bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of the receptor tyrosine kinase p145c‐kit with the p210bcr/abl kinase in myeloid cells
Authors: Hallek Michael , Danhauser‐Riedl Susanne , Herbst Ronald , Warmuth Markus , Winkler Almut , Kolb Hans‐Jochen , Druker Brian , Griffin James D. , Emmerich Bertold , Ullrich Axel
Published: October 2003

The chimaeric bcr/abl oncogene is detected in virtually all cases of chronic myelogenous leukaemia (CML). It encodes a constitutively active tyrosine kinase of 210kDalton, p210bcr/abl, which stimulates a variety of cytosolic signalling intermediates. The effects of bcr/abl on the activity of growth factor receptors are less well known. In order to investigate interaction of p210bcr/abl with the receptor tyrosine kinase p145c‐kit, we used two myeloid, factor‐dependent cell lines, MO7 and 32D, to generate bcr/abl positive sublines, MO7p210 and 32Dp210, by transfection with the bcr/abl gene. Since 32D and 32Dp210 cells did not express p145c‐kit, a c‐kit retrovirus was used to generate c‐kit positive cell lines (32Dkit, 32Dp210kit). In contrast to MO7 and 32Dkit cells, MO7p210 and 32Dp210kit cells were factor independent and did not respond to the growth‐promoting effects of recombinant human Steel factor (rhSF). Preincubation with a monoclonal antibody (MAb) neutralizing the binding of SF to p145c‐kit did not affect the growth of MO7p210 cells, thus eliminating the possibility of an autocrine SF secretion. 32Dkit cells transfected with bcr/abl containing an inactivating point mutation (Lys→Arg271) in the Abl kinase domain (32Dp210(Arg271)kit) retained their responsiveness to the effects of rhSF. Immune complex kinase assays showed that the kinase activity of p145c‐kit was several‐fold higher in MO7p210 and 32Dp210kit cells than in MO7, 32Dkit and 32Dp210(Arg271)kit cells, suggesting that Abl kinase activity was necessary to activate p145c‐kit. Co‐immunoprecipitation experiments with anti‐Kit and anti‐Abl MAbs demonstrated that p145c‐kit and p210bcr/abl were associated in an intracellular complex in human bcr/abl positive, c‐kit positive cell lines (MO7p210; GM/SO). Finally, colony assays with bone marrow from bcr/abl positive CML patients showed that the haemopoietic progenitors of three of four patients did not respond to rhSF. Taken together, the results suggest that p145c‐kit can be activated by p210bcr/abl via an Abl‐kinase dependent mechanism involving the complex formation of both proteins. These findings could explain some clinical features (basophilia, increase of immature myeloid cells) of chronic‐phase CML.
Prognostic factors in myelodysplastic syndromes: critical analysis of the impact of age and gender and failure to identify a very‐low‐risk group using standard mortality ratio techniques
Authors: Morel Pierre , Declercq Christophe , Hebbar Mohamed , Bauters Francis , Fenaux Pierre
Published: October 2003

We studied the prognostic value of age and gender by survival, and by standard mortality ratio (SMR) analyses, in 203 untreated patients with myelodysplastic syndromes (MDS); 57 refractory anaemia (RA), 23 refractory anaemia with ringed sideroblasts, 41 refractory anaemia with excess blasts (RAEB), 3 RAEB in transformation (RAEB‐T) and 79 chronic myelomonocytic leukaemia (CMML), aged 23–89 years (median 69, M/F 0.5), who were all karyotyped. Median survival was 36 months. Adverse prognostic factors were: high bone‐marrow blast percentage, complex karyotype, low platelet count, age>60 years, low or high WBC count, haemoglobin<10g/dl, male gender. However, the standard mortality ratio (i.e. mortality compared to that of an age‐ and sex‐adjusted population) was not different between male and female patients. Patients<60 had a higher SMR than older patients. Therefore the prognostic values of age and gender for survival in MDS patients may reflect, at least in part, a characteristic of the population. Furthermore, even in low‐risk groups defined by scoring system we were unable to define a subgroup of patients with a mortality similar to that of the normal population, especially in MDS patients aged<60.
Heparin induces apoptosis in human peripheral blood neutrophils
Authors: Manaster J. , Chezar J. , Shurtz‐Swirski R. , Shapiro G. , Tendler Y. , Kristal B. , Shasha S. M. , Sela S.
Published: October 2003

Programmed cell death, by apoptosis, has been shown to play an important role in the regulation of haemopoiesis. Using trypan blue exclusion for distinguishing intact membranes, flow cytometry for detection of sub G1 peak and in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL), this study shows that heparin induces apoptosis in vitro in human peripheral blood neutrophils. The known anti‐proliferative effect of heparin in several in vitro cell systems has therefore to be interpreted in the light of apoptosis. In addition, apoptosis may help explain the anti‐inflammatory effects resulting from the interaction between vessel wall heparan sulphate and chemoattracted peripheral blood neutrophils.
Interleukin‐6 is Required for Pristane‐Induced Plasma Cell Hyperplasia In Mice
Authors: Dedera Douglas A. , Urashima Mitsuyoshi , Chauhan Dharminder , LeBrun David P. , Bronson Roderick T. , Anderson Kenneth C.
Published: October 2003

