bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of two mutations in platelet glycoprotein (GP) Ibα in two Finnish Bernard–Soulier syndrome families
Authors: Koskela S. , Partanen J. , Salmi T. T. , Kekomäki R.
Published: July 2010

Abstract:  Bernard‐Soulier syndrome (BSS) is a rare hereditary bleeding disorder and macrothrombocytopenia which is caused by a defect in the platelet glycoprotein Ib/IX/V (GP Ib/IX/V) complex, the receptor for von Willebrand factor and thrombin. Here we report the molecular basis of the classical form of BSS in two unrelated Finnish patients, both with a life‐long history of severe bleeding. Flow cytometry and immunoblotting showed no expression of GP Ib/IX, GP Ibα, GP Ibβ or GP IX (less than 10%) in the patients' platelets. No expression of GP V (<10%) was observed in propositus 1, but a residual amount was found in propositus 2 (24%). DNA sequencing analysis revealed that propositus 1 was compound heterozygous for a two‐base‐pair deletion at Tyr505(TAT) and a point mutation Leul29(CTC)Pro(CCC) in the GP Ibα gene. Propositus 2 was homozygous for the Tyr505(TAT) deletion. The nine relatives who were heterozygous for either of the mutations also had low levels of GP Ibα (74–90%). Hence, Bernard‐Soulier patients homozygous or compound heterozygous for Tyr505(TAT) are severely affected. Interestingly, both mutations have independently been found in three other families in previous reports, suggesting their ancient age or mutational ‘hot spot’.
Selectivity of von Willebrand factor triplet bands towards heparin binding supports structural model
Authors: Fischer Bernhard E. , Thomas Kathy B. , Schlokat U. , Dorner F.
Published: July 2010

Abstract:  Human plasma‐derived von Willebrand factor (hp‐vWF) and recombinant von Willebrand factor (r‐vWF) have been fractionated by heparin affinity chromatography followed by multimer analysis using SDS‐agarose gel electrophoresis. Because heparin binding sites are contained in each vWF subunit, high molecular weight multimers of r‐vWF and hp‐vWF, respectively, were eluted with higher salt concentration, in comparison to r‐vWF and hp‐vWF molecules with a low degree of multimerization. Heparin affinity chromatography did not affect the multimer composition of r‐vWF. By contrast, faster migrating satellite bands and slower migrating satellite bands of hp‐vWF exhibited reduced and increased heparin affinity, respectively, compared to the intermediate band of the same triplet. Because heparin binding sites are localised in the N‐terminal domain of the hp‐vWF subunit, this result confirms a structural model of hp‐vWF (Fischer et al., Biochem. J. 1998;331:483–488) suggested recently, in which the slower migrating satellite bands have excess of one N‐terminal fragment and the faster migrating satellite bands lack one N‐terminal fragment, respectively, in comparison with the corresponding intermediate triplet band.
A comparative study of CD34+ cells, CD34+ subsets, colony forming cells and cobblestone area forming cells in cord blood and bone marrow allografts
Authors: Theilgaard‐Mönch Kim , Raaschou‐Jensen Klas , Heilmann Carsten , Andersen Helle , Bock Johannes , Russel Charlotte A. , Vindeløv Lars , Jacobsen Niels , Dickmeiss Ebbe
Published: July 2010

Abstract:  Cord blood (CB) has become an alternative source of hematopoietic progenitor cells (HPCs) for allogeneic transplantation. We have developed a new efficient protocol for CB collection. Using this method an average of 17.7 times 108 [range (6.8–29.6) × 108, n = 13] total nucleated cells (TNCs) were harvested. Based on recent Eurocord data, which have shown safe engraftment using a threshold dose of 0.37 times 108 CB TNCs/kg body weight (BW), we calculated that six out of thirteen CB grafts collected by this method were sufficient to engraft adults. The CB derived CD34+ population contained two‐fold higher numbers of committed HPCs (CFU‐GM, BFU‐E) and six‐fold higher numbers of pluripotent HPCs [CD34+/CD38− cells, wk 5 and wk 8 cobblestone area forming cells (CAFCs)] than the CD34+ population of BM. Extrapolation revealed that BM grafts providing the threshold dose for allogeneic transplantation of 2 times 108 TNCs/kg BW contained nearly 3 times more pluripotent HPCs than CB grafts providing the Eurocord threshold dose. The assessment of CD34+/CD38− cell numbers in CB grafts was highly reproducible and correlated well with the in vitro performance of pluripotent HPCs, i.e. numbers of CAFCs. We conclude that CB grafts providing high numbers of TNCs have the potential to engraft adults and that the enumeration of pluripotent HPCs by flow cytometry may be a useful tool to define the ultimate threshold dose for CB transplantation.
Prognostic implications of cytogenetic aberrations in diffuse large B‐cell lymphomas
Authors: Jerkeman M. , Johansson B. , Åkerman M. , Cavallin‐Ståhl E. , Kristoffersson U. , Mitelman F.
Published: July 2010

A single institution series of 81 consecutive, cytogenetically analyzed, diffuse large B‐cell lymphomas (DLBL), the majority of which treated with anthracycline‐containing combination chemotherapy, were reviewed retrospectively to investigate whether the karyotypic pattern or certain abnormalities correlate with survival. Clonal chromosome changes were found in 79 of the 81 cases. The prognostic impact of the following aberrations, all suggested in previous studies to be associated with either shorter or longer survival, were tested: 1q21–23 breakpoints, +2/dup(2p), +3/dup(3p), +5, +6, 6q21–25 breakpoints, monosomy 7/der(7p)/i(7q), trisomy 7, 14q11–12 breakpoints, monosomy 17/der(17p)/i(17q), trisomy 18, >4 marker chromosomes, >4 breakpoints, and ≥10 abnormalities. Univariate analysis showed that a breakpoint at 1q21–23 or trisomy 6 was associated with a shorter survival. However, when adjusted for age, stage, performance status and lactate dehydrogenase level, none of the cytogenetic aberrations had an independent prognostic value. Thus, the present investigation provides no support for any of the above‐mentioned abnormalities being of prognostic importance in DLBL.
In vitro effects of insulin‐like growth factor‐1 (IGF‐1) on proliferation and constitutive cytokine secretion by acute myelogenous leukemia blasts
Authors: Frostad S. , Bruserud Ø.
Published: July 2010

