bluuebunny/crossref_metadata_2025 huggingface dataset:
Adhesion molecule CD11a/CD18‐deficient Burkitt's lymphoma cells lack the transcript for the β, but not the α, integrin subunit
Authors: Neira M. , Rincon J. , Arias H. , Law S. K. A. , Patarroyo M.
Published: July 2010

Abstract:  Adhesion to cells and matrices participates in the regulation of lymphocyte proliferation, maturation and tissue localization. Consequently, abnormal patterns of adhesion molecule expression may contribute to the pathophysiology of lymphoprohferative disorders. Integrins are major cell‐surface adhesive proteins composed by a and β subunits. In contrast to normal lymphocytes, Burkitt's lymphoma (BL) cells lack the β2 integrin CD11a/CD18. To study the molecular mechanism underlying this deficiency, presence of the transcript for each subunit was analysed by Northern blotting in group I BL lines (BL biopsy‐like) and, for comparison, Epstein‐Barr virus (EBV)‐transformed lymphoblastoid cell lines (LCLs). While transcripts for both CD11a (α subunit) and CD18 (β subunit) were readily detected in LCLs, BL lines contained the transcript for the α subunit only. Treatment of BL cells with phorbol ester for 72 h induced expression of the β subunit mRNA and the CD11a and CD18 antigens on the cell surface. The results indicate that the CD11a/CD18 deficiency of BL is due to absence of the β subunit transcript and that this defect is restored by stimulation of the cells.
2‐Chlorodeoxyadenosine in the treatment of relapsed/refractory chronic lymphoproliferative disorders
Authors: Rondelli D. , Lauria F. , Zinzani P. L. , Raspadori D. , Ventura M. A. , Galieni P. , Birtolo S. , Forconi F. , Algeri R. , Tura S.
Published: July 2010

Abstract:  2‐Chlorodeoxyadenosine (2‐CdA) is a purine analog with cytotoxic activity on both resting and cycling lymphocytes which has been used as salvage therapy in advanced/resistant chronic lymphoproliferative disorders. In our study 39 patients (19 B‐CLL, 5 B‐PLL, 9 low‐grade B‐NHL, 5 CTCL and 1 high‐grade T‐NHL) who relapsed or became resistant after 1–4 chemotherapy regimens were treated with 2‐CdA 6 mg/m2 per day by 2 h infusion for 5 d every 28 d. The overall clinical response rate, including complete remission (CR) and partial remission (PR), was 66%. Two of 19 (10%) B‐CLL patients achieved a CR lasting 9 months, while 11/19 B‐CLL (58%) and 4/5 B‐PLL (3 B‐PLL/B‐CLL and 1 B‐PLL) (80%) achieved a PR. Interestingly, 5 of 6 patients who had been previously treated with fludarabine obtained a clinical response (2 CR and 3 PR). One of 9 (11%) low‐grade B‐NHL patients achieved a CR and relapsed after 26 months, and 5/9 (55%) achieved a PR. One of 5 (20%) CTCL achieved a CR lasting 32 months, while 2/5 (40%) achieved a PR. The overall mean duration of PR was 7.4 months and no differences were observed among different groups of patients. Toxicity was acceptable, as only a transient severe hematological impairment was observed in 20% of the patients while nonhematological toxicity was not documented. Two patients died because of bacterial pneumonia, 1 of meningitis due to Listeria and 9 from progression of the disease. In conclusion, treatment with 2‐CdA in heavily pretreated patients with chronic lymphoproliferative disorders is well tolerated and obtains high response rates, even in patients relapsed after treatment with fludarabine.
Elevated levels of soluble thrombomodulin in plasma from children with Arg 506 to Gln mutation in the factor V gene
Authors: Nowak‐GÖttl U. , Vielhaber H.
Published: July 2010

Abstract:  To find out to what extent the Arg506 to Gln mutation in the factor V gene affects the defence system against thromboemboüsm we investigated soluble thrombomodulin, protein C, protein S along with thrombin generation and d‐dimer formation in 188 children. Children with the Arg506 to Gln mutation in the factor V gene (n = 48) showed significantly elevated thrombomodulin concentrations compared to nonaffected brothers and sisters (n = 50; p = 0.001) and age‐matched healthy controls (n = 90; p < 0.0001). In addition, thrombin generation and d‐dimer formation were significantly elevated in children with the mutation. In contrast, protein C and total protein S antigen levels were no different in the populations tested. Thus, with respect to thrombomodulin being a potent inhibitor of coagulation activation, the present data might be interpreted as a counter‐regulatory mechanism in infants and children with the Arg506 to Gln mutation in the factor V gene, maintaining the coagulation balance. The role of TM and other proteins involved in the coagulation balance in children and adults homozygous for the Arg506 to Gln mutation in the factor V gene remains to be clarified.
Ultrastructural localization of Charcot – Leyden crystal protein in human eosinophils and basophils
Authors: Calafat J. , Janssen H. , Knol E. F. , Weller P. F. , Egesten A.
Published: July 2010