Intraperitoneal injection of pristane induces production of interleukin‐6 (IL‐6) and either plasmacytosis or plasmacytoma in mice, depending upon the genetic background. Pristane does not induce plasmacytoma in IL‐6 knockout (IL‐6−/−) mice, suggesting that IL‐6 is required for this process. In the present study we determined whether IL‐6 is also required for pristane‐induced hyperplasia of normal plasma cells. Pristane was injected intraperitoneally into IL‐6−/− and IL‐6 wild‐type (IL‐6+/+) mice. Overall there were more deaths in IL‐6+/+ mice (85%) than in IL‐6−/− mice (40%), P=0.024. Hyperplastic lymph node and spleen weight did not differ (P=0.82 and P=0.15, respectively) in IL‐6−/− versus IL‐6+/+ mice. Lymphocytosis with similar patterns of expression of B‐cell (B220) and T‐cell (Thy‐1) antigens was noted in both IL‐6−/− and IL‐6+/+ mice. However, morphological studies, dual fluorescent staining for Syn‐1 and B220 antigens (syn‐1+ B220+ cells), and intracytoplasmic Ig staining revealed plasma cell hyperplasia in lymph node and spleen from IL‐6+/+, but not IL‐6−/−, mice. These plasma cells from IL‐6+/+ mice were polyclonal and unable to induce tumour formation in severe combined immunodeficient mice. These data demonstrate that IL‐6 is required for pristane‐induced hyperplasia of polyclonal plasma cells in mice.
Molecular diagnosis of t(11;14) in mantle cell lymphoma using two‐colour interphase fluorescence in situ hybridization
Authors: MONTEIL MICHÈLE , CALLANAN MARY , DASCALESCU CRISTINA , SOTTO JEAN‐JACQUES , LEROUX DOMINIQUE
Published: October 2003

t(11;14) is observed in up to 70% of mantle cell lymphoma (MCL) cases and is therefore an important diagnostic element. In routine practice, detection of t(11;14) by conventional cytogenetic techniques is hindered by the low yield and quality of tumour metaphases. Molecular techniques (Southern blot, PCR) are unable to detect a large number of 11q13 breakpoints due to scattering over distances up to 1 Mb. Using 23 MCL patients with karyotypically determined t(11;14) and eight negative controls, we have devised a two‐colour interphase FISH assay for detection of the 14q+ chromosome. We chose an 11q13 probe telomeric to the major 11q13 translocation cluster sites and an IGH probe centromeric of the 14q32 breakpoints. This method detected the translocation in all 23 t(11;14) positive patients, with an overall average of 60% nuclei showing colocalized signals. Widespread application of this technique will constitute an important diagnostic aid in clinical management of MCL patients. Since FISH is a convenient method for retrospective analysis of large numbers of patient specimens, this method should contribute to an accurate estimation of t(11;14) frequency in MCL and other chronic B‐cell malignancies and consequently to their better nosological characterization.
CD34 selections from myeloma peripheral blood cell autografts contain residual tumour cells due to impurity, not to CD34+ myeloma cells
Authors: Willems P. , Croockewit A. , Raymakers R. , Holdrinet R. , Van Der Bosch G. , Huys E. , Mensink E.
Published: October 2003

Malignant cells in haemopoietic autografts can contribute to post‐transplant relapse. Engraftment of myeloma patients with CD34+ peripheral blood progenitors selected from total autografts reduces the number of tumour cells infused by 2.7–4.5 logs. Residual tumour cells detected in CD34+ selected cells may be due to selection impurity or the existence of malignant CD34+ progenitors. In three patients we evaluated the CD34 purity and tumour load of total autografts, CD34+ progenitors selected with immunomagnetic beads and highly purified CD34+ progenitors obtained in two rounds of selection (combining magnetic with flow cytometry activated cell sorting) to determine the cause of residual tumour cells in CD34 selections.

Using allele‐specific oligonucleotides (ASO) complementary to the unique Ig heavy chain sequence (CDRIII region) of the malignant clone, semi‐quantitative ASO‐PCR was capable of detecting one malignant cell in 104–105 normal white blood cells. Selection of CD34+ cells from bone marrow (BM) with approximately 20% malignant plasma cells resulted in a 1.4 log reduction of tumour burden. Using two‐colour flow‐cytometry we observed CD34−, BB4+ malignant plasma cells contaminating this CD34 selection. Prior to sorting, peripheral blood cell autografts (PBCA) contained approximately 0.1% malignant cells. Selection of >99% pure CD34+ cells using immunomagnetic beads (Dynal) resulted in an approximate 2 log reduction of malignant cells, but residual tumour cells were still detectable. ASO‐PCR detected no malignant cells in >99.9% pure CD34+ peripheral blood progenitors obtained with two rounds of selection (combining magnetic with flow cytometry activated cell sorting). We conclude that CD34+ malignant cells are not detectable in myeloma PBCA and that residual tumour cells in CD34 selections are due to contaminating CD34‐negative cells.
Characterization of an acquired IgG inhibitor of coagulation factor XIII in a patient with systemic lupus erythematosus
Authors: Ahmad Ferhaan , Solymoss Susan , Poon Man‐Chiu , Berube Caroline , Sullivan Arthur K.
Published: October 2003

