jilma B. , Eichler H. G. , Becherer A. , Brugger S. , Kalhs P. , Stohlawetz P. , Kapiotis S. , Kapiotis S.
Published: July 2010

Abstract: We have studied the kinetics of plasma levels of circulating (c)selectins in 8 patients undergoing bone marrow or stem cell transplantation to gain estimates for the distribution and half‐life of (c)selectins and to potentially identify an endothelial source of cp‐selectin in patients who are deprived of platelets and megakaryocytes. Blood was sampled just before conditioning treatment and immediately after, at 2 occasions under bone marrow aplasia (1 and 2 wk after BMT), on 2 separate days (3–8 d apart) before and at 60 min after platelet transfusion, and 30–72 d after BMT. All (c)selectins showed a strikingly parallel decrease during bone marrow aplasia: cp‐selectin decreased by a median of 70% (range: 59–95%), CE‐selectin by 63% (range: 27–78%), and cl‐selectin by 75% (range: 66–90%) compared to baseline (p = 0.012 for all comparisons). Estimates for plasma half‐lives of all (c)selectins were obtained from individual time vs. concentration profiles of the maximal decreases and ranged from 2 to 4 d. cp‐selectin increased by 23% (p = 0.003), ce‐selectin by 5% (p = 0.041) and cl‐selectin by 19% (p = 0.009) 1 h after the platelet transfusions. Based on these results the calculated volume of distribution (Vd) of transfused (c)selectins was 2.5‐fold higher than the plasma volume, which supports the concept of the in‐vivo expression of binding sites, i.e. ligands, for CE‐selectin and CL‐selectin. In summary, while our study cannot provide any evidence for endothelial cells as a source of cp‐selectin, we have found for the first time evidence for the existence of ligands for soluble CE‐selectin and cL‐selectin in humans.
Within‐subject biological variation of reticulocytes and reticulocyte‐derived parameters
Authors: Sandberg Sverre , Rustad Pål , Johannesen Berit , Stølsnes Berit
Published: July 2010

Abstract: Automation of reticulocyte counting has decreased the analytical imprecision of this parameter. This has made it possible to use the number of reticulocytes and its derived parameters (reticulocyte maturity and reticulocyte cell indices) in new diagnostic and monitoring situations. For rational use of these parameters, it is important to have knowledge of their biological variability. The biological variability of reticulocytes and its derived parameters was studied in 13 healthy people during a period of 7 wk on 2 different instruments. The within‐subject coefficient of variation for the reticulocyte count was about 11%, for the mean reticulocyte volume, mean reticulocyte haemoglobin content and mean reticulocyte haemoglobin concentration it was between 1 and 2%, whereas the coefficient of variations for the subpopulations of reticulocytes with different maturity varied depending on the method used for the measurements. The critical difference, that is the change in a result making it significantly different from the previous result, was about 35% for the reticulocyte count and 5–8% for the reticulocyte cell indices, making these indices excellent to follow changes in erythropoiesis. With a possible exception for the mean reticulocyte volume, the within‐subject variation was small compared to the between‐subject variation.
Acute phase proteins and interleukins in steady state sickle cell disease
Authors: Bourantas Konstantinos L. , Dalekos Georgios N. , Makis Alexandres , Chaidos Aristidis , Tsiara Stavroula , Mavridis Anestis
Published: July 2010

Abstract: To identify a possible acute phase response during the steady state of sickle cell disease, we estimated the serum alterations of acute phase proteins, β2‐microglobulin (β2M), κ and λ light chains, interleukins (ILs) and tumor necrosis factor‐α (TNFα) in 21 patients. Increased concentrations of C‐reactive protein (CRP) were found in 5 patients, alpha‐1‐acid‐glycoprotein (AGP) in 3, alpha‐1‐antitrypsin (AAT) in 8, ceruloplasmin (CER) in 2, alpha‐2‐macrogiobulin (AMG) in 14 and decreased haptoglobin (HPT) and transferrin (TFR) in 11 and 9, respectively. Increased β2M was found in 10 patients and κ and λ light chains in 11. IL‐1β, IL‐2, IL‐4, IL‐10 and TNFα were not detected in any of the patients. However, significantly increased values of IL‐6 and sIL‐2r were found. This study has demonstrated increased serum levels of some of the acute phase proteins in patients during the steady state of sickle cell disease. This may be a result of a subclinical vaso‐occlusion which in turn leads to a covert inflammatory response. Cytokines, and in particular IL‐6, produced after this response, seem to be responsible for the high levels of acute phase proteins in the steady state of this disease.
X‐linked sideroblastic anaemia due to a mutation in the erythroid 5‐aminolaevulinate synthase gene leading to an arginine170 to leucine substitution
Authors: Edgar A. J. , Vidyatilake H. M. S. , Wickramasinghe S. N.
Published: July 2010