Abstract:  The Charcot–Leyden crystal (CLC) protein with lysophospholipase activity and carbohydrate‐binding properties is a characteristic constituent of eosinophils and basophils. We investigated its subcellular distribution using immunoelectron microscopy. Eosinophil progenitors, mature eosinophils and basophils all contained CLC protein in their cytosol and in the euchromatin of the nucleus. A minor population of granules in eosinophils, increasing in number with maturation, and a more abundant granule‐population in basophils, were found to contain CLC protein. Double‐labeling experiments showed, in eosinophils, that CLC protein‐containing granules contain also eosinophil peroxidase, a characteristic specific granule protein. This suggests a relationship between the CLC protein‐containing organelle and the specific granule. In basophils both the CLC protein positive and the negative granules showed the same characteristic particulate‐like structure of the granular matrix and both share the same membrane marker CD63. In nasal polyps, macrophages were observed phagocytosing necrotic eosinophils. In these macrophages CLC protein‐containing vesicles were observed, probably representing late endosomes. The dual (cytosolic/nuclear and granular) localization of CLC protein suggests that this protein enters both a secretory and a nonsecretory pathway during its biosynthesis, indicating functional roles for this protein both within the cell and extracellularly.
Venous thrombosis: factor V G1691A genotyping related to APC resistance as measured by 2 methods
Authors: Alderborn Anders , Siegbahn Agneta , Wadelius Claes
Published: July 2010

Abstract:  Blood samples were collected from consecutive unrelated patients with venous thrombosis. The patients originate from the middle part of Sweden. We investigated the presence of the reported point mutation at nt 1691 of factor V which renders the protein resistant to cleavage by activated protein C (APC). Thirty‐seven per cent of the patients were heterozygote carriers, and 4.5% were homozygotes for a mutated factor V gene. In addition, resistance to coagulation induced by APC was measured, both by the conventional APTT assay and by a modified APTT assay which has been reported to have an increased resolution. Compared to a control group of healthy people, the mean value was significantly lower in the patient group. A strong correlation between low APC ratio and presence of the factor V mutation was found. By using the modified method, a complete resolution of carriers of the factor V mutation and people with normal factor V alleles was found. However, there was still an overlap between heterozygote carriers and people homozygous for the mutation. The modified method was also found useful in patients treated with warfarin. Among 40 healthy blood donors 7% were found to be heterozygous.
Outcome of a multicenter treatment program including autologous or allogeneic bone marrow transplantation for de novo acute myeloid leukemia
Authors: Wahlin Anders , Brinch Lorentz , Hörnsten Per , Evensen Stein A. , Öberg Gunnar , Simonsson Bengt , Hedenus Michael
Published: July 2010

Abstract:  The results of an intensive treatment program for patients 16–60 yr of age with de novo acute myeloid leukemia are presented. The patients were given conventional induction treatment with daunorubicin and cytarabine. Patients not entering complete remission (CR) after 1 course of daunorubicin/cytarabine were given 1 course of amsacrine/etoposide/cytarabine. Those entering complete remission received 3 consolidation courses using mitoxantrone, etoposide, amsacrine and cytarabine. One hundred and eighteen patients were enrolled. Complete remission was attained after 1–2 courses in 90 patients (76%). Another 6 patients reached CR after 3–4 induction courses for a total CR rate of 81%. If feasible, patients were offered either allogeneic or unpurged autologous bone marrow transplantation. Twenty‐four patients underwent allogeneic bone marrow transplantation; 15 in first remission, 8 in second remission, 1 in early relapse. Thirty patients below 56 yr of age underwent autologous bone marrow transplantation in first remission. The overall probability of survival at 4 yr was 34%, and for patients below 40 yr of age 50%. Leukemia‐free survival was 35% for the whole cohort of patients; 52% for patients below 40 yr of age. Patients undergoing allogeneic or autologous bone marrow transplantation in first remission had an overall survival of 86% and 47%, respectively, while the probability of leukemia‐free survival in these groups was 87% vs. 40% at 4 yr. The CR rate and long‐term results of this intensive treatment program compare favorably with other recent studies using intensive consolidation with allogeneic or autologous bone marrow transplantation or high dose cytarabine.
Differences in cell lineage involvement between MDS‐AML and de novo AML studied by fluorescence in situ hybridization in combination with morphology
Authors: Bernell Per , Arvidsson Ingrid , Hast Robert , Jacobsson Björn , Stenke Leif
Published: July 2010

Abstract:  We have employed fluorescence in situ hybridization (FISH) in combination with standard morphology (MGG/FISH) to identify the clonal involvement of different bone marrow cell lineages in 20 AML patients (14 MDS‐AML, 6 de novo AML). Even though the number of cells belonging to the abnormal clone varied between individual cases, the percentage of clonal blasts was similar in MDS‐AML and de novo AML patients. The erythropoietic cells appeared to be part of the abnormal clone in 13 of 14 patients with MDS‐AML, but only in 1 of 6 with de novo AML. Similarly, clonal granulocytes were detected in 13 of 14 patients with MDS‐AML, compared to 2 of 6 with de novo AML. Lymphocytes consistently displayed normal, diploid karyotype. The results suggest that it is possible to distinguish between MDS‐AML and de novo AML by the use of MGG/FISH; in de novo AML the abnormal chromosomal clone is generally confined to the immature myeloid cells, while in MDS‐AML mature granulocytes and erythroid cells are of clonal origin. It is, however, not possible to conclude that MDS‐AML is a “multipotent” type of leukaemia, since it cannot be ruled out that the chromosomally aberrant erythroid cells and granulocytes represent surviving cells from the original MDS clone
Recombinant human erythropoietin in the treatment of cancer‐related anaemia
Authors: Kasper C. , Terhaar A. , Fosså A. , Welt A. , Seeber S. , Nowrousian M. R.
Published: July 2010