An acquired IgG inhibitor to factor XIII was identified in a 30‐year‐old Vietnamese woman with systemic lupus erythematosus. The IgG fraction was isolated from plasma by chromatography on an agarose gel column and by protein G adsorption. The anti‐factor XIII activity, identified within the IgG fraction, inhibited factor XIII from both normal plasma (a2b2) and platelets (a2). A normal level of the b subunit was measured by immunoelectrophoresis of the patient’s plasma, but the a subunit was not detected. These results suggested the presence of an IgG immunoglobulin which recognized the a subunit and interfered with its activity.
Detection of human parvovirus B19 DNA in plasma pools and blood products derived from these pools: implications for efficiency and consistency of removal of B19 DNA during manufacture
Authors: Saldanha J. , Minor P.
Published: October 2003

The polymerase chain reaction (PCR) assay was used to detect human parvovirus B19 DNA in 38 blood products and start plasma pools from five different manufacturers. The products examined were albumin, factor VIII, intravenous (i.v.) and intramuscular (i.m.) immunoglobulin batches. The majority of pools from all the manufacturers had detectable B19 DNA (64/75: 85%; ranging from 60% to 100% for individual manufacturers). B19 DNA was found in 3/12 albumin samples, in 7/7 factor VIII samples, 3/15 IVIG samples and 3/4 IMIG samples. The levels of B19 DNA in pools varied from 102 to 109 genome equivalents/ml, whereas the levels in products varied from 102 to 106 genome equivalents/ml, but there was no clear relationship between the levels of B19 DNA in start pools and final products.  The levels of B19 DNA varied between different batches of the same product from a single manufacturer, possibly due to small variations in the processing parameters. In addition, there was some indication from the study of IVIG samples that treatment at low pH may result in removal of PCR‐detectable B19 DNA.
A comparison of busulphan versus total body irradiation combined with cyclophosphamide as conditioning for autograft or allograft bone marrow transplantation in patients with acute leukaemia
Authors: Ringdén O. , Labopin M. , Tura S. , Arcese W. , Iriondo A. , Zittoun R. , Sierra J. , Gorin N. C.
Published: October 2003

We retrospectively compared the outcome in patients in the EBMT database transplanted for acute leukaemia from January 1987 to January 1994 who received busulphan and cyclophosphamide (BU/CY) as a pretransplant regimen versus those who received cyclophosphamide and total‐body irradiation (CY/TBI). The patients were matched for type of transplant (autologous bone marrow transplantation (ABMT) versus allogeneic (BMT)), diagnosis (acute lymphoblastic leukaemia (ALL) or acute myeloid leukaemia (AML)), status (early (first complete remission, CR‐1) versus intermediate (second or later remission, first relapse)), age, FAB classification for AML, prevention of graft‐versus‐host disease and year of transplantation. In ABMT recipients (matched paired 530 × 2) with ALL CR‐1, AML CR‐1 and AML intermediate disease, transplant‐related mortalities (TRM), relapse incidence (RI) and leukaemia‐free survival (LFS) did not differ significantly in patients treated with BU/CY or CY/TBI. However, in ABMT recipients with ALL intermediate disease, the probability of relapse was 82 ± 5% (±95% confidence interval) in the BU/CY group compared to 62 ± 6% in the CY/TBI group (P = 0.002) and the 2‐year leukaemia‐free survival 14 ± 4% and 34 ± 6%, respectively (P = 0.002). In BMT recipients of bone marrow from HLA‐identical siblings (matched paired 391 × 2), the TRM, RI and LFS did not differ significantly between the two treatments in all groups. In particular, the 2‐year LFS in patients with AML CR‐1 was 64 ± 3% in those treated with BU/CY (n = 237) compared to 66 ± 3% in those given CY/TBI (n = 237). In all groups the findings were confirmed in a multivariate analysis of prognostic factors. Veno‐occlusive disease (VOD) of the liver (P < 0.05) and haemorrhagic cystitis (P < 0.001) was more common in the BU/CY group compared to the CY/TBI group for ABMT and BMT patients.

In conclusion, BU/CY and CY/TBI as pretransplant regimens gave similar results in all situations, except ABMT for ALL intermediate stages with more than 2 years from diagnosis to transplantation, where a lower RI and a higher LFS were associated with CY/TBI.
Molecular alterations of IL‐6R, lck and c‐myc genes in transforming monoclonal gammopathies of undetermined significance
Authors: GERNONE ANGELA , DAMMACCO FRANCO
Published: October 2003

We studied the molecular alterations of IL‐6R, lck and c‐myc genes in the tumour cells of 50 patients with multiple myeloma (MM) and 20 patients with monoclonal gammopathies of undetermined significance (MGUS). Southern blot analysis revealed a 5.3 kb IL‐6R amplified band by digestion with EcoRI and HindIII in three MGUS patients, but no IL‐6R gene alteration was found in MM patients. BamHI digestion revealed a 6.2 kb rearranged band of the lck gene in two MGUS patients with IL‐6R amplification. In one MGUS patient we detected a rearrangement upstream of the lck coding region. Myc rearrangement was observed in three MM and two MGUS patients who showed coexistent lck rearrangement and IL‐6R amplification. These molecular alterations were detected in the MGUS patients with an IgA monoclonal component, who showed a rapid progression into aggressive MM. Myc rearrangement seems to be associated with a small group of a clinically aggressive MM at diagnosis, secreting IgA or k light chains. Multiple rearrangements and/or molecular alterations are likely to occur at the time of MGUS‐IgA transformation into aggressive MM and myc rearrangement may promote a positive pressure for transformation and progression. MM tumourigenesis remains a heterogenous multistep process involving the deregulation of many genes and gene products.
p53 tumour suppressor gene and RAS oncogenes: molecular analysis in the chronic and leukaemic phases of essential thrombocythaemia
Authors: NERI ANTONINO , FRACCHIOLLA NICOLA STEFANO , RADAELLI FRANCA , BOLETINI ALKETA , RIBERA SONIA , MIGLIORINI CHIARA , TRECCA DINO , MAIOLO ANNA TERESA
Published: October 2003