Abstract:  The effects of insulin‐like growth factor 1 (IGF‐1) on autonomous proliferation, cytokine‐dependent proliferation and constitutive cytokine secretion by acute myelogenous leukemia (AML) blasts were investigated using serum‐free in vitro conditions. IGF‐1 enhanced AML blast proliferation independent of the presence of other exogenous cytokines only for 2 of 21 patients, but for 10 additional patients IGF‐1 altered blast proliferation in the presence of certain exogenous cytokines or cytokine combinations. IGF‐1 had minor effects on AML blast cytokine secretion only for a subset of the patients (decreased levels for 1 patient, increased levels for 7 patients). Our in vitro observations indicate that IGF‐1 can modulate AML blast proliferation and/or cytokine secretion for a subset of patients.
Serum levels of soluble vascular cell adhesion molecule‐1 (sVCAM‐1) are elevated in advanced stages of non‐Hodgkin's lymphomas
Authors: Christiansen Ilse , Sundström Christer , Kälkner Karl‐Mikael , Bring Johan , Tötterman Thomas H.
Published: July 2010

Abstract:  The serum levels of soluble vascular cell adhesion molecule‐1 (sVCAM‐1) were measured in 116 patients with non‐Hodgkin's lymphomas (NHL) tested previously for soluble intercellular adhesion molecule‐1 (sICAM‐1). In contrast to Hodgkin's disease and chronic lymphocytic leukaemia, the sVCAM‐1 levels in NHL patients were not significantly different from the levels of healthy controls (n = 31). However, sVCAM‐1 was elevated in advanced stage disease, i.e. stages III + IV. Elevated serum levels of sVCAM‐1 were associated with significantly poorer disease‐free (p = 0.024) and overall (p = 0.02) survival. sVCAM‐1 correlated poorly with other known prognostic variables (LDH, sTK and β3m) and with sICAM‐1. None of the tested markers added prognostic information for disease‐free survival independently of Ann Arbor stage and B‐symptoms. The expression of VCAM‐1 and ICAM‐1 in tumour biopsies from 15 patients representing 7 different histologies were examined and compared with the serum levels of the soluble adhesion molecules. No correlation was found between the adhesion molecule expression by vascular endothelium and the corresponding serum levels.
Refractory anemia with excess of blasts: increased survival when treated with cyclophosphamide, methotrexate and 6‐mercaptopurine
Authors: Majluf‐Cruz Abraham , Hurtado Rafael , Vargas‐Vorackova Florencia , Labardini‐Méndez Juan
Published: July 2010

Abstract:  Owing to the lack of efficacious treatments for refractory anemia with an excess of blasts (RAEB), evaluation of other therapeutic strategies is necessary, especially in elderly patients. We report herein our experience with an oral triple drug regimen with cyclophosphamide 200 mg/m2 and methotrexate 20 mg/m2 once a week, and 6‐mercaptopurine 50 mg/m2 daily for the treatment of RAEB. Eighteen patients with a median age of 62 yr (range 17–80) received a triple drug regimen (TDR), and they were compared with 6 patients who received oxymetholone (2 mg/m2/d) and 9 who received supportive therapy only. Partial response was achieved in 45% of patients receiving TDR. In 77% of patients treated with TDR the number of bone marrow blasts decreased to <5%; however, they persisted with trilineage dyspoietic morphologic changes. Median survival for TDR was 23 months (range 1–96), which was longer than that for the other groups. A slight rise in liver enzymes was the only side effect of TDR. TDR seems to be a useful alternative in patients with RAEB, a finding to be confirmed in further prospective studies.
Long‐term follow‐up of autoimmune thrombocytopenic purpura (ATP) patients submitted to splenectomy
Authors: Mazzucconi Maria Gabriella , Arista Maria Cristina , Peraino Monica , Chistolini Antonio , Felici Carla , Francavilla Vittorio , Macale Elvira , Conti Laura , Gandolfo Giuseppe Maria
Published: July 2010

Abstract:  We studied the general outcome in 94 adult patients with autoimmune thrombocytopenic purpura (ATP) submitted to splenectomy. Of 84/94 patients who presented a complete or partial response 30 d after splenectomy, 16 (19%) showed one or more relapses. The clinical situation of the 81 patients still under observation is as follows: 13 unrensponsive, 60 completely or partially responsive, without relapses during the follow‐up, 8 completely or partially responsive after one or more relapses. No correlation was found between the favourable splenectomy outcome and age at splenectomy, the diagnosis‐splenectomy interval and initial response to corticosteroids. The probability of disease‐free survival is 83%, projected at 10 yr, while the overall survival is 93%, projected at 10 yr.