Abstract:  The efficacy and safety of recombinant human erythropoietin (rhEPO) were tested when given subcutaneously (s.c.) in an escalating dose of 2000–10,000 units (U) daily in 60 patients with cancer‐related anaemia (CRA). A positive response, defined as an increase in haemoglobin more than 2 g/dl and independence of blood transfusions, was observed in 23 of 48 evaluable patients (48%) within a median of 8 wk. In detail, rhEPO corrected anaemia in 11 of 14 patients (79%) with malignant lymphoma, in 8 of 15 patients (53%) with multiple myeloma and in 4 of 10 patients (40%) with a solid tumour. The median dose of rhEPO in successful cases was 5000 U daily. Four patients with agnogenic myeloid metaplasia and 5 with myelodysplastic disorder failed to respond to rhEPO. No patient had any severe side effects. Pretreatment serum erythropoietin levels appeared to be a weak predictor for response to rhEPO treatment. In conclusion, rhEPO seems to be safe and effective in correcting CRA in certain groups of patients.
Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c‐kit protein
Authors: Sonoda Yoshiaki , Kimura Takafumi , Sakabe Hideaki , Tanimukai Shigeatsu , Ohmizono Yoshikazu , Nakagawa Shuichi , Yokota Shouhei , Lyman Stewart D. , Abe Tatsuo
Published: July 2010

Abstract:  We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34+ blood progenitors which express different levels of c‐kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU–G) formation in the presence of granulocyte‐colony stimulating factor (G–CSF) by peripheral blood (PB)‐derived CD34+c‐kit− cells which contained a large number of CFU–G. In addition, FL could synergistically increase the number of CFU–G supported by a combination of interleukin (IL)‐3 and G–CSF, as did SCF. As we reported previously, SCF showed a significant burst‐promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB‐derived CD34+c‐kithigh cells in which erythroid‐burst (BFU‐E) was highly enriched. However, FL could synergize with IL‐3 or GM–CSF in support of erythrocyte‐containing mixed (E‐Mix) colony by PB‐derived CD34+c‐kithigh or low cells in the presence of Epo. Replating of E‐Mix colonies derived from CD34+c‐kithigh cells supported by IL‐3+Epo+SCF yielded more secondary colonies than those supported by IL‐3+Epo or IL‐3+Epo+FL. When PB‐derived CD34+c‐kitlow cells which represent a more immature population than CD34+c‐kithigh cells were used as the target, number of secondary colonies supported by IL‐3+Epo, IL‐3+Epo+SCF or IL‐3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL‐3+Epo+FL than in that containing IL‐3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.
Treatment of the anemia of aplastic anemia patients with recombinant human erythropoietin in combination with granulocyte colony ‐ stimulating factor: a multicenter randomized controlled study
Authors: Bessho Masami , Hirashima Kunitake , Asano Shigetaka , Ikeda Yasuo , Ogawa Nobuya , Tomonaga Masao , Toyama Keisuke , Nakahata Tatsutoshi , Nomura Takeo , Mizoguchi Hideaki , Yoshida Yataro , Niitsu Yoshiro , Kohgo Yutaka ,   Multicenter Study Group
Published: July 2010

Abstract:  A multicenter randomized controlled study was undertaken in order to determine whether epoetin beta (EPO) ameliorates the anemia in aplastic anemia (AA) patients treated with granulocyte colony‐stimulating factor (G–CSF). Enrolled patients were randomized into 3 groups: group C receiving G–CSF alone as the control; group L receiving G–CSF and 200 IU/kg of EPO; group H receiving G–CSF and 400 IU/kg of EPO. Throughout the study, the dose and the administration interval of G–CSF were adjusted to maintain neutrophil counts between 1000 and 5000 μl. EPO was administered subcutaneously for 12 wk as the first step in treatment and when favorable effects were observed over this period, treatment was continued for another 12 wk as the second step in treatment. Significant erythroid responses were defined as increases in untransfused hemoglobin values >1.0 g/dl or >50% decreases in RBC transfusion requirements over the treatment period. Of 131 patients enrolled, 88 patients allocated to groups L and H were evaluated for toxicity to EPO and 110 were evaluated for erythroid responses. Four of the 31 patients (12.9%) in group C, 6 of the 41 patients (14.6%) of group L, and 14 of the 38 patients (36.8%) of group H showed erythroid responses in the first step in treatment. The erythroid responses of group H were significantly higher than those of the other 2 groups (p<0.05). The significant effects of EPO were due to erythroid responses in non‐severe AA. Responding patients were significantly different from non‐responders with regard to disease severity, hemoglobin concentration, reticulocyte count, serum endogenous erythropoietin levels and serum transferrin receptors; non‐severe AA patients were more likely to respond to EPO, and responding patients had lower serum EPO and higher hemoglobin concentration, reticulocyte count and serum transferrin receptors than non‐responders. The response rate increased in the second step in treatment, suggesting that long‐term treatment improved the efficacy of EPO. No serious side‐effects were observed. From these results, we conclude that EPO given in combination with G–CSF is a safe and effective alternative for the treatment of anemia of a subset of AA patients.
Leucocyte and platelet‐derived bioactive substances in stored blood: effect of prestorage leucocyte filtration
Authors: Nielsen H. J. , Skov F. , Dybkjær E. , Reimert C. M. , Pedersen A. N. , Brünner N. , Skov P. S.
Published: July 2010