A panel of 51 cases of essential thrombocythaemia (ET), in chronic or leukaemic phase, was investigated for p53 gene and RAS oncogenes mutations by PCR‐SSCP‐direct sequencing. No RAS oncogenes mutations were detected, but p53 mutations were identified in three cases: 1/27 cases (≈4%) in chronic phase not undergoing chemotherapy, 1/19 cases (≈5%) in chronic phase undergoing chemotherapy, and 1/5 cases (20%) which had progressed to leukaemia. Our results suggest that: (1) p53 gene mutations occur sporadically in the chronic phase of ET, independent of chemotherapy, and may contribute to the progression to the leukaemic phase in a limited number of ET patients; (2) the RAS genes family does not seem to be involved in the pathogenesis of ET, unlike other bcr/abl negative chronic myeloproliferative diseases (CMPDs).
Effect of the protein tyrosine kinase inhibitor genistein on normal and leukaemic haemopoietic progenitor cells
Authors: CARLO‐STELLA CARMELO , REGAZZI ESTER , GARAU DANIELA , MANGONI LINA , RIZZO MARIA TERESA , BONATI ANTONIO , DOTTI GIANPIETRO , ALMICI CAMILLO , RIZZOLI VITTORIO
Published: October 2003

Receptor and nonreceptor protein tyrosine kinases (PTKs) play a key role in the control of normal and neoplastic cell growth. The availability of PTK inhibitors prompted us to evaluate the effects of genistein, a natural inhibitor of PTKs, on in vitro colony formation by normal multilineage colony‐forming units (CFU‐Mix), erythroid bursts (BFU‐E), granulocyte‐macrophage colony‐forming units (CFU‐GM), long‐term culture‐initiating cells (LTC‐IC) and acute myelogenous leukaemia colony‐forming units (CFU‐AML). Continuous exposure of normal marrow and blood mononuclear non‐adherent cells, blood CD34+CD45RA− cells, and leukaemic blasts to increasing doses of genistein (1–100 μM) resulted in a statistically significant (P ≤ 0.05) dose‐dependent suppression of CFU‐Mix, BFU‐E, CFU‐GM and CFU‐AML growth. Regression analysis showed that growth inhibition was linearly related to genistein concentration. Genistein dose causing 50% inhibition (ID50) of CFU‐AML was significantly lower compared to CFU‐GM ID50 for marrow (19 v 32 μMP  ≤0.017), unseparated blood (19 v 44 μMP ≤ 0.028) or CD34+CD45RA− blood (19 v 36, P ≤ 0.04). Preincubation of leukaemic blasts with genistein (200 μM) for 1–2 h confirmed that CFU‐AML were significantly more sensitive than normal marrow and blood CFU‐GM to genistein. Preincubation conditions which maximally suppressed leukaemic and normal colony growth spared a substantial percentage of marrow (29 ± 4%) and blood (40 ± 3%) LTC‐IC. In conclusion, our data demonstrate that: (a) genistein strongly inhibits the growth of normal and leukaemic haemopoietic progenitors; (b) growth inhibition is dose‐ and time‐dependent; (c) leukaemic progenitors are more sensitive than normal progenitors to genistein‐induced growth inhibition; (d) genistein exerts a direct toxic effect on haemopoietic cells while sparing a substantial proportion of LTC‐IC. The potent CFU‐AML growth inhibition associated with the relative resistance of normal LTC‐IC strongly supports the use of genistein for marrow purging.
Kell typing by allele‐specific PCR (ASP)
Authors: Avent N. D. , Martin P. G.
Published: October 2003

The Kell blood group system is important in transfusion medicine, and the Kell antigen (K1) is probably second in importance to Rh D as an immunogen in alloimmunized pregnancies which cause haemolytic disease of the newborn. The K/k (K1/K2) blood group polymorphism has been recently defined. A point mutation changes Thr193 (k) to Met193 (K) in the Kell glycoprotein. The mutation which creates K destroys a consensus N‐glycan addition site.  We describe a simple PCR test for K blood group typing. The test is based on the use of an allele‐specific K‐primer. We have shown the test to give results in complete concordance with serologically defined Kell blood group status using 65 genomic DNA samples derived from both amniocytes and peripheral blood lymphocytes. The test is suitable for the prenatal determination of Kell type.
The effect of granulocyte colony‐stimulating factor (G‐CSF) on the degranulation of secondary granule proteins from human neutrophils in vivo may be indirect
Authors: Xu Shengyuan , Höglund Martin , Venge Per
Published: October 2003