Abstract: DNA sequencing of the coding region of the erythroid 5‐aminolaevulinate synthase (ALAS2) cDNA from a male with pyridoxine‐responsive sideroblastic anaemia revealed a missense mutation, a G561T transversion in exon 5 of the gene. Previously, the mutation G561A has been shown to be responsible for sideroblastic anaemia in females and thought to be lethal in males (1). The mutation G561T results in the loss of an MspA1‐I cutting site. Analysis of MspA1‐I restriction enzyme digests of amplified exon 5 genomic DNA from other family members revealed that the proband's mother, aunt and youngest sister, who were not anaemic, were heterozygous carriers of the mutation. The G561T mutation results in an arginine to leucine substitution at amino acid residue 170. This arginine residue is conserved in both the erythroid and housekeeping ALAS in vertebrates as well as in all other known ALAS proteins and is located in a predicted alpha‐helix region close to the amino‐terminus of the enzymatic region of the protein.
Risk of relapse and clinico‐pathological features in 103 patients with diffuse large‐cell lymphoma in complete response after first‐line treatment
Authors: Sanz Llúcia , López‐Guillermo Armando , Martínez Carmen , Bosch Francesc , Esteve Jordi , Cobo Francesc , Montoto Silvia , Perales María , Bladé Joan , Cervantes Francisco , Nomdedeu Benet , Campo Elías , Montserrat Emilio
Published: July 2010

Abstract: Patients with diffuse large‐cell lymphoma (DLCL) achieve a complete response (CR) in most cases, but at least one‐third of them eventually relapse. Such an event occurs most frequently within 2 yr from CR achievement. The aim of the present study was to analyse the risk and pattern of relapse of patients with DLCL in CR. One hundred and three patients with DLCL (53 male/50 female; median age: 55 yr) in CR after doxorubicin‐containing first‐line treatments were included in the study. Main clinicobiological characteristics at diagnosis and at relapse were analysed. Uni‐ and multivariate studies were performed. Forty‐one patients (40%) eventually relapsed, in 27 cases within 2 yr from CR and 14 thereafter. Histological subtype was the same at diagnosis and at relapse in all the early relapsing patients and in 8 of 10 late relapsing patients with available biopsy. The most important variables at diagnosis for predicting relapse were advanced stage (p < 0.01) and bone marrow infiltration (p = 0.05), with stage (I–II vs. III–IV) (p = 0.009; relative risk = 2.28) being the only predictive variable in the multivariate analysis. No differences were found according to the treatment given. The second CR rate obtained in the late relapsing patients after salvage therapies was higher that in early relapsing (50% vs. 37%). Median survival from relapse was 1.4 yr for patients early relapsing and it was not achieved for those with late relapses (p = 0.09). Late relapse is a quite common event in DLCL lymphomas, with those patients achieving more frequently a second CR and having better survival than early relapsed patients.
Cisplatin inhibits erythroid committed progenitor (BFU‐E) mobilization in peripheral blood
Authors: Pannacciulli Ivo , Castello Giorgio , Lerza Roberto , Mencoboni Manlio , Botta Marco , Arboscello Eleonora , Bogliolo Giuseppe
Published: July 2010

Abstract: Circulating myeloid (CFU–GM) and erythroid (BFU–E) progenitor levels were evaluated weekly throughout 3 courses of treatment with vinorelbine (VNB) ifosfamide (IFO) and filgrastim (G‐CSF) with or without addition of cisplatin (DDP) in 20 stage IIIB or IV non small‐cell lung cancer patients. In IFO, VNB, DDP‐treated patients, BFU‐E mobilization in peripheral blood following chemotherapy and G‐CSF was completely lacking, in contrast with the patients treated with IFO, VNB plus G–CSF. CFU–GM release, however, was of the same order in the 2 groups of patients. Further investigations are needed to explain why presence of DDP in this chemotherapeutic protocol hinders erythroid progenitor release in peripheral blood.
Anagrelide, a novel platelet lowering option in essential thrombocythaemia: treatment experience in 48 patients in Germany
Authors: Petrides Petro E. , Beykirch Maria K. , Trapp Oliver M.
Published: July 2010