Abstract:  Adverse reactions to transfusion of allogeneic blood may depend on content of leucocytes and platelets and on storage‐time of the erythrocyte suspensions. Therefore, we studied the efficacy of prestorage leucocyte reduction by filtration on total content and extracellular accumulation of histamine, eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO), plasminogen activator inhibitor type‐1 (PAI‐1) and interleukin‐6 (IL‐6) in samples obtained from 5 units of SAGM blood, 7 units of plasma‐reduced whole‐blood and 6 units of whole‐blood before and after filtration, respectively. In addition, we analysed supernatants from the same units after storage at +4°C for 0, 21 and 35 d, respectively. The filtration was performed at room temperature within 2–4 h after donation. The substances were analysed by ELISA and RIA methods and we also analysed the donor plasma levels of the same bioactive substances. The total content of histamine, ECP, EPX, and MPO were 10–70‐fold higher in all unfiltered erythrocyte products compared to donor plasma concentrations, while PAI‐1 content was 15–20‐fold higher only in plasma‐reduced whole‐blood and whole‐blood. Prestorage leucocyte filtration significantly reduced the total histamine, ECP, EPX, MPO and PAI‐1 content to levels similar to donor plasma levels in plasma‐reduced whole‐blood and whole‐blood, while PAI‐1 was still low in filtered SAGM blood. In addition, the levels of extracellular bioactive substances at d 0 after donation and filtration were within the range of concentrations in donor plasma, and there was no time‐dependent accumulation during storage for 35 d at +4°C. IL‐6 was not detected in either plasma or samples obtained from the blood bags. These results suggest prestorage leucocyte filtration to deplete leucocyte contents to levels, which prevent the previously shown time‐dependent accumulation of leucocyte derived bioactive substances in various erythrocyte suspensions. In addition, the PAI‐1 results suggest leucocyte filters to reduce the obligatory platelet content in whole‐blood products.
The platelet proaggregating and potentiating effects of unfractionated heparin, low molecular weight heparin and heparinoid in intensive care patients and healthy controls
Authors: Burgess J. K. , Chong B. H.
Published: July 2010

Abstract:  Heparin binds to platelets and can cause platelet proaggregating and potentiating effects, possibly causing thrombocytopenia, particularly in patients in intensive care with hyperaggregable platelets. In this study we compared the platelet proaggregating and potentiating effects of unfractionated heparin (UH), 2 low molecular weight (LMW) heparins, enoxaparin and dalteparin, and a heparinoid, danaparoid sodium (orgaran), to platelets of an ICU patient population and a normal control group. In both populations UH caused platelet aggregation in a dose‐dependent manner. This occurred in the therapeutic range of the drug, with as little as 0.5 U/ml UH. The LMW heparins caused less and the heparinoid least platelet aggregation. Generally, the aggregation observed in ICU patients was greater than in the normal population. The potentiating effects of the 4 drugs in association with physiological agonists was examined. Similar patterns of potentiation were observed in both populations, with UH causing significant enhancement of platelet aggregation, the LMW heparins intermediate and heparinoid least enhancement. There was substantial variability in the individuals' platelets' reactions to the drugs, in particular to UH. Our findings suggest that UH has the greatest effect, the low molecular weight heparins an intermediate effect and the heparinoid the least propensity to cause platelet activation.
In vitro improvement of chlorambucil‐induced cytotoxicity by deflazacort and 6‐methylprednisolone in B‐cell chronic lymphocytic leukaemia
Authors: Morabito F. , Callea I. , Irrera G. , Cartisano G. , Dattola A. , Rodinò A. , Brugiatelli M.
Published: July 2010

Abstract:  We examined whether CLL cell chemosensitivity to in vitro exposure to chlorambucil (CLB) might be improved by the presence of deflazacort (DFZ) in comparison to 6‐methylprednisolone (PDN). The PDN lethal dose (LD)50 values were low in 5 samples, intermediate in 4 and high in 7. Low, intermediate and high DFZ‐LD50 values were detected in 3, 2 and 11 samples, respectively. The CBL‐LD50 mean values were significantly reduced at all PDN and at the 4 highest DFZ concentrations. However, a dose‐response effect was seen only in the DFZ group. Both CLB‐DFZ and CLB‐PDN interactions were analysed in 16 samples at 25 different dose‐combinations, resulting in 400 comparisons between expected and observed leukaemic cell survival (LCS) values for each group. In particular, 45.75% and 40% dose combinations were synergistic in CLB‐DFZ and CLB‐PDN groups, respectively. A relatively higher number of antagonistic interactions were observed among CLB‐PDN dose combinations, while analogous number of additive interactions were detected. At concentrations of CLB × 1 μg/ml the phenomenon of synergism, regardless of the steroid concentration, did occur more frequently. On the other hand, a more elevated number of antagonistic interactions were counted at CLB 100 μg/ml. In conclusion, both DFZ and PDN synergize in vitro with CLB, especially at low concentrations of the alkylating agent.
Evidence for a NO synthase in porcine platelets which is stimulated during activation/aggregation
Authors: Berkels R. , Bertsch A. , Zuther T. , Dhein S. , Stockklauser K. , Rösen P. , Rösen R.
Published: July 2010

Abstract: We tried to characterize the porcine platelet nitric oxide (NO) synthase and its L‐arginine (L‐arg)/NO metabolism. Using RT‐PCR we could show a constitutive endothelial NOS (ecNOS) and an inducible NOS (iNOS) similar mRNA in platelets. The NOS protein could be evidenced by an ecNOS specific antibody which also bound in platelets. This finding could be confirmed by Western blot showing an ecNOS in the membrane but not the cytosolic fraction; iNOS protein could not be detected. Using NADPH‐diaphorase staining we could show NO synthase in preactivated platelets but not in resting platelets, indicating that the platelet NOS may be activated during platelet activation/aggregation. Porcine L‐arg plasma levels (9.31 × 10–5mol/l ± 10%) could be shown to be in the same range as human plasma levels. Moreover, we could show that the NO precursor L‐arg and hydroxy‐L‐arginine (OHarg) concentration dependently inhibited collagen induced platelet aggregation. Summarizing these results confirm the existence of and further characterize porcine platelet NO synthases.
Clinical results of recombinant erythropoietin in transfusion‐dependent patients with refractory multiple myeloma: role of cytokines and monitoring of erythropoiesis
Authors: Musto Pellegrino , Falcone Antonietta , D'Arena Giovanni , Scalzulli Potito Rosario , Matera Rosella , Minervini Maria Marta , Lombardi Gina Francesca , Modoni Sergio , Longo Antonio , Carotenuto Mario
Published: July 2010