Granulocyte colony‐stimulating factor (G‐CSF) was administered at a dose of 7.5 or 10 μg/kg s.c. once daily for 6 d (days 1–6) to two groups consisting of eight and six healthy volunteers. The administration of G‐CSF resulted in a rapid decrease in neutrophil counts and serum levels of the secondary granule protein, human neutrophil lipocalin (HNL) after 30 min, followed by a recovery and gradual increase within 180 min. The number of circulating neutrophils and plasma and serum levels of neutrophil secondary granule proteins were dramatically elevated on day 2 (1 d after the administration of G‐CSF) and stayed so until day 7. The plasma levels of HNL and lactoferrin (LF) showed a biphasic pattern with peaks at day 2 and days 5–7, and remained highly elevated at day 12. The serum levels of HNL and LF increased rapidly (about 8‐fold and 6‐fold, respectively) on day 2 and stayed elevated until day 7, subsequently returning to baseline levels. At day 5, neutrophil release induced in vitro by f‐MLP was significantly enhanced. The cellular contents of HNL and LF were reduced to about 50% of levels before G‐CSF administration at day 5. The release of lactoferrin and HNL, but not of myeloperoxidase (MPO), was slightly enhanced after preincubation of isolated normal neutrophils with G‐CSF in vitro, but no obvious release of these proteins was observed with G‐CSF alone. The administration of G‐CSF resulted in a dramatic increase in the alkaline phosphatase (AP) activity in the plasma membrane, with maximal activity occurring at day 5. Furthermore, during administration of G‐CSF, TNF‐α in plasma increased about 25‐fold. TNF‐α started to rise at day 2 and peaked at day 6. After discontinuation of G‐CSF the levels of TNF‐α gradually decreased. The elevated levels of TNF‐α (tumour necrosis factor‐α) were temporally correlated to the other signs of neutrophil activation. GM‐CSF and IL‐8, however, were not detected in plasma. Our data suggest that G‐CSF affects the neutrophils not only directly but also indirectly by the induction of the production of other cytokines such as TNF‐α.
High incidence of anti‐FVIII antibodies against non‐coagulant epitopes in haemophilia A patients: a possible role for the half‐life of transfused FVIII
Authors: Dazzi F. , Tison T. , Vianello F. , Radossi P. , Zerbinati P. , Carraro P. , Poletti A. , Girolami A.
Published: October 2003

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post‐transfusion antibodies may also arise against non‐coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII‐induced antibodies by use of an enzyme‐linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non‐inhibitory antibodies was evaluated in three patients; although half‐life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti‐FVIII antibodies by ELISA when routinely assessing haemophilia A patients.
GROWTH FACTOR INDUCTION OF CYTOSOLIC PROTEIN TYROSINE KINASE ACTIVITY IN HUMAN HAEMOPOIETIC PROGENITOR CELLS ISOLATED BY FLOW CYTOMETRY
Authors: Harvey Kevin A. , Siddiqui Rafat A. , Jansen Jan , Akard Luke P. , Thompson James M. , Cui Yi , Chang Qing , English Denis
Published: October 2003

We employed a highly sensitive method to assay protein tyrosine kinase activity in extracts of subpopulations of CD34+ bone marrow progenitor cells isolated by fluorescence activated cell sorting in an attempt to better define how growth‐factor induction of enzymatic activity relates to progenitor cell maturation. FACS analysis confirmed that, under the conditions employed, essentially all of the CD34+ cells in adult human marrow that lacked the CD38 antigen were devoid of the myeloid maturation marker CD33 as well as the lineage antigens: CD10, 13, 14, 15, 16, 19, 71 and glycophorin A. A variable portion (50–90%) of these CD34+, CD38− progenitor cells expressed HLA‐DR. CD34+, CD38− cells that did not express HLA‐DR were found to lack detectable levels of either membrane or cytosolic tyrosine kinase activity. HLA‐DR+ progenitor cells that lacked CD38 possessed elevated levels of cytosolic tyrosine kinase activity but only low levels of plasma membrane activity. In contrast, CD34+ cells that expressed CD38 (and HLA‐DR) possessed high levels of membrane‐associated tyrosine kinase activity. A cocktail of haemopoietic growth factors that included IL‐3, IL‐6 and stem cell factor effectively induced tyrosine kinase activity in CD34+, CD38−, HLA‐DR− progenitor cells. Growth factor induction of tyrosine kinase activity in these cells was not inhibited by actinomycin D or cyclohexamide. Most of the tyrosine kinase activity induced by these growth factors was recovered from the cytosolic fraction of disrupted cells. Thus, induction of cytosolic tyrosine kinase activity is an early event in the response of uncommitted haemopoietic cells to haemopoietic growth factors. Subsequent activation of membrane tyrosine kinases may initiate key transduction processes as these cells begin to differentiate.
Tretinoin induces bone marrow collagenous fibrosis in acute promyelocytic leukaemia: new adverse, but reversible effect
Authors: Hatake Kiyohiko , Ohtsuki Tetsuya , Uwai Masaya , Takahashi Hironori , Izumi Tohru , Yoshida Minoru , Kanai Nobuyuki , Saito Ken , Harigaya Ken‐ichi , Miura Yasusada
Published: October 2003