Abstract: We report on our treatment experience in Germany with anagrelide, a novel platelet lowering agent, in 48 patients (27 females, 21 males) with essential thrombocythaemia. Their age was between 19 and 79 yr when anagrelide therapy was initiated. Sixteen patients were previously untreated, 15 pretreated with hydroxyurea and 17 had multiple pretreatments. Forty‐one of the 48 patients had either microvascular, thromboembolic or bleeding complications. About 50% received low dose acetylsalicylic acid as an adjunct. Their platelet count prior to therapy ranged from 850,000/μl to 3,100,000/μl. Eighty‐seven per cent of the patients treated with anagrelide were complete hematological responders, while 13% responded only partially or failed to respond. Twelve of our patients (25%) developed short‐term (from a few days to a maximum of 4 wk) side effects including headache (most frequent), palpitations, tachycardia or nausea. Eight patients reported long‐term (more than 1 month) adverse effects. However, in only 5 of all 48 patients (10%) were these side effects not acceptable so that treatment had to be discontinued. We have now treated patients for up to 7 yr (median maintenance dose 2.5 mg/d). Preliminary evidence suggests that anagrelide mediated platelet count reduction also prevents recurrence of thromboembolic complications. Hence, anagrelide has the potential to become the first‐line platelet‐lowering treatment in myeloproliferative disorders with high platelet counts.
Glycocalicin in the diagnosis and management of immune thrombocytopenia
Authors: Steffan A. , Pradella P. , Cordiano I. , Girolami A. , De Marco L. , Fabris F.
Published: July 2010

Abstract: We studied glycocalicin (GC), expressed as plasma GC concentration and as GC index (ratio to platelet count), in 129 thrombocytopenic patients (platelet count <100×109/1) and 60 sex‐ and age‐matched controls. Seventy‐two patients had idiopathic immune thrombocytopenia, 32 secondary immune thrombocytopenia, 8 microangiopathic thrombocytopenia and 17 thrombocytopenia secondary to bone marrow aplasia. Patients with immune thrombocytopenia (ITP) were also subclassified, according to their clinical behaviour, as having active disease or being in spontaneous or therapy‐induced partial remission. A significant correlation was found between glycocalicin levels and platelet count both in normals and in patients with bone marrow aplasia (r = 0.75). ITP patients showed a GC index significantly higher than controls (6.02±7.87 vs. 0.9±0.2, p<0.001). When ITP patients with similar platelet count (30–50×109/l) were studied, the mean level of GC and the GC index were significantly higher in those patients with active disease than in those in remission (0.97±0.38 vs. 0.58±0.17 μg/ml, p<0.05; 6.41±2.64 vs. 3.44±0.94, p<0.05, respectively). A longitudinal study performed in 10 patients with different subtypes of ITP suggested a positive correlation between GC index and the activity of the disease. The GC value and GC index were significantly higher in patients with microangiopathic thrombocytopenia than in controls (1.44±0.73 vs. 0.8±0.16 μg/ml, p<0.01; and 18.77±22.23 vs. 0.9±0.2, p<0.001, respectively). The GC value was significantly lower in bone marrow failure (0.15±0.04 μg/ml, p<0.01) compared to controls, while no difference was observed in the GC index. Our data confirm that the GC index is helpful in differentiating thrombocytopenia due to increased platelet destruction from the one due to impaired production. In addition, the assay has been proven useful in the differential diagnosis of different ITP subtypes and their follow‐up.
Phenotypic, morphologic changes and Ig secretion induced on B‐NHL cells in vitro by interferon alpha and all‐trans‐retinoic acid: possible progression toward a more differentiated state
Authors: Bonnefoix Thierry , Sotto Marie‐France , Gressin Remy , Martin Isabelle , Garban Frederic , Leroux Dominique , Renversez Jean‐Charles , Sotto Jean‐Jacques
Published: July 2010