PAIgG levels of the normal subjects and of responding patients were found to be similar, while in the groups of non‐responding/relapsing patients, significantly higher values of PAIgG were detected, as compared to the control group.
Ifosfamide‐induced stomatocytosis and mesna‐induced echinocytosis: influence on biorheological properties of blood
Authors: Reinhart W. H. , Baerlocher G. M. , Cerny T. , Owen G. Rh. , Meiselman H. J. , Beer J. H.
Published: July 2010

Abstract:  Ifosfamide is an alkylating agent which has poorly understood toxic side effects such as encephalopathy. We hypothesized that ifosfamide and concomitantly applied mesna could have an influence on the flow properties of blood, and thus carried out an in vitro study. Whole blood was incubated in vitro with increasing concentrations of ifosfamide (0–50 mg/ml), mesna (0–20 mg/ml) and combinations thereof. Chloroacetaldehyde, a major metabolite of ifosfamide, was also studied (0–5 mmol/l). Ifosfamide led to a dose‐dependent stomatocytic shape transformation and mesna to an echinocytic shape transformation of erythrocytes. These shape changes were reversible upon removal of the causing agent. Both shape changes increased whole blood viscosity. Erythrocyte aggregation was decreased by both drugs at high concentration. Erythrocyte deformability, as measured with the transit time through 5‐μm pores, was decreased by mesna and remained unaffected by ifosfamide. These effects were seen at concentrations which may be reached in vivo at the infusion site of the drugs into a vein and in the urinary tract. We conclude that ifosfamide and mesna interact with the lipid bilayer of the cell membrane, which may contribute to the toxicity of the compounds.
Low‐dose subcutaneous interleukin‐2 in patients with minimal residual lymphoid neoplasm disease
Authors: González‐Barca E. , Grañena A. , Fernández‐Sevilla A. , Moreno V. , Salar A. , Rueda F. , García J.
Published: July 2010

Abstract:  Interleukin‐2 (IL‐2) is a cytokine that became available for clinical use with the development of recombinant DNA technology. Patients with resistant or relapsed lymphoid neoplasm have been treated with high‐dose IL‐2 with some responses. The aim of the present study is to determine whether there may be a biological justification for the use of low dose subcutaneous (s.c.) IL‐2 as maintenance therapy in patients with lymphoid neoplasm in complete remission with high risk of relapse. We treated 15 patients with sc IL‐2, 4.5 Million International Units (MIU) daily, 5 days per week for 12 consecutive weeks, in the outpatient clinic. This therapy was well tolerated and could be administered in an outpatient regimen. It increased the eosinophil count (p = 0.009), but the number of granulocytes, monocytes, T‐lymphocytes and B‐lymphocytes did not change. The number of natural killer (NK) cells increased from 11% to 35% of all lymphocytes during IL‐2 therapy (p = 0.0006). Effector lymphokine‐activated killer activity (eLAK) also increased from 6 × 10‐3 Lytic Units (LU)/ml to 80 × 10‐3 LU/ml (p = 0.02). All these changes reached a “plateau” after the 4th week of therapy. The increase in the number of NK cells correlated strongly with the increase in eLAK activity (r = 0.96, p <0.0001). Disease‐free survival was determined in 14 patients who completed the treatment and compared with historical controls. Patients treated with IL‐2 had the same relapse risk (median time to relapse 11.1 months, 95% confidence interval 5.5–16.6) as did controls (median time to relapse 9.7 months, 95% confidence interval 1–27.7) (p = 0.9). Low dose s.c. IL‐2 stimulated NK proliferation, which generated cytotoxic activity in vivo in patients with lymphoid neoplasms. However, these patients did not have a lower risk of disease relapse compared to historical controls.
Involvement of SHP‐1, a phosphotyrosine phosphatase, during myeloid cell differentiation in acute promyelocytic leukemia cell lines
Authors: Uesugi Yumiko , Fuse Ichiro , Toba Ken , Kishi Kenji , Furukawa Tatsuo , Koike Tadashi , Aizawa Yoshifusa
Published: July 2010

Abstract:  We investigated the modulation of the expression and phosphatase activity of SHP‐1 during granulocytic differentiation of human myeloid leukemia cell line, HL60 and t(15;17) positive APL cell line, HT93, in response to all‐trans retinoic acid (ATRA) or dimethylsulfoxide (DMSO). ATRA induced differentiation in both cell lines which was associated with marked growth inhibition and S‐phase reduction. On the other hand, DMSO induced it only in HL60 without obvious growth inhibition and S‐phase reduction. The expression and phosphatase activity of SHP‐1 were upregulated only when the 2 cell lines were differentiated to granulocytes. Furthermore, the changes were not dependent on the inducers or the growth inhibition. These findings suggest that SHP‐1 is involved in common myeloid differentiation, and that upregulation of SHP‐1 is not always related to myeloid cell growth.
Propionate and butyrate esters of camptothecin and 9‐nitrocamptothecin as antileukemia prodrugs in vitro
Authors: Han Z. , Cao Z. , Chatterjee D. , Wyche J. , Pantazis P.
Published: July 2010

Abstract:  Six camptothecin (CPT) alkyl esters and four 9‐nitrocamptothecin (9NC) alkyl esters were assayed for ability to inhibit proliferation and induce programmed cell death (apoptosis) in human leukemia HL‐60 and U‐937 cells, which exhibit differential sensitivity to CPT and 9NC. In general, CPT‐propionate and CPT‐butyrate demonstrated activities, while the other esters were practically inactive. Similarly, 9NC‐propionate and 9NC‐butyrate were active, while the other 9NC esters exhibited little or no activity. The biologically active esters required metabolic conversion (i.e., de‐esterification) to their parental compounds as demonstrated by the conversion of CPT‐propionate to CPT in mouse liver homogenate, and the topoisomerase I‐inhibition assay. In conclusion, the propionate and butyrate esters of CPT and 9NC are CPT and 9NC prodrugs, that can develop to important chemotherapeutic agents for the effective treatment of human leukemias and other malignancies.
Variant Bernard‐Soulier syndrome due to homozygous Asn45Ser mutation in the platelet glycoprotein (GP) IX in seven patients of five unrelated Finnish families
Authors: Koskela Satu , Javela Kaija , Jouppila Jussi , Juvonen Eeva , Nyblom Olle , Partanen Jukka , Kekomäki Riitta
Published: July 2010