In 11/13 acute promyelocytic leukaemia (APL) cases treated with tretinoin (RA) we observed RA‐induced inaspirable collagenous fibrosis of the bone marrow. To study the mechanism of RA on collagen production, we cultured a human bone marrow derived stromal cell line and an osteoblastic cell line with RA in vitro. 10−7 and 10−6 M of RA stimulated collagen production. Clinical and experimental observation may be important to understand this adverse effect of RA as it is useful in cancer chemoprevention as well as treatment for APL. This adverse effect is spontaneously reversible after stopping RA or following chemotherapy.
Protein kinase C isozymes in human megakaryoblastic leukaemia cell line, MEG‐01: possible involvement of the isozymes in the differentiation process of MEG‐01 cells
Authors: Nagata Koh‐ichi , Okano Yukio , Nozawa Yoshinori
Published: October 2003

The expression of the different protein kinase C (PKC) isozymes in various states of differentiation of the human megakaryoblastic leukaemia cell line MEG‐01 were analysed using thermocycle amplification of mRNA and immunoblotting. MEG‐01 expressed mRNAs of PKCα, ‐βI, ‐βII, ‐δ, ‐ε, ‐η, ‐θ and ‐ζ, but not PKCγ. At the protein molecule level, MEG‐01 was observed to express PKCα, ‐βI, ‐βII, ‐ε, ‐θ and ‐ζ, but lack ‐γ, ‐δ and ‐η. When differentiation of MEG‐01 was induced by 100 nM 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), rapid translocation from cytosol to membrane fraction and down‐regulation of PKCα, ‐ε and ‐θ was observed in 1–2 h. On the other hand, PKCβI and ‐βII were observed to translocate not only to the membrane fraction but also to the cytoskeletal fraction in a different manner, and their down‐regulation, especially βII, was very slow. The myristoylated, alanine‐rich C kinase substrate (MARCKS) in the membrane fraction of MEG‐01 cells was observed to decrease gradually throughout the differentiation process. Additionally, time‐course study of TPA treatment indicated that incubation of the cells for 30 min is sufficient for differentiation. These results strongly suggest that the activation of PKCα, ‐ε and ‐θ is involved in the initiation of differentiation, and that PKCβI and ‐βII have important roles in the maintenance of differentiation. Although PKCζ was resistant to TPA treatment and its translocation was reduced, the amount of this isozyme in the cytosol fraction decreased throughout the differentiation process.
MOBILIZATION OF PHILADELPHIA‐NEGATIVE PERIPHERAL BLOOD MONONUCLEAR CELLS IN CHRONIC MYELOID LEUKAEMIA USING HYDROXYUREA AND G‐CSF (FILGRASTIM)
Authors: JOHNSON R. J. , OWEN R. G. , CHILD J. A. , MORGAN G. J. , BARNARD D. L. , DICKINSON H. , RICKETTS S. , RAWSTRON A. , EVANS P. , WOODHEAD V. , MAJOR K. , ROBINSON F. , SMITH G. M.
Published: October 2003

A relatively simple and non‐toxic out‐patient‐based regimen for the mobilization of Philadelphia‐negative (Ph − ve) mononuclear cells in chronic myeloid leukaemia (CML) was evaluated in 10 patients, nine in stable chronic phase and one in accelerated phase. They received oral hydroxyurea at a mean dose of 3.5 g/m2 daily for 7 d, followed by 300 μg of G‐CSF daily until the last day of harvesting. In the nine chronic‐phase patients the mean number of days from the end of hydroxyurea to the commencement of harvesting was 14.5 (range 10–18). The patient in accelerated phase recovered and was harvested after 6 d. The mean number of aphereses performed was 3.4. Adequate numbers of stem cells were obtained in 9/10 patients judged by our usual criteria. Side‐effects were mild in comparison to published intravenous schedules. No patients lost their hair. Five (50%) patients required admission with neutropenic fever which responded to antibiotics in all cases. Four (40%) patients developed a transient rash and four (40%) experienced mild oral mucositis. This level of toxicity enabled half of the patients to be treated entirely on an out‐patient basis. The harvest products were analysed for cells belonging to the leukaemic clone by conventional cytogenetics, FISH and PCR. All were PCR positive. The mean Ph positivities by cytogenetics and FISH were comparable at 18.1% and 15% respectively. Half the patients had >% normal metaphases. We conclude that this approach is comparable in efficacy to published intravenous regimens and significantly less toxic. It can be safely used at diagnosis before interferon therapy commences.
Haemophagocytic lymphohistiocytosis associated with constitutional inversion of chromosome 9
Authors: H asle Henrik , Brandt Carsten , Kerndrup Gitte , Kjeldsen Eigil , Grethe SØrensen Anne
Published: October 2003

Familial haemophagocytic lymphohistiocytosis (HLH) is considered an autosomal recessive disease, although the putative gene responsible for the disease has not yet been localized. Identification of the involved gene may elucidate the pathogenesis of the disease and is essential for prenatal testing in affected families. We present an infant with HLH and constitutional inversion 9 (p23q31) in cells from bone marrow, lymphocytes and fibroblasts. The parents had normal karyotypes. It may be speculated that one of the parents was a carrier of HLH and a de novo inversion occurred in chromosome 9 from the non‐carrier parent. This would imply that the putative HLH‐related gene is located at one of the two breakpoints on chromosome 9.
Expression of Fas antigen in acute myeloid leukaemia is associated with therapeutic response to chemotherapy
Authors: Hong Min Yoo , Lee Seok , Woon Lee Jung , Young Chong So , Sook Hahn Jee , Woong Ko Yun
Published: October 2003