Abstract:  Recombinant erythropoietin (r‐EPO) was administered to 37 patients with advanced, transfusion‐dependent and chemo‐resistant multiple myeloma (MM), at the fixed dose of 10,000/U s.c, 3 times a week, for 2 months. Thirteen patients (35.1%) achieved a significant response in terms of complete abolition of red cell transfusions. Factors significantly predictive of response were: a) inappropriate production of endogenous EPO, as expressed by a reduced observed/predicted ratio; b) presence of a consistant number of circulating erythroid precursors BFU–E; c) low serum levels of tumor necrosis factor (TNF) and interleukin‐1 (IL‐1), cytokines with inhibitory activity on erythropoiesis; d) a single line of previously received chemotherapy. Renal failure, bone marrow plasma cell infiltration, serum levels of IL‐6 and other main clinical and laboratory parameters did not affect significantly the response to r‐EPO. High fluorescence reticulocytes (HFR) and soluble transferrin receptor (sTfR) values were useful to detect an early stimulation of erythropoiesis in responders, while a high percentage of circulating hypochromic erythrocytes (HE), as assessed by an automated counter, identified those patients developing functional iron deficiency during r‐EPO treatment. We conclude that about one‐third of severely anemic patients with advanced MM, unresponsive to chemotherapy, may benefit by r‐EPO therapy. The clinical management of these patients can be accomplished using non‐invasive parameters, such as sTfR, HFR and HE.
Maximal γ‐globin expression in the compound heterozygous state for –175 Gγ HPFH and β°39 nonsense thalassaemia: a case study
Authors: Pistidda P. , Frogheri L. , Guiso L. , Manca L. , Dore F. , Mura L. , Longinotti M.
Published: July 2010

The –175 (T→C) Gγ hereditary persistence of fetal haemoglobin is a very rare promoter mutation occurring in Caucasians as well as in African‐Americans. Heterozygotes for this non‐deletional HPFH show 20% HbF, mostly of Gγ type. We describe here a healthy Sardinian man who coinherited –175 (T→C) Gγ HPFH with the β‐thalassaemia codon 39 nonsense mutation in trans; he showed 64% HbF, 100% of Gγ type. Although the β‐globin haplotype pattern (II/II) was indicative of the presence of the AγT allele on both chromosomes, the AγT expression was undetectable by HPLC even in red cell populations separated by age. The proband was, moreover, homozygous for the –4 bp deletion at position ‐225 to ‐222 of Aγ promoter which has recently been associated with decreased AγT globin expression. These findings suggest that this maximal overexpression of Gγ‐globin probably reflects intensified stimulation of the mutated Gγ promoter in this hitherto undescribed genetic condition.
Acute toxicity and effectiveness of idarubicin in childhood acute lymphoblastic leukemia
Authors: Neuendank Andrea , Hartmann Reinhard , Bührer Christoph , Winterhalter Bernd , Klumper Edwin , Veerman Anjo J. P. , Henze Günter
Published: July 2010

Abstract:  Anthracyclines have become important components of multi‐agent remission induction and continuation therapy of acute lymphoblastic leukemia (ALL). New anthracycline derivatives are being investigated in an attempt to shift the balance of side effects and antileukemic potency. To evaluate the toxicity and efficacy of idarubicin (IDA) in childhood ALL, a prospective multicenter phase‐II study was performed. A total of 51 children with prognostically poor recurrences of ALL were enrolled, all of whom had been exposed to anthracyclines during front‐line treatment. A single 48‐h continuous infusion of IDA at 24 mg/m2 was started on the first day of salvage treatment without concomitant systemic cytostatic agents. The response was assessed by reduction of leukemic blasts in the bone marrow and other compartments 2 wk later. IDA monotherapy caused complete and partial remissions in 5 and 20 patients, respectively (49%). Delays of treatment with subsequent polychemotherapy courses were frequent and mainly caused by prolonged intervals of myelosuppression and high rates of systemic infection. Non‐hematological toxicities including acute cardiac reactions were transient and moderate. Our findings suggest that IDA is an effective drug for remission induction in children with ALL, with acute hematological toxicity being dose‐limiting.
Circadian cell cycle variations of erythro‐ and myelopoiesis in humans
Authors: Abrahamsen Jenny Foss , Smaaland Rune , Sothern Robert B. , Laerum Ole Didrik
Published: July 2010

Abstract:  By use of a multiparameter flow cytometric method with specific surface markers, circadian (24‐h) variations in cell cycle distribution have been studied in 19 healthy male volunteers by sampling bone marrow (BM) every 4–5 h during 24‐h periods. Admixture of peripheral blood during the sampling was specifically adjusted for, and the fractions of cells in DNA synthetic phase were measured for different hemopoietic cell lineages. Significant circadian variations in DNA S‐phase were demonstrated both in myelo‐ and erythropoiesis of the human BM, with 75% (myeloid) and 80% (erythroid) of the volunteers showing highest activity (values) of DNA S‐phase during the day and lowest activity (values) between midnight and 04:00 h. A temporal relationship in the circadian variation of S‐phase and G2/M‐phase was demonstrated between the myeloid and erythroid cell lineages. The highest fractions of S‐phase cells were found in erythropoiesis, while the highest circadian stage dependent variation was found in myelopoiesis. The existence of a similar phasing in DNA synthetic activity for myelopoietic and erythropoietic cells in the human bone marrow indicates that the circadian rhythmicity of hemopoiesis may be caused by a common regulatory mechanism. These findings may be relevant with regard to optimizing the use of cytotoxic drugs and hemopoietic growth factors by taking into consideration the intrinsic (endogenous) circadian variation in proliferative activity of human BM subpopulations.
Dominant‐negative mutations of the Wilms' tumour predisposing gene (WT1) are infrequent in CML blast crisis and de novo acute leukaemia
Authors: Carapeti Melina , Goldman John M. , Cross Nicholas C. P.
Published: July 2010