Abstract:  Bernard‐Soulier syndrome (BSS), a rare bleeding disorder with macrothrombocytopenia, is caused by a defect of the platelet glycoprotein (GP) Ib/IX/V complex. Here we report a variant form of BSS in eleven patients of five unrelated families who originate from a particular area of Finland. The differential diagnosis from idiopathic thrombocytopenic purpura was difficult. Bleeding symptoms were epistaxis and haematomas debuting in childhood, but no spontaneous, severe bleeding episodes were reported. The platelet count varied from 43 to 81 × 109/l. Screening the entire GP Ibα, GP Ibβ, GP IX and GP V genes revealed a recurrent homozygous Asn45Ser mutation in GP IX in all probands. Flow cytometry showed markedly reduced expression of GP Ib (<10%), and only moderately reduced expression of GP IX (24–36%) and GP V (38–49%). The expression of subunits seemed to vary independently from the normal polymorphisms. Heterozygotes did not differ significantly from controls by their GP Ib/IX/V expression. Since the Asn45Ser mutation has also been reported in three other kindreds of northern and central European origin, this study reveals that instead of being a mutation hot spot, it may be ancient and scattered in Europe. Moderate, chronic thrombocytopenia should be carefully studied to diagnose variant BSS correctly from treatment resistant idiopathic thrombocytopenia.
Early establishment of hematopoietic chimerism following allogeneic peripheral blood stem cell transplantation in comparison with allogeneic bone marrow transplantation
Authors: Nakao Shinji , Zeng Weihua , Yamazaki Hirohito , Wang Honbo , Takami Akiyoshi , Sugimori Naomi , Miura Yuji , Shiobara Shintaro , Matsuda Tamotsu , Shinagawa Yuji , Harada Mine
Published: July 2010

Abstract:  To characterize the process of the establishment of complete chimerism after allogeneic peripheral blood stem cell transplantation (allo‐PBSCT), we determined the origin of leukocytes in peripheral blood (PB) obtained from 23 patients in the very early period after allo‐PBSCT using amplification of mini‐ or microsatellite regions of genomic DNA. Donor‐specific alleles were amplified from the PB obtained at day 8 post‐transplant for 19 allo‐PBSCT patients. Among the 19 patients, 12 showed only donor‐specific alleles (complete chimerism) while 7 did both donor and host‐specific alleles (mixed chimerism). Although donor specific alleles were amplified in 10 of 12 patients who received allogeneic bone marrow transplantation (allo‐BMT) similarly to allo‐PBSCT, all of these ten showed mixed chimerism. When the chimeric state was examined in PB samples obtained serially at 2–3‐day intervals post‐transplant, host‐specific alleles in allo‐PBSCT patients were not detectable in the PB much earlier than those in allo‐BMT patients. These findings indicate that the appearance of donor‐derived cells associated with the disappearance of host‐derived cells in the circulation occurs earlier after allo‐PBSCT as compared with allo‐BMT, leading to the rapid establishment of complete chimerism.
Survival in conventionally treated younger (<60 years) multiple myeloma patients: no improvement during two decades
Authors: Hjorth Martin , Holmberg Erik , Rödjer Stig , Turesson Ingemar , Westin Jan , Wislöff Finn ,   for the Nordic Myeloma Study Group(NMSG)
Published: July 2010

Abstract:  The patient registers of five prospective population based Nordic studies were reviewed for patients <60 yr. A total of 313 patients with symptomatic multiple myeloma were identified. Thirty‐nine of them were judged retrospectively to have been ineligible for intensive chemotherapy regimens. The remaining 274 patients were considered appropriate as a historical control group for comparison with patients treated with high‐dose chemotherapy and autologous stem cell support. Of these, 32 had been diagnosed during the period 1970–83, 101 during the period 1984–89 and 141 during the period 1990–92. The median age was 54 yr. Six percent were Durie/Salmon stage I, 38% stage II and 56% stage III. Melphalan‐prednisone was used for initial therapy in 87%. Median survival for all patients with symptomatic myeloma was found to be 41 months, and for those selected for the control group 44 months, with no noted differences between the aforementioned diagnostic periods. We conclude that the expected median survival is 44 months for myeloma patients <60 yr who may be considered for high‐dose therapy protocols. New developments in chemotherapy and supportive therapy, achieved during the two decades which preceded the use of high‐dose chemotherapy with stem cell rescue, have not changed the overall prognosis in multiple myeloma.
In aplastic anemia progenitor cells have a reduced sensitivity to the effects of growth factors
Authors: Novitzky Nicolas , Jacobs Peter
Published: July 2010

Abstract:  We have recently shown that in patients with aplastic anemia (AA) recovering following immunosuppressive therapy, the persistent reduction in the bone marrow clonogenic potential is unrelated to suppressive effects of the myeloid stroma and intrinsic to the hematopoietic progenitors. We examined the mechanisms of this defect by determining the response of the aplastic CD34+ clonogenic precursors to proliferative signals induced by hematopoietic growth factors and comparing their results with those of a control population. Light density bone marrow mononuclear cells were lymphocyte and monocyte depleted and enhanced for the CD34+ progenitors by immunomagnetic selection. Selected progenitors were then cultured in the mixed colony assay with incremental concentrations of combinations containing erythropoietin (Epo), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin‐3 (IL‐3) and c‐kit ligand. Bone marrow from aplastic patients had significantly fewer light density cells displaying the CD34 antigen (mean 0.65%, SD 0.35 vs. 1.62%, SD 1.4; p = 0.002). Dose response studies on aplastic CD34+ cells demonstrated that at low concentrations of Epo, IL‐3 and GM‐CSF, clonogenic growth was significantly impaired but achieved normal values at concentrations giving plateau growth in control cultures. However, for all colony types, responses to effective concentrations of c‐kit ligand corresponded with those of controls. These data suggest abnormalities at the receptor or signal transduction levels.
All‐trans retinoic acid at low concentration directly stimulates normal adult megakaryocytopoiesis in the presence of thrombopoietin or combined cytokines
Authors: Visani Giuseppe , Ottaviani Emanuela , Zauli Giorgio , Tosi Patrizia , Pellacani Annalisa , Isidori Alessandro , Pierpaoli Sabina , Tura Sante
Published: July 2010