Flow cytometric immunofluorescent analysis was used to assess Fas antigen (CD95) expression in blasts obtained from the bone marrow of 30 patients with acute myeloid leukaemia. The percentage of positive cells in each sample was highly variable. Fas antigen expression did not correlate with age, FAB subtype, white blood cell counts, or CD34 expression. Low expression of Fas was associated with a low complete remission rate after induction chemotherapy (62.5% in cases with <20% positive cells v 92.9% in cases with ≥ 20% positive cells, P<0.01). The main cause for not achieving remission was resistant disease. Our results suggest that the quantitation of Fas expression can be predictive of treatment outcome in acute myeloid leukaemia.
THE ROLE OF SHEEP STROMA IN HUMAN HAEMOPOIESIS IN THE HUMAN/SHEEP CHIMAERAS
Authors: Almeida‐Porada Graça , Ascensão João L. , Zanjani Esmail D.
Published: October 2003

We have previously reported on the successful engraftment and long‐term expression of human HSC (haemopoietic stem cells) in sheep following transplantation into pre‐immune fetuses. The mechanism(s) underlying the support/maintenance of human haemopoiesis in sheep is not understood. In these studies we examined the ability of sheep stroma to support human haemopoiesis in vitro and compared this with support provided by human stroma. We found that, overall, human stroma was significantly more effective than sheep stroma in supporting human haemopoiesis in vitro. Nevertheless, stroma established from bone marrow of normal sheep was capable of lower‐level support of the proliferation and multilineage differentiation of highly purified (CD34+, CD38−) preparations of human HSC in vitro. At lower concentrations, sheep stroma conditioned media (CM) induced human bone marrow mononuclear cells enriched for the CD34‐positive fraction to enter the cell cycle at levels comparable to that of human stroma CM. However, unlike human CM, at higher concentrations, sheep CM inhibited human cells from initiating the cell cycle. These results provide a possible explanation for the long‐term maintenance of low levels of human haemopoietic cell activity in human/sheep chimaeras.
ACUTE‐PHASE RESPONSE AND THE HYPERCOAGULABLE STATE IN PULMONARY TUBERCULOSIS
Authors: Robson Simon C. , White Neil W. , Aronson Ingrid , Woollgar Roderick , Goodman Hillel , Jacobs Peter
Published: October 2003

In our experience, severe pulmonary tuberculosis (PTB) is often complicated by deep venous thrombosis (DVT). Because of the association between inflammation and haemostatic changes that can result in a hypercoagulable state, we have prospectively examined such predisposing factors in representative patients.  Sequential analyses in a control group with active PTB showed anaemia, thrombocytosis, elevations in plasma fibrinogen, fibrin(ogen) degradation products (FDP), tissue plasminogen activator (t‐PA) and inhibitor (PAI‐1) with depressed antithrombin III levels. Age, sex and disease matched individuals with venographically proven DVT had higher FDP (15.8 ± 14.3 v 3.2 ± 1.7 μg/ml : P < 0.01), t‐PA (19.4 ± 14.9 v 11.3 ± 0.8 ng/ml : P < 0.01), and functional PAI‐1 activity (11.6 ± 6.3 v 4.2 ± 4.1 : P < 0.01) with lower platelet counts (347 ± 110 v 563 ± 230 × 109/l : P < 0.01). Fibrinogen levels in all patients rose during the first 2 weeks of therapy and, together with related disturbances, corrected within 12 weeks.  In conclusion, elevated plasma fibrinogen with impaired fibrinolysis coupled with a decrease in antithrombin III and reactive thrombocytosis would appear to favour the development of DVT in PTB.
The reduction of large von Willebrand factor multimers in plasma in essential thrombocythaemia is related to the platelet count
Authors: Van Genderen Perry J. J. , Budde Ulrich , Michiels Jan J. , Van Strik Roel , Van Vliet Huub H. D. M.
Published: October 2003

We have investigated the relationship between platelet count and large VWF (von Willebrand factor) multimers in the plasma of 36 patients with essential thrombocythaemia (ET) and 26 patients with reactive thrombocytosis (RT). In both ET and RT patients an inverse relationship could be established between platelet count and large VWF multimers in plasma as well in relatively decreased ristocetin cofactor/von Willebrand factor antigen and collagen binding activity/von Willebrand factor antigen ratios. A normalization of the platelet count was accompanied by restoration of a normal plasma VWF multimeric distribution. Our data suggest that increasing numbers of platelets circulating in blood result in increased removal of large VWF multimers from plasma.
Evaluation of cytogenetic conversion to Ph− haemopoiesis in long‐term bone marrow culture for patients with chronic myeloid leukaemia on conventional hydroxyurea therapy, on pulse high‐dose hydroxyurea and on interferon‐alpha
Authors: Coutinho L. H. , Brereton M. L. , Santos A. M. W. , Ryder W. D. J. , Chang J. , Harrison C. J. , Liu Yin J. A. , Dexter T. M. , Testa N. G.
Published: October 2003