Abstract:  To determine if mutations of the Wilms' tumour predisposing gene (WT1) are associated with haematological malignancies, we have investigated 65 cases of acute leukaemia, including 39 patients in blast crisis of chronic myeloid leukaemia (CML), by amplification of WT1 exons 7, 8 and 9 followed by single‐strand conformation polymorphism analysis. WT1 transcripts were detected by RT‐PCR in all samples. An exon 7 silent polymorphism (A→G; Arg 313) was identified in 17 individuals, 5 of whom were homozygous, but no other lesions were found. In 1 sample from a patient with acute lymphoblastic leukaemia a smaller size transcript missing exon 9 was detected; a similar abnormality has been described previously in a patient with Wilms' tumour and the resultant protein shown to act in a dominant‐negative manner. No mutations of the exon 9 donor or acceptor splice sites were found in this patient and the basis of the abnormal transcript remains obscure. We conclude that dominant‐negative mutations of the zinc finger region of the WT1 gene are uncommon in CML blast crisis. Abnormalities of this gene may, however, contribute to a small proportion of cases of de novo acute leukaemia.
Platelet membrane fluidity and intraplatelet Ca2+ mobilization are affected in uraemia
Authors: Walkowiak B. , Borkowska E. , Koziolkiewicz W. , Michalec L. , Sobol A. , Cierniewski C. S.
Published: July 2010

Abstract:  In present investigations, platelet membrane fluidity and intraplatelet Ca2+ mobilization were analysed in uraemic platelets by fluorescence techniques. Thirteen non‐dialyzed uraemic patients and 16 control subjects were examined. Anisotropy of DPH‐probe, measured at 37°C, was significantly higher in control (0.2236 ± 0.0050) than in uraemic platelets (0.1969 ± 0.0082; p < 0.01). There was no difference between control (109.8 ± 6.0 nm) and uraemic platelets (100.0 ± 7.3 nm) when the basal [Ca2+]i in resting platelets was determined. Activation of platelets by ADP (12.5 μm) or by thrombin (0.1 U/ml) resulted in an increase in [Ca2+]i. It was significantly higher (p* < 0.003 for ADP and p* < 0.009 for thrombin, respectively) in control platelets (383.6 ± 56.3 nm and 2031.0 ± 298.8 nm, respectively) than in uraemic ones (191.0 ± 21.3 nm and 838.7 ± 144.1 nm, respectively). The amount of released Ca2+ was higher in control platelets activated by both ADP and thrombin (157.6 ± 21.4 nm and 409.3 ± 71.0 nm, respectively) than in uraemic platelets (76.7 ± 15.7 nm and 203.0 ± 29.3 nm, respectively) and the differences were significant (p < 0.01 and p* < 0.01, respectively). These results indicate an abnormal intracellular Ca2+ mobilization in uraemic platelets. Both increased membrane fluidity and decreased Ca2+ mobilization should be considered as a possible reason of reduced fibrinogen receptor exposure on uraemic platelets.
Interleukin 4 responses in acute leukaemia patients with severe chemotherapy‐induced leucopenia
Authors: Bruserud Ø. , Ulvestad E. , Halstensen A. , Berentsen S. , Bergheim J. , Nesthus I.
Published: July 2010

Abstract:  T lymphocyte functions in acute leukaemia patients with severe chemotherapy‐induced leucopenia were investigated using 3 different approaches: (i) analysis of serum concentrations of the T cell cytokine interleukin 4 (IL4) demonstrated that serum IL4 levels increased during complicating bacterial infections. However, this response was modulated by a concomitant increase in serum levels of the potential IL4 antagonist soluble IL4 receptor α chain (sIL4Rα). (ii) Even during leucopenia a subset of T lymphocytes derived from leucopenic patients expressed the activation markers CD25 (IL2 receptor), CD71 (transferrin receptor) and HLA‐DR. (iii) Subsets of circulating CD4+ and CD8+ T lymphocytes could undergo clonogenic proliferation in vitro, and a majority of these clones secreted IL4. CD4+ clones showed higher IL4 levels than CD8+ clones. Our results indicate that T lymphocytes can be activated and contribute to cytokine responses in acute leukaemia patients with severe chemotherapy‐induced cytopenia.
Different expression of adhesion molecules on myeloid and B‐lymphoid CD34+ progenitors in normal bone marrow
Authors: De Waele Marc , Renmans Wim , Damiaens Serge , Flament Jocelyne , Schots Rik , Van Riet Ivan
Published: July 2010

Abstract:  The expression of adhesion molecules was studied on B lymphoid and myeloid CD34+ precursors in normal bone marrow. Bone marrow aspirates were labelled in a double fluorescence procedure with the CD34 monoclonal antibody 43A1 and with antibodies directed against maturation and differentiation antigens and adhesion molecules. Three clusters of CD34+ cells could be distinguished by their light scatter characteristics in flow cytometry. The population with the lowest forward scatter contained B‐lymphoid precursors while the two others showed phenotypic characteristics of, respectively, early and late myeloid precursors. Nearly all CD34+ cells in the 3 subpopulations expressed VLA‐4, VLA‐5, LFA‐3 and H‐CAM. B‐lymphoid progenitors showed a higher density of VLA‐4 and VLA‐5 than the myeloid progenitors. Myeloid precursors, and particularly the late subset, expressed more HCAM than the B‐lymphoid progenitors. The majority of the CD34+ cells also expressed LFA‐1 and L‐selectin. Higher numbers of positive cells were found in the myeloid subset. The early myeloid subset showed the highest positivity for L‐selectin. We conclude that B lymphoid and early and late myeloid CD34+ precursors in normal bone marrow show a different profile of adhesion molecules. These profiles could reflect a higher tendency of the myeloid CD34+ precursors to circulate.
Immunoglobulin G subclasses of anti‐human platelet antigen la in maternal sera: relation to the severity of neonatal alloimmune thrombocytopenia
Authors: Mawas F. , Wiener E. , Williamson L. M. , Rodeck C. H.
Published: July 2010