Abstract:  In order to investigate the direct effects of retinoids on normal adult hematopoietic progenitors, purified CD34+ cells were seeded in serum‐free cultures in the presence of pharmacological (10‐6) M or physiological (10‐12) M concentrations of all‐trans retinoic acid (ATRA) and 9‐cis retinoic acid (9‐cis RA) plus combinations of specific cytokines. 10‐6 M ATRA and 9‐cis RA significantly decreased the number of granulomacrophagic, erythroid and megakaryocytic (CFU‐meg) progenitors. On the other hand, 10‐12 M ATRA significantly promoted the growth of CFU‐meg, in the presence either of thrombopoietin or of IL‐3 + GM‐CSF, and induced a reproducible stimulation of the immature CD34+ DR− subset. In conclusion, our findings suggest that retinoic acids probably play a direct role in normal adult hematopoietic development at both physiological and pharmacological concentrations. The stimulatory effect on megakaryocytopoiesis should be considered in the perspective of a potential use of low‐dose ATRA, combined with thrombopoietin or other cytokines, in pathological conditions where the megakaryocytic compartment is impaired and the stimulation of megakaryocytopoiesis is requested.
Low frequency of myeloid progenitor cells in chronic idiopathic neutropenia of adults may be related to increased production of TGF‐β1 by bone marrow stromal cells
Authors: Papadaki Helen A. , Giouremou Kalliopi , Eliopoulos George D.
Published: July 2010

Abstract:  Previous studies in our laboratory have shown that patients with chronic idiopathic neutropenia of adults (CINA) have increased serum levels of inflammatory cytokines including IL‐1β. Since IL‐1β may affect bone marrow stromal cell function, a study was designed to investigate the capacity of patients' stromal cells to produce adequate amounts of haemopoietic growth factors or excessive amounts of inhibitors of myelopoiesis in long‐term bone marrow cultures (LTBMCs). The study was carried out on 52 CINA patients and 19 normal controls. We found that CINA patients had significantly low numbers of marrow lineage‐specific CD34+ cells, including CFU‐GM and CD34+/CD33+ cells. Stromal cells from patients' LTBMCs failed to stimulate CFU‐GM colony formation by normal marrow cells in a manner comparable to that of stromal cells of controls. Patients' LTBMC supernatants had normal or increased amounts of G‐CSF. Detectable amounts of supernatant GM‐CSF were found in 35% of patients and 19% of controls. IL‐3 and MIP‐1α were not detected in any supernatant fluid. Moreover, supernatants from patients' LTBMCs had increased concentrations of IL‐6 and TGF‐β1, which strongly correlated with serum IL‐1β. About 82% of our patients had TGF‐β1 values higher than the upper limit of values found in the controls. Individual TGF‐β1 values inversely correlated with the number of circulating neutrophils and the frequency of marrow CD34+/CD33+ cells. We suggest that increased levels of serum IL‐1β, resulting from an underlying low‐grade chronic inflammatory process, may stimulate marrow stromal cells to produce both haemopoietic growth factors and inhibitors of myelopoiesis. Since steady‐state myelopoiesis results from a balance between negative‐ and positive‐acting cytokines, it seems very probable that the increased production of TGF‐β1 by bone marrow microenvironment in CINA patients may suppress myelopoiesis and contribute, to some extent, to the pathogenesis of neutropenia in affected subjects.
The influence of interferon‐α on the pharmacokinetics of cyclophosphamide and its 4‐hydroxy metabolite in patients with multiple myeloma
Authors: Hassan Moustapha , Nilsson Christina , Olsson Hanna , Lundin Jeanette , Österborg Anders
Published: July 2010

Abstract: The pharmacokinetics of cyclophosphamide (CP) and its cytotoxic metabolite 4‐hydroxycyclophosphamide (4‐OHCP) have been studied in multiple myeloma patients treated with the CIB (CP, interferon‐α (IFN‐α) and betamethasone) regimen. In the present investigation we aimed to determine whether exposure to CP and its cytotoxic metabolite 4‐OHCP is influenced by the concomitant administration of IFN‐α. Ten patients with previously untreated multiple myeloma entered the study. Each patient received two courses of CIB in randomized order. Interferon was administered either 2 h before the CP infusion in one course or 24 h after the CP infusion in the other course. A cyclophosphamide dose of 750–900 mg/m2was given as a 2 h constant infusion. Interferon‐α (10–15 times 106IE) was given subcutaneously. All patients received betamethasone 24 h after CP or later. The elimination of CP was described by monoexponential decay. The administration of IFN‐α before CP caused a decrease in CP clearance to 63% (P= 0.004), a 137% longer half‐life (P= 0.004) and a 137% higher peak plasma concentration (P= 0.006) compared to the results obtained when IFN‐α was administered 24 h after CP. The formation of 4‐OHCP was also affected by the administration of IFN‐α prior to CP, 45% less exposure to 4‐OHCP expressed as AUC (P= 0.002) and a 61% lower peak plasma concentration (P= 0.002) compared with that observed when IFN‐α was administered 24 h after CP. The administration of IFN‐α after CP resulted in a greater (45%,P= 0.02) decrease in leukocyte count compared with results when IFN‐α was given before CP. This study demonstrates that the administration of IFN‐α prior to CP significantly impairs pharmacokinetics of CP and 4‐OHCP. When IFN‐α was administered after CP, a higher exposure to the cytotoxic metabolite 4‐OHCP was observed and reflected by a significant decrease in leukocyte count compared to that when IFN‐α was given before CP. In conclusion, the time of administration of IFN‐α in relation to concomitant chemotherapy (CP) has to be considered to obtain a higher efficacy of IFN‐α/alkylating agent combining regimens for induction in multiple myeloma and related disorders.
Somatic mutations and intraclonal variations in the rearranged Vk genes of B‐non‐Hodgkin's lymphoma cell lines
Authors: Gabay C. , Ben‐Bassat H. , Schlesinger M. , Laskov R.
Published: July 2010