Long‐term bone marrow culture (LTBMC) has been used successfully in autologous transplantation in chronic myeloid leukaemia (CML). However, variation between patients in the recovery of Ph− cells in culture limits the application of this procedure to a minority. Treatment that effectively reduces in vivo tumour burden prior to initiation of LTBMC may improve the selection of Ph− cells in culture. To test this hypothesis we evaluated the frequency and degree of cytogenetic conversion to Ph− haemopoiesis in LTBMC from four independent groups of CML patients: Untreated (n = 19); conventional dosage of hydroxyurea (HU) (n = 10); pulse high‐dose HU (P‐HU) (n = 22) and interferon (IFN)‐α (n = 12). In this study IFN‐α therapy resulted in a significantly higher incidence of patients with detectable Ph− clonogenic cells in the marrow (P = 0.01) and with ≥50% Ph− haemopoiesis in LTBMC as compared to newly diagnosed patients (P = 0.05). Also, sequential culture studies undertaken in 14 CML patients at diagnosis and following the start of pulse high‐dose HU therapy showed that in eight patients the average proportion of Ph− metaphases detected in LTBMC substantially increased from 1.7% (range 0–7) at diagnosis to levels of 71% (range 14–100) after treatment. Therefore we conclude that the use of IFN or pulse high‐dose HU in early stage disease appears to create an opportunity to harvest the marrow for long‐term culture (LTC) purging with reduced leukaemic burden.
A sensitive sandwich ELISA for measuring thrombopoietin in human serum: serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders
Authors: TAHARA TOMOYUKI , USUKI KENSUKE , SATO HIROAKI , OHASHI HIDEYA , MORITA HARUHIKO , TSUMURA HARUHIKO , MATSUMOTO ATSUSHI , MIYAZAKI HIROSHI , URABE AKIO , KATO TAKASHI
Published: October 2003

A sensitive sandwich enzyme‐linked immunosorbent assay (ELISA) has been established to estimate serum thrombopoietin (TPO) concentrations in healthy volunteers and patients with haemopoietic disorders. The ELISA uses a mouse monoclonal antibody (Ab) as the capture Ab and a biotinylated rabbit polyclonal Ab as the detector. The ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intra‐ and inter‐assay variation were from 3.0% to 4.9% and from 5.9% to 6.1%, respectively. The quantitative limit of the ELISA was 0.09 fmol/ml in serum. The quantitative limit was lower than the normal level. The dose–response curves of serum samples from healthy volunteers and patients with haemopoietic disorders were parallel to the standard curves. The ELISA did not cross‐react with a variety of blood components and cytokines to produce false‐positive results.

The serum TPO concentrations from 29 normal males and 21 females were 0.79 ± 0.35 and 0.70 ± 0.26 fmol/ml, respectively. Serum TPO levels in patients with aplastic anaemia (AA), acute lymphocytic leukaemia (ALL) and essential thrombocythaemia (ET) were measured using the ELISA. The serum TPO levels in the patients with ET (n = 6, 2.80 ± 1.55 fmol/ml) were higher than the normal level. The patients with AA (n = 7, 18.53 ± 12.37 fmol/ml) and ALL (n = 5, 10.36 ± 5.57 fmol/ml) had significantly higher serum TPO levels than normal individuals. These results indicate that the ELISA specific to TPO should prove useful in measuring the TPO concentration in serum samples.
IN VITRO RESISTANCE TO CYTOSINE ARABINOSIDE, NOT TO DAUNORUBICIN, IS ASSOCIATED WITH THE RISK OF RELAPSE IN DE NOVO ACUTE MYELOID LEUKAEMIA
Authors: Klumper Edwin , Ossenkoppele Gert J. , Pieters Rob , Huismans Dieuwke R. , Loonen Annemarie H. , Rottier Antoinette , Westra Guus , Veerman Anjo J. P.
Published: October 2003

The efficacy of chemotherapy in acute myeloid leukaemia (AML) is limited by clinical drug resistance. We determined in vitro resistance to cytosine arabinoside (ARA‐C), daunorubicin (DNR), mitoxantrone (MITOX), m‐amsacrine (AMSA) and etoposide (VP16) in 49 adults with de novo AML using the MTT assay. Results showed that non‐ responders to chemotherapy were, in vitro, 2.9‐fold more resistant to DNR, but not more resistant to ARA‐C, compared to complete responders. However, complete responders who were in vitro resistant to ARA‐C had a 4‐fold higher risk of relapse (95% CI 1.3–12.5‐fold) compared to complete responders in vitro sensitive to ARA‐C. With a mean follow‐up of 12 months the probability of continuous complete remission (CCR) for patients in vitro sensitive to ARA‐C was 61% at 34 months (95% CI 28–82%), whereas all patients in vitro resistant to ARA‐C relapsed within 18 months from diagnosis. This difference appeared to be independent of other clinical features such as sex, age, white blood cell count, FAB classification, and CD34 expression. In vitro resistance to DNR was not related to the probability of CCR. We conclude that in vitro drug resistance assessed with the MTT assay appears to be associated with short‐ and long‐term clinical outcome in AML. Confirmatory studies comprising a sufficient number of patients for multivariate analyses should prove whether in vitro resistance to ARA‐C will appear to be an independent risk factor.
A novel delivery system for continuous desferrioxamine infusion in transfusional iron overload
Authors: ARAUJO A. , KOSARYAN M. , MACDOWELL A. , WICKENS D