Abstract: Twenty‐five B‐cell‐nonHodgkin's lymphomas (B‐NHL): 6 lymphocytic, 2 centrocytic, 13 follicular, centrocytic/centroblastic, 2 lymphoplasmocytoid and 2 centroblastic were tested for their ability to acquire features of mature plasma cells under the effect of interferon alpha (final concentration, 600 Ul/ml), all‐trans‐retinoic‐acid (ATRA) (final concentration, 10−6 mol/1) and the association of both. B‐NHL cells were negatively purified (>99%) by an immunomagnetic method, cultured for 7 d with or without interferon and ATRA, then stained with anti‐CD 19, CD20, surface Ig, DR, CD38 and with anti‐CD138 (syndecan‐1) antibody‐recognizing plasma cells. Ig production was estimated in culture supernatants by an ELISA method and changes in cell morphology were investigated on May–Grünwald–Giemsa‐stained cytospin preparations. In all cases interferon and ATRA, alone or in association, were able to induce changes in the immunophenotypic profile, associated or not with morphologic changes and induction of Ig secretion. All changes were greatly variable from one to the other B‐NHL sample and no relationship could be found between a particular pattern of change and the histological subtype. Interferon alpha was more potent than ATRA in inducing changes. In favour of a differentiation process, we observed a concomitant decrease of DR expression and increase of CD38 expression in 8 cases with interferon alpha, and in 4 cases with ATRA. Although interferon‐ or ATRA‐treated cells did not display cytologic, functional features and changes of the immunophenotypic profile fully compatible with those of terminally differentiated cells, these results suggest a possible transition toward more differentiated elements, especially with interferon alpha.
Cytokines released in vitro by stromal cells from autologous bone marrow transplant patients with lymphoid malignancy
Authors: Roingeard Françoise , Binet Christian , Lecron Jean‐Claude , Truglio Danielle , Colombat Philippe , Domenech Jorge
Published: July 2010

Abstract: Marrow stromal cells of patients treated by autologous bone marrow transplantation (ABMT) for malignancies have been assessed for their ability to secrete granulocyte colony‐stimulating factor (G–CSF), granulocyte‐macrophage colony‐stimulating factor (GM–CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), interleukin‐6 (IL‐6), transforming growth factor β1 (TGFβ1) and macrophage inflammatory protein‐1α (MIP‐1α). Long‐term marrow cultures were established from 10 patients prior to and 3 months after ABMT, from 7 patients 1 yr after ABMT and from 11 controls. Cytokines in culture supernatants of stromal layers (SL) were evaluated by enzyme‐linked immunosorbent assay (ELISA). Significant differences between patient groups and controls were apparent in baseline production of GM–CSF, SCF, MlP‐1α and TGFβ1. After IL‐1β addition in cultures, G–CSF production was reduced in pretransplant and post‐transplant patients compared to controls. The production of TGFβ1, LIF, IL‐6 and more particularly SCF were reduced in post‐transplant patients, while elevated levels of GM–CSF and MIP‐1α were observed in these patients only when the values were corrected for the number of cells growing in the SL. These results indicate a prolonged stromal defect in growth factor production following ABMT for the early‐stage acting cytokines IL‐6, LIF and SCF as well as for G–CSF, but not for GM–CSF, while the production of the 2 inhibitors shows different pathways.
In vitro effect of stem cell factor on human clonogenic marrow progenitors after myeloablative treatments
Authors: Hérault O. , Binet C. , Degenne M. , Desbois I. , Héraud N. , Colombat P. , Domenech J.
Published: July 2010

Abstract: Abnormal hematopoiesis, including a deficiency of marrow progenitors and particularly of erythroid progenitors, has been described after autologous stem cell transplantation (ASCT), persisting for several years. In order to explain this deficiency, a resistance of marrow progenitors to stem cell factor (SCF) after ASCT was investigated. Marrow samples were harvested from pregraft patients at graft collection prior to ASCT, transplanted patients 6–24 months after high‐dose therapy and control patients. CD34+ cells were cultured in a serum‐free clonogenic assay with increasing doses of SCF. The clonogenic efficiency without SCF was lower for BFU‐E in treated groups than in controls, whereas it was not different for CFU‐GM. With increasing doses of SCF a dose‐dependent effect was found on the numbers of both CFU–GM and BFU–E in all groups, although the maximal number of BFU–E remained lower in treated groups. However, the SCF dose that induced 50% of maximal BFU–E growth (D50) was similar in all groups. Furthermore, a dose‐dependent effect on the size of BFU–E was found in all groups, with no difference in the proportion of large colonies. Thus, clonogenic erythroid progenitors from patients who have received myelotoxic treatments remain sensitive to SCF, with no evidence for a chemotherapy‐related resistance.
Impact of endogenous thrombopoietin levels on the differential diagnosis of essential thrombocythaemia and reactive thrombocytosis
Authors: Hou Ming , Carneskog Jan , Mellqvist Ulf‐Henrik , Stockelberg Dick , Hedberg Martin , Wadenvik Hans , Kutti Jack
Published: July 2010