Abstract:  Three established Burkitt's lymphoma (BL) cell lines (Daudi, Raji and DG‐75) and three B‐non‐Hodgkin's lymphoma (B‐NHL) of other types (Pfeiffer, Farage and Toledo) were analyzed with respect to the presence of somatic point mutations in their rearranged immunoglobulin Vk genes. Two of the Vk sequences of BL and two of those of the B‐NHL were heavily mutated (up to 11%), when compared with their closest germline variable region counterparts (“clonal mutations”). Only one of the six cell lines contained an unmutated germline Vk sequence. The clonal mutations have features characteristic of the mutation machinery operating in the course of the T‐dependent immune response, such as a preference of mutations in purine bases, more transitions than transversions and targeting to CDR and to known “hotspot” motifs. Sequence variations among different Vk PCR clones isolated from each of the cell lines (“intraclonal mutations”) showed that the Vk of Toledo exhibited about 5‐fold higher mutation frequency (MF) than the background level of Taq polymerase error (˜0.12% mut/bp). Similarly, the MF of Vk of two of the BL cell lines was 3–4‐fold higher than the Taq polymerase misincorporation rate. In contrast, the mutation frequencies of the Vk of DG‐75, Farage and Pfeiffer did not significantly exceed the level of Taq polymerase error.

Our combined results show that 5 out of the 6 B‐cell lines studied originated from B‐cells that have already somatically mutated in vivo their rearranged Vk genes. Moreover, two of the Burkitt's and one of the B‐NHL cell lines exhibit intraclonal variation indicating that the process of somatic hypermutation continued following the neoplastic event, either in vivo or in culture. These results are in accord with the presumed origin of the majority of the BL and some types of the B‐NHL, from centrocytes or centroblasts of the germinal centers in which the process of somatic hypermutation is taking place.
Different expression of adhesion molecules on CD34+ cells in AML and B‐lineage ALL and their normal bone marrow counterparts
Authors: De Waele M. , Renmans W. , Jochmans K. , Schots R. , Lacor P. , Trullemans F. , Otten J. , Balduck N. , Gucht K. Vander , Van Camp B. , Van Riet I.
Published: July 2010

Abstract:  The expression of adhesion molecules on CD34+ cells in acute myeloid leukemia (AML) and B‐lineage acute lymphoblastic leukemia (B‐lineage ALL) was compared with that on the myeloid and B‐lymphoid CD34+ cells in normal bone marrow. Bone marrow aspirates of 10 patients with AML, 8 patients with B‐lineage ALL and of 6 healthy volunteers were examined. The phenotype of the CD34+ cells was determined with a double immunofluorescence method and flow cytometry. CD34+ cells in AML and B‐lineage ALL showed a lower expression of VLA‐2 and VLA‐3 and a higher expression of ICAM‐1 and LFA‐3 than their normal bone marrow counterparts. AML CD34+ cells had less L‐selectin but more VLA‐5 on their surface membrane than normal myeloid CD34+ cells. B‐lineage ALL CD34+ cells showed an overexpression of LFA‐3. In individual patients deficiencies or over‐expression of the β1 integrin chain, VLA‐4, PECAM‐1 or HCAM also occurred. An abnormal adhesive capacity of the leukemic cells may influence their proliferation, their localisation and apoptosis. An aberrant expression of adhesion molecules may be used for the detection of minimal residual leukemia in these patients.
Occurrence of cytokine receptors on different lymphoid leukaemic cells
Authors: Koubek Kristian , Starý Jan , Kumberová Andrea , Klamová Hana , Filipec Martin
Published: July 2010

Abstract: We have studied the expression of cytokine receptors CD25 (IL‐2Rα, 55 kD), CD116 (hGM‐CSFR, 145 kD), CD117 (CSFR, 145 kD), CD120a (TNFR, 55 kD), CD120b (TNFR, 75 kD), CD121a (IL‐1R, type I, 80 kD), CDw123 (IL‐3R), CD124 (IL‐4R, 140 kD), CD126 (IL‐6R, 80 kD), CD127 (IL‐7R, 75 kD), CDw128 (IL‐8R), CD130 (gp130 subunit), CDw131 (common β), CD132 (IL‐2Rγ), CD134 (OX40) and also CD95 (Fas antigen) on the lymphoid leukaemic cells. Cells from peripheral blood or bone marrow of 24 patients with disorders in lymphoid lineage mostly included acute lymphoid leukaemias (with a high leukocyte count and percentage of blasts) were analysed for the expression of surface membrane molecules by the immunofluorescence method evaluated by flow cytometry. The findings indicate that some monoclonal antibodies have a reactivity against cytokine receptors of pathological cells in individual cases, but with very variable qualitative and quantitative expression (number copies/cell). The lymphoid leukaemic cells demonstrate unique cytokine receptor profiles, which reveal the great diversity of immunophenotypes within the main functional characterisation of T and B lymphoproliferative malignancies.
In vitro effect of granulocyte‐colony stimulating factor and all‐trans retinoic acid on the expression of inflammatory cytokines and adhesion molecules in acute promyelocytic leukemic cells
Authors: Hsu Hui‐Chi , Tsai Wen‐Hui , Chen Pai‐Gene , Hsu Ming‐Ling , Ho Chi‐Kuan , Wang Sheng‐Yuan
Published: July 2010