Abstract:  The monoclonal antibody immobilization of platelet antigen (MAIPA) technique was employed to detect and semiquantitatively assess total IgG anti‐HPA‐1a and its subclasses in sera of mothers who gave birth to severely thrombocytopenic (<50×109 platelets/L) (n = 14) or mildly thrombocytopenic/unaffected (>50×109 platelets/L) (n = 13) neonates. There was no statistically significant difference between the IgG anti‐HPA‐1a subclass composition of the 2 groups of sera. The majority of sera (26/27,96%) showed IgG1+IgG3 while 17/27 (63%) had all 4 subclasses of the antibody. No significant differences between the severely thrombocytopenic and mildly thrombocytopenic/unaffected groups were detected in the levels of IgG, IgG1, IgG2 or IgG4 of the antibody. However, the values of IgG3 anti‐HPA‐1a were significantly higher in the severely thrombocytopenic than in the mildly thrombocytopenic/unaffected group of sera with only little overlap (median 2.94 vs. 1.68, range 1.36–9.71 vs. 1.50–2.84, respectively; p <0.01). The results suggest that maternal IgG3 anti‐HPA‐1a has predictive value for severe thrombocytopenia of the neonate. However, a prospective study of IgG HPA‐1a subclasses in a greater number of maternal sera at different times of pregnancy is needed to test if IgG3 anti‐HPA‐1a is predictive of the degree of fetal/neonatal thrombocytopenia.
Decrease in arterial oxygen partial pressure within the first 24 h of rhGM–CSF administration in AML patients
Authors: Zühlsdorf Michael , von Eiff Michael , Thomas Michael , Büchner Thomas
Published: July 2010

Abstract:  GM–CSF may induce pulmonary complications, such as dyspnea with temporary decreases in oxygen saturation described as first dose effect for higher dosages of intravenous rhGM–CSF. This study investigated possible pulmonary disturbances in adult de novo AML patients receiving yeast rhGM–CSF 24 h prior to chemotherapy under phase II/III conditions. Eighteen patients were monitored for 22 treatment episodes. GM–CSF was given s.c. 1 q.d., 2 q.d. or continuously i.v. at 250 μg/m2/d 24 h prior to induction chemotherapy (TAD9, n = 18) and consolidation (TAD9, n = 4). Spirometry, bodyplethysmography, single breath‐diffusion capacity (DLCO) and arterial blood gas analyses were obtained prior to GM–CSF, and repeated after 24 h. Pulse oxymetric oxygen saturation (saO2) was registered continuously for the first 16 h within day 1 of rhGM–CSF treatment. Patients were aged 21–75 years. The saO2 monitoring did not reveal any first dose effect. PaO2 values decreased from 78.9 mmHg before GM–CSF to 72.8 mmHg after 24 h (p <0.01, maximum shift 15 mmHg). PaO2 shifts occurred mainly with pre‐existing lowered paO2, but otherwise were independent of age, the route of GM–CSF administration, leukocyte levels, or increase of leukocytes with GM–CSF. Increases in AaDO2 reflected the paO2 shifts (p <0.05). No dyspnea corresponded to these changes. DLCO values did not decrease significantly after 24 h. Summarily, contemporary dosage of yeast rhGM–CSF avoids short‐term oxygen desaturations, but leads to clinically benign impairment in oxygen tension, based on ventilation/perfusion mismatches. This should be taken into account for patients starting at subnormal paO2.
Observations on the relationship between γ‐globin chain content and globin chain precipitation in thalassaemic erythroblasts and on the composition of erythroblastic inclusions in HbE/β‐thalassaemia
Authors: Wickramasinghe S. N. , Lee M. J.
Published: July 2010

Abstract:  Electron microscope immunocytochemical studies were performed on the bone marrow from 2 patients each with β‐thalassaemia major and β‐thalassaemia intermedia and 4 patients with HbE/β‐thalassaemia, using the immunogold technique. Studies of sections that were reacted with mouse monoclonal antibodies against human γ‐globin chains showed the presence of large quantities of γ‐chains in several erythroblasts and marrow reticulocytes containing large amounts of precipitated globin chains. At least in these cells, substantial γ‐chains synthesis did not protect against the formation of erythroblastic inclusions, indicating that factors other than the rate of γ‐chain synthesis influence the extent of globin‐chain precipitation. Studies of sections of marrow that were reacted with a rabbit polyclonal antibody against human α‐globin chains and a mouse monoclonal antibody against human β‐globin chains showed that the erythroblastic inclusions in HbE/β‐thalassaemia did not contain detectable quantities of HbE and consisted only of precipitated α‐globin chains. Thus, the generally milder phenotype of HbE/β‐thalassaemia cannot be attributed to co‐precipitation of HbE and excess free α‐globin chains.
No evidence for an altered mRNA expression or protein level of haematopoietic cell phosphatase in CD34+ bone marrow progenitor cells or mature peripheral blood cells in polycythaemia vera
Authors: Andersson P. , LeBlanc K. , Eriksson B‐Å. , Samuelsson J.
Published: July 2010