Abstract: By using the newly commercialized QuantikineTM human TPO immunoassay, plasma thrombopoietin (TPO) concentrations were measured in 12 patients with essential thrombocythaemia (ET), 13 patients with reactive thrombocytosis (RT) and 11 healthy volunteers. For the healthy volunteers the mean plasma TPO concentration was 21.1±11.0 pg/ml. The mean plasma TPO concentration in the group of RT was slightly lower (16.4±8.6 pg/ml) but did not differ significantly from the control group. The mean plasma TPO concentration in ET patients (44.1 ±45.2 pg/ml) was significantly (p<0.05) higher than the mean for RT patients, but did not differ statistically from the mean of healthy volunteers. These data suggest a defective clearance of plasma TPO in patients with ET.
Resistance to activated protein C in thalassaemic patients: an underlying cause of thrombosis
Authors: Giordano P. , Del Vecchio G. C. , Altomare M. , Coppola B. , Schettini F. , Lolascon A. , De Mattia D.
Published: July 2010

Abstract: We evaluated 81 thalassaemia major and 4 thalassaemia intermedia patients (48 M, 37 F), median age 17 years; 62/85 patients were HCV‐positive, 3/85 HIV‐positive, 19/85 were splenectomized. Forty normal healthy children were recruited as the control group. The number of thrombotic events was studied retrospectively. Platelet poor plasma was filtered and quick‐frozen at ‐70°C until time of assay. APC resistance was measured in an activated thromboplastin time and results were expressed as normalized ratio. All tests were done with diluted 1 in 5 (v/v) factor V deficient plasma and with undiluted plasma. Molecular genetic investigation of factor V gene was performed with polymerase chain reaction, followed by digestion of amplified products with restriction enzyme Mnl I. Data obtained with molecular investigation revealed the presence of 4 heterozygous subjects for factor V Leiden (4.7%). Functional tests were able to detect all heterozygotes for factor V Leiden both with undiluted and with diluted plasma, and there were no false negative subjects. However, undiluted plasma revealed a greater number of false positive subjects (n = 15) than did diluted plasma. Therefore, tests done with undiluted and diluted plasma revealed a 100% sensitivity, while specificity was 81 % for undiluted plasma and 97% for diluted plasma. Only one thrombotic event was observed in one of the 85 studied patients, as a case of stroke in a thalassaemia intermedia patient with APC resistance. In the same patient an additional thrombogenic risk factor was represented by a pronounced haematocrit increase at the beginning of her tranfusion regimen.
Long‐term oral pamidronate treatment inhibits osteoclastic bone resorption and bone turnover without affecting osteoblastic function in multiple myeloma
Authors: Abildgaard N. , Rungby J. , Glerup H. , Brixen K. , Kassem M. , Brincker H. , Heickendorff L. , Eriksen E. F. , Nielsen J. L.
Published: July 2010