Differentiation therapy with all‐trans retinoic acid (ATRA) represents a landmark approach in the treatment of acute promyelocytic leukemia (APL). However, a potentially fatal complication of retinoic acid (RA) syndrome occurs in about a quarter of patients and its pathophysiology is still unclear. In order to investigate whether or not the treatment with ATRA leads to increased elaboration of inflammatory cytokines and adhesion molecules by the APL cells, the expression of interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, IL‐8, L‐selectin and intercellular adhesion molecule‐1 (ICAM‐1) was examined in the APL cells after induction of differentiation with ATRA in the presence or absence of granulocyte‐colony stimulating factor (G‐CSF) or IL‐3 in the present study. Cytokine elaboration by the treated cells was detected using both Northern blotting and enzyme‐linked immunosorbent assay. Our results have shown that ATRA induces an increased expression of IL‐8, IL‐1β, TNF‐α and ICAM‐1 in APL cells, which can be amplified by the addition of G‐CSF. These data imply that the induction of inflammatory cytokines in APL cells may play an important role in the pathogenesis of RA syndrome. Furthermore, G‐CSF, through its potent differentiating activity, may increase the risk of such complications during ATRA treatment.
Serum beta‐2‐microglobulin, TNF‐α and interleukins in myeloproliferative disorders
Authors: Bourantas Konstantinos L. , Hatzimichael Eleftheria Chr. , Makis Alexandres Chr. , Chaidos Aristidis , Kapsali Eleni D. , Tsiara Stavroula , Mavridis Anestis
Published: July 2010

Whereas beta‐2‐microglobulin (β2M) has mainly been used as a prognostic factor in patients with lymphoproliferative disorders, some studies have reported the value of β2M in myeloproliferative disorders (MPD). In order to investigate a potential role in the pathogenesis of MPD and to find a possible value as indicators in monitoring the course of the disease, we measured β2M, TNF‐α, IL‐1α, IL‐1β, IL‐2, sIL‐2R, IL‐6 and IL‐10 in 55 patients with MPD, at diagnosis and during the course of the disease. In progressive disease and particularly when transformation to acute leukemia occurred, high levels of β2M, IL‐2 and sIL‐2R were found in all patients; the elevation was progressive, which suggests a potential prognostic usefulness in the individual patient.
Pathogenetic factors underlying juvenile deep vein thrombosis (DVT) in Indians
Authors: Saxena Renu , Mohanty Sujata , Srivastava Anurag , Choudhry Ved Prakash , Kotwal Jyoti
Published: July 2010

Abstract: The role of hereditary antithrombotic protein defects in juvenile deep vein thrombosis (DVT) was evaluated. Fifty six young patients (age <45 yr) with doppler‐proven DVT were investigated for the presence of resistance to activated protein C (APC‐R), lupus anticoagulant (LA), anticardiolipin antibodies and deficiencies of protein C, protein S, ATIII activities. Fifty nine normal healthy individuals served as controls. APC‐R was observed to be the commonest defect underlying the Indian DVT as seen in 39.2% of patients followed by elevated ACA (5.3%), PAI (2.8%), presence of LA (2.8%) and reduced ATIII levels (2.8%). None of the subjects had protein C or S deficiency. APC‐R was associated with ATIII deficiency in one case, and elevated ACA in two cases. In two subjects, APC‐R was associated with elevated PAI levels. Patients with more than one prothrombotic factor had a higher prevalence of pulmonary thromboembolism, suggesting that the thrombogenic potential of APC‐R is enhanced by the presence of coexisting hereditary or acquired prothrombotic defect.
Reduction of bioactive substances in stored donor blood: prestorage versus bedside leucofiltration
Authors: Hammer Janne H. , Mynster Tommie , Reimert Claus M. , Pedersen Anders N. , Nielsen Hans J.
Published: July 2010

Leucocyte filtration has been suggested to improve transfusion products. We studied the effect of prestorage versus bedside leucofiltration on reduction of bioactive substances and leucocyte content in donor blood. Forty‐five units of whole blood from healthy blood donors were studied. Of these units, 9 were stored under standard conditions for 35 d, 9 were leucofiltered after donation and then stored for 35 d, and 3×9 units were stored for 7, 21 and 35 d, respectively, before leucofiltration. Samples were collected from blood units immediately after donation, and before and after leucofiltration, and analysed by ELISA and RIA methods for extracellular content of myeloperoxidase (MPO), eosinophil cationic protein (ECP), histamine (HIS) and plasminogen activator inhibitor‐1 (PAI‐1). Leucocyte content was counted in all samples. In non‐filtered blood extracellular MPO, ECP, HIS and PAI‐1 were accumulated in a storage time‐dependent manner, while prestorage leucofiltration prevented this accumulation. Leucofiltration after storage for 7, 21 or 35 d did not significantly reduce the accumulated bioactive substances, which were similar to levels in non‐filtered blood stored for the same period of time. Prestorage and bedside leucofiltration on day 7 reduced the leucocyte content to less than 0.5×106/L, whereas the median content in blood stored for 21 or 35 d was only reduced to 32.0 and 52.2×106/L, respectively. Prestorage leucofiltration may thus be advantageous to bedside leucofiltration.
Effect of a 2‐hour infusion of 2‐chlorodeoxyadenosine in the treatment of refractory or previously untreated Waldenström's macroglobulinemia
Authors: Hellmann A. , Lewandowski K. , Zaucha J.M. , Bieniaszewska M. , Hałaburda K. , Robak T.
Published: July 2010