Abstract:  Polycythaemia vera (PV) is a myeloproliferative disorder characterized by haematopoietic progenitor cells being hypersensitive to cytokines such as erythropoietin, interleukin‐3, stem cell factor and insulin‐like growth factor 1, which results in an increased production of mature blood cells. The pathogenetic cellular mechanism(s) behind this hypersensitivity to cytokines is unknown, but the number of cytokine receptors and the interaction between ligand and receptor are normal in PV. Interest has therefore focused on post‐receptor mechanism(s). Haematopoietic cell phosphatase (HCP) is an intracellular tyrosine phosphatase that has been demonstrated to regulate proliferative signals negatively induced by the cytokines mentioned above. Moreover, motheaten mice that genetically lack HCP have an increased amount of erythroid progenitors that are hypersensitive to Epo, and patients with familial polycythaemia have been shown to exhibit a mutation of the Epo receptor gene that includes the docking site for HCP. We therefore studied mRNA expression of HCP in pure populations of CD34+ cells, granulocytes, platelets and lymphocytes from patients with PV, chronic myeloid leukaemia (CML) or essential thrombocythemia (ET), as well as healthy controls. Using a polymerase chain reaction analysis employing specific primers for HCP, we failed to detect any abnormalities of HCP expression in PV in any of the cell populations that were examined. Moreover, HCP mRNA expression was similar in ET and CML compared to controls. Finally, Western blot analysis revealed a normal HCP protein content in PV granulocytes and platelets. We therefore conclude that neither an impaired expression of the HCP gene nor a defect in HCP protein synthesis is present in PV, and does not seem to play a role in the aetiology of this disorder.
A review of the use of teicoplanin in haematological malignancy
Authors: Davies J. M.
Published: July 2010

Abstract:  Factors determining the change in patterns of antibiotic use in patients with haematological malignancies include the growing numbers of infections caused by Gram‐positive pathogens, the increasing reliance on indwelling catheters, and strategic movement away from inpatient therapy towards day‐case and non‐inpatient therapy. The suitability of teicoplanin in this context is examined. The indications for teicoplanin use are now established as: early use in clinically infected patients; as a second‐line agent for sensitive pathogens; and in primary antibiotic failures. Teicoplanin should not be used as empirical therapy in fevers of unknown origin; neither should teicoplanin be used as prophylaxis (the situations of infection in chemotherapy patients or for Hickman catheter insertion in particular are discussed). Comparative studies versus vancomycin have shown teicoplanin to be equally effective and to be well tolerated. Teicoplanin's good safety profile and ease of use make it particularly useful for non‐inpatient therapy. Problems are posed for both glycopeptides by specific resistant pathogens, although these pathogens are currently not common. Haematologists must be aware of, and guard against, abuse of the glycopeptides in their own and other fields, including oral gut decontamination, the treatment of Clostridium difficile and veterinary use.
Experience with teicoplanin in non‐inpatient therapy in children with central line infections
Authors: Kinsey S. E.
Published: July 2010

Abstract:  Indwelling intravenous catheters are an invaluable part of the curative therapy or terminal care of children with haematological malignancies. The increase in their use has been paralleled by an increase in Gram‐positive infections, however. This article provides an overview of non‐inpatient treatment of central line infections using teicoplanin. The main drivers for considering non‐inpatient therapy were to increase the quality of the patient's life by reducing the amount of time spent in hospital, and to prolong the life of the catheter. A large proportion (95%) of the children in the unit described have indwelling catheters in situ, including Port‐a‐caths, Hickman catheters and Vascaths. The indications suitable for non‐inpatient antibiotic therapy of line infections were those patients near the end of their chemotherapy courses, during terminal care, in non‐neutropenic patients to complete an antibiotic course, and in patients with chronically neutropenic aplastic anaemia. Persistent line infections are not always eradicated but usually controlled. Care can take place in the home, in the general practitioner (GP) surgery or in the outpatient clinic. Care can be undertaken by nurses, older patients and parents. Follow‐up procedures are in place to ensure safe, effective therapy.
Central venous catheter infections treated with teicoplanin
Authors: McCarthy A. , Rao J. S. , Byrne M. , Breatnach F. , O'Meara C. A.
Published: July 2010

Abstract:  A novel way of using teicoplanin in situ to treat central venous catheters is described. Profound immunosuppression and the fact that the lines remain indwelling for long periods are two of the main reasons for these infections. In children it is also difficult to prevent these lines being played with, which increases the likelihood of infection. The different types of infection that can occur in a central venous catheter are described and the clinical definition of a catheter infection is provided. In an initial study, infective episodes in a small group of 11 children were treated successfully with in situ amikacin. Most pathogens were Gram‐negative cocci. None of the catheters had to be removed, and catheter life was prolonged by a mean of 118 d. Due to the high incidence of Staphylococcus epidermidis in the initial study, in situ teicoplanin was assessed in a subsequent study. Over the course of 1 yr, 20 line infections occurred in 12 children. Empirical amikacin therapy was instituted and switched to teicoplanin once the pathogen was confirmed as Gram‐positive. An antibiotic–heparin mixture was introduced into the line and left in place for 24 h, after which time it was replaced with fresh mixture until cultures were sterile. All pathogens were sensitive to teicoplanin, all infections were treated successfully and no catheters had to be removed. Overall, catheter life was prolonged by a mean of 136 d. It was concluded that in situ teicoplanin was effective and well tolerated for line infections (no side‐effects were reported during the study). A minimum of 6 d therapy was recommended. The patients with less severe infections would have been suitable for treatment at home by their parents, district nurse or general practitioner (GP).