Abstract: This study was performed as a cross‐sectional substudy to the Danish–Swedish Pamidronate Study, a randomized placebo‐controlled multicentre trial in multiple myeloma. The purpose was to evaluate the biological effects of long‐term treatment with oral pamidronate 300 mg daily on bone metabolism by using histomorphometry and analysis of cytokines and biochemical markers of bone turnover. Sixteen patients were included after median 27.5 months of protocolized treatment; 10 patients received active treatment and 6 patients placebo. When compared with placebo, pamidronate treatment was associated with: (a) marked decreased osteoclastic resorption rate (0.86±0.59 μm/d vs. 5.7±5.0 μm/d, p=0.002), and diminished activation frequency (0.20±0.18 yr−1 vs. 0.72±0.55 yr−1, p=0.014); (b) compensatory reduced volume referent bone formation rate (0.17±0.21 yr−1 vs. 0.71±0.54 yr−1, p=0.007), but unaltered mineral appositional rate; (c) neutral (–0.66±5.6 mm) vs. negative (–2.15±2.2 μm, p=0.013) bone balance per remodelling cycle; (d) higher trabecular bone volume (21.0±6.2% vs. 13.0±3.7%, p=0.01); (e) suppressed urinary excretion and serum levels of some of the biochemical markers of bone metabolism; and (f) significant reduction of circulating soluble interleukin‐6 receptor (IL‐6sR) (25.9±4.1 ng/ml vs. 32.1±6.6 ng/ml, p=0.04), and (g) a uniform tendency of lower serum and marrow plasma levels of IL‐6, IL‐1β, and TNFα. Thus oral pamidronate was absorbed in biologically active amounts, and reduced overall bone resorption and bone turnover without impairing osteoblastic bone formation. The observation that cytokine and cytokine receptor levels were reduced extends the possible and potential beneficial actions of bisphosphonates in multiple myeloma.
Effect of chlorambucil on bone mineral density in the course of chronic lymphoid leukemia
Authors: Leone J. , Vilque J.‐P. , Jolly D. , Pignon B. , Blaise A.‐M. , Pennaforte J.‐L , Eschard J.‐P. , Etienne J.‐C.
Published: July 2010

Abstract: The purpose of this work was to study the effects of chronic lymphoid leukemia (CLL) and its treatments on bone mineral density (BMD). Lumbar and femoral BMD was measured by X‐ray absorptiometry in 50 (32 M, 18 F, median age 65, range age: 47–87 yr) CLL patients. In order to gauge the respective effects of CLL and corticoids on bone mass, 31 CLL patients under treatment were compared with 31 controls on cortisone. Nineteen untreated patients with CLL were compared with controls devoid of osteopenia risk factor. There was no significant difference regarding lumbar and femoral BMD between the untreated patients with CLL and the healthy controls. An increase in lumbar and femoral BMD was noted in the treated CLL group compared with the controls on cortisone (lum BMD: 1.018 vs. 0.861 g/cm2, p = 6.10−4; fern BMD: 0.773 vs. 0.699 g/cm2, p = 0.037). This increase was observed only in patients who had received chlorambucil (lum BMD: 1.066 vs. 0.861 g/cm2, p=0.10−4; fern BMD: 0.806 vs. 0.699 g/cm2, p = 4.10−3), whereas there was no difference between the CLL patients treated without chlorambucil and the controls on cortisone. Multiple linear regression analysis confirmed the marked effect of chlorambucil (r = 0.3715, p < 10−3) on BMD increase in the course of CLL.
A new candidate mutation (N528S) within the von Willebrand factor propeptide identified in a Japanese patient with phenotype IIC of von Willebrand disease
Authors: Gaucher C. , Uno H. , Yamazaki T. , Mashiba H. , Mazurier C.
Published: July 2010

Abstract: Phenotype IIC of von Willebrand disease (vWD) is a subtype of type 2A vWD characterized by recessive inheritance and an impaired multimerization of von Willebrand factor (vWF) molecules beyond dimers. The 5 patients with phenotype IIC whose vWF gene defect has been characterized so far are either homozygous or double heterozygotes for mutations localized in exons 11, 12, 14 or 15. We report here the identification of a new candidate mutation in a previously described Japanese patient affected with phenotype IIC vWD. The propositus is homozygous for the A1833G nucleotide substitution, in exon 14 of vWF gene, responsible for the N528S mutation within the vWF propeptide. This finding is in agreement with the consanguineous origin of the propositus, whose parents are first cousins. Six patients' relatives who are asymptomatic were studied and found heterozygous for the N528S mutation. The screening of the whole vWF gene, either by SSCP or sequencing, did not reveal any other deleterious sequence alteration in the patient. Furthermore, the N528S nonconservative substitution identified is located in the vWF propeptide region, where the other phenotype IIC mutations described so far are clustered. The N528S candidate mutation characterized is, therefore, most probably responsible for the multimerization defect of vWF observed in this patient.
Hepatic and extrahepatic clearance of circulating human lactoferrin: an experimental study in rat
Authors: Peen Elisabeth , Johansson Anders , Engquist Markus , Skogh Thomas
Published: July 2010