2‐Chlorodeoxyadenosine (2‐CdA) is a new purine analogue active in indolent lymphoid malignancies. In this retrospective study 22 patients with Waldenström's macroglobulinemia (MW) were treated with 2‐CdA given in 2‐h intravenous infusions. Nine of them were untreated and 13 relapsed or were refractory to previous therapeutic modalities with chlorambucil/prednisone (11 patients) or COP (2 patients). The patients were given 1–11 (median 4) courses of 2‐CdA at the dose of 0.14 mg/kg daily in 2‐h intravenous infusion for 5 consecutive days. The courses were repeated every 28–35 d. If severe myelosuppression or infection developed, 2‐CdA therapy was stopped until the haematological parameters increased. The effectiveness of the treatment was evaluated after the 3 cycles and after completion of therapy. None of the patients has achieved complete response (CR) after 3 courses of treatment and only one (4.5%) has obtained CR after 5 courses. Partial response (PR) was achieved in 8 (36.4%) patients, giving an overall response rate of 40.9%. Ten further patients (45.4%) responded to the treatment with less than 50% decrease in monoclonal protein (defined as stabilisation). There was no significant difference between the response rate in previously pretreated (38.5%) and untreated (44.4%) patients (p >0.05). Mean observed decrease in monoclonal protein was 41%. In the group of 9 patients responding to 2‐CdA treatment mean duration of response was 12 months (range 3–34). Myelosuppression was the most prominent side‐effect. Neutropenia was present in 17 (77.3%) and thrombocytopenia in 7 (31.8%) patients. In 6 patients myelosuppression was the reason for treatment discontinuation after 1 or 2 courses without significant therapeutic effect. Seven patients died, including 4 from the responding group and all three non‐responding patients. Treatment‐related thrombocytopenia and fatal haemorrhage was the course of death in 1 patient. In conclusion, the results of our study show that 2‐CdA given in 2‐h infusions is an effective agent in WM and may be given on an outpatient basis. However, myelosuppression is frequent and the drug must be administered with caution.
A cloned CD15s‐negative variant of HL60 cells is deficient in expression of FUT7 and does not adhere to cytokine‐stimulated endothelial cells
Authors: Weston Brent W. , Hiller Kara M. , Mayben John P. , Manousos George , Nelson Curt M. , Klein Marina B. , Goodman Jesse L.
Published: July 2010

The initial steps of leukocyte adhesion depend on selectin/ligand interactions. Surface ligands on leukocytes are often modified by addition of the sialyl Lewis x (CD15s) determinant. Biosynthesis of CD15s is dependent upon α(2,3)sialyltransferases and α(1,3)fucosyltransferases. We report the isolation of an HL60 cell line variant, HL60A2, that no longer expresses CD15s. HL60A2 cells do not adhere to cytokine‐stimulated endothelial cells. Enzymatic assays reveal that this cell line has normal α(2,3)sialyltransferase activity but is deficient in the α(1,3)fucosyltransferase responsible for biosynthesis of CD15s (FUT7). The fucosyltransferase that constructs the non‐sialylated antigen, Lewis x (CD15), is expressed at high levels (FUT4). Transcript analyses show that FUT7 and FUT4 are inversely expressed in HL60 and variant cell lines. HL60A2 cells provide a tool to study the regulation of selectin ligands and corresponding human fucosyltransferase genes.
Effects of hTNFα expression in T cells on haematopoiesis in transgenic mice
Authors: Glosli H. , Veiby O. P. , Fjerdingstad H. , Mehlum A. , Probert L. , Kollias G. , Gjernes E. , Prydz H.
Published: July 2010

Abstract: A transgenic line of mice carrying one copy of the hTNFα gene under the control of its own promoter and the CD2 locus control region has been analysed for the effects of TNFα on haematopoiesis. A low level constitutive expression of hTNFα in lymphoid tissue was observed. Human TNFα binds to and activates the murine p55 receptor, but not the p75 receptor. This implies that the observed effects of hTNFα in mice were mediated only through the p55 receptor. Various lymphoid tissues were depleted of lymphocytes, especially thymus, spleen and peripheral blood. Effects on thymus development were detected already at 3 wk of age, more general effects on haematopoiesis were evident by 5 wk: a drop in total blood leukocytes, mainly due to a 67% decline in lymphocytes. At 16 wk the mice had developed anaemia, whereas platelets, neutrophils and monocytes had increased. The fall in lymphocytes was due to lowered levels of T cells as well as B cells. The cause of the shortened lifespan of the transgenic mice was probably not the haematological effects of hTNFα directly. Absence of trophic factors supplied by the normal T cell population remains possible.
A longitudinal study of serum ferritin in 319 adolescent Danish boys and girls examined in 1986 and 1992
Authors: Milman Nils , Byg Keld‐Erik , Backer Vibeke , Ulrik Charlotte , Graudal Niels
Published: July 2010

Abstract: This study examined trends in iron status in adolescents. Serum ferritin was measured in 1986 and 1992 in 319 Danes (161 males) stratified into 5 groups: I. median age 9 yr in 1986 vs. 15 yr in 1992; II. 11 vs. 17 yr; III. 13 vs. 19 yr; IV. 15 vs. 21 yr; V. 17 vs. 23 yr. Males in group I demonstrated no change in ferritin or estimated iron stores in mg/kg; groups II‐V displayed an increase in iron status parameters. All groups showed an increase in estimated total iron stores. Changes in iron status parameters were inversely correlated with height velocity in group III, and positively correlated with height velocity in group V. Females in age groups I and II demonstrated a fall in ferritin and estimated iron stores in mg/kg in association with menarche; values were unchanged in groups III and IV, and increased in group V. All groups showed an increase in estimated total iron stores. Changes in iron status parameters were inversely correlated with height velocity in groups I and II. In conclusion, ferritin levels in adolescents display great variation during growth spurt and at menarche. Changes in ferritin showed no consistent association with growth velocity. In both genders, estimated total iron stores increased with age.