Abstract: Lactoferrin, unlabelled or 125I‐labelled by 2 different methods, was given intravenously to rats. Blood, tissue and liver cell radioactivity was measured. Both of the radiolabelled preparations were eliminated by the liver, and some deposited extrahepatically. One preparation formed large aggregates – here 90% of the hepatic uptake occurred in the Kupffer cells. The other preparation, consisting mostly of protein monomers but also dimers/oligomers/microaggregates, was taken up by hepatocytes (63% of total liver uptake), liver endothelial cells (22%) and Kupffer cells (15%). On a per cell volume basis, lactoferrin uptake was much more efficient by nonparenchymal cells compared to hepatocytes, which explains why immunomorphological staining only revealed lactoferrin in the nonparenchymal liver cells. The study demonstrates that radio‐iodination of lactoferrin can affect its properties and handling, which may be important regarding contradictory reports on hepatic lactoferrin uptake. We conclude that both hepatocytes and nonparenchymal liver cells are involved in the blood clearance of lactoferrin, probably to a great extent owing to nonspecific mechanisms. Extrahepatic deposition and exposure (for instance on vessel walls/glomeruli) suggests that lactoferrin can be available to circulating anti‐lactoferrin autoantibodies in autoimmune disease.
Red cell folate in elderly patients with myelodysplastic syndrome
Authors: Cafolla A. , Dragoni F. , Girelli G. , Tosti M. E. , Monarca B. , Kendall R. , Scott C. S.
Published: July 2010

Abstract: During a 7‐month period a prospective study of 71 anaemic patients (29 males and 42 females) over the age of 50 was undertaken in order to identify patients with myelodysplastic syndrome (MDS). The mean values of mean corpuscular volume (MCV), serum ferritin, folate, vitamin B12 and red cell folate (RCF) of patients grouped according to the diagnosis were compared to those observed in age‐matched blood donors. Forty‐four of the 71 elderly patients showed macrocytic anaemia: 21 of them had gastric disease and the remaining 23 MDS. Two further patients with MDS showed microcytic anaemia. The 25 patients diagnosed with MDS were subclassified according to the FAB nomenclature: 9 had a refractory anaemia with excess of blasts and 16 refractory anaemia. The mean values of MCV, serum folate, ferritin, vitamin B12 and RCF were statistically different between patients with macrocytic anaemia due to gastric disease and patients with MDS. Among patients with MDS, the RCF level pathologically high was inversely correlated to the haemoglobin level (r=‐0.39; p<0.05). Thus the RCF and serum folate may represent useful parameters for the diagnosis of MDS in elderly anaemic patients.
Low rate of somatic hypermutations characterize progressive B‐cell lymphomas
Authors: Rosenquist Richard , Lindström Anita , Li Ai‐Hong , Roos Göran , Lindh Jack , Holmberg Dan
Published: July 2010

Abstract: Immunoglobulin heavy (IgH) chain gene rearrangements were characterized in 40 samples from 15 patients with B‐cell lymphomas at different time points during tumour progression. Using polymerase chain reaction (PCR) amplification and single strand conformation polymorphism (SSCP) analysis of variable heavy (VH) chain gene segments, we found that 6 cases displayed alterations in their IgH chain rearrangements at relapse. These alterations were mainly observed in follicular or transformed lymphomas, but no association to clinical features was found. Nucleotide sequence analysis revealed a low frequency of mutations in 3 cases, whereas 1 case displayed an extensive mutation rate in a compartment with transformed morphology at relapse. The mutations observed most probably resulted from somatic hypermutations. Further, the mutations were scattered randomly over the V11 gene segment and no significant bias favouring amino acid substitutions was observed in 3 cases, suggesting that the tumour cells had not been subjected to antigen‐driven selection. In 1 case, however, the mutation pattern indicated that the tumour cells had been affected by an antigen selection process. In the 2 remaining cases, the original VHDJH rearrangement could no longer be detected by VH gene family specific PCR at relapse, but using primers specific for the framework region 2 or 3 altered rearrangements were demonstrated, implying that mutations had been introduced in framework region 1. However, the majority of the tumour cell clones analysed were relatively stable during tumour progression, which make them eligible for analysis of minimal residual disease using the VH gene regions as molecular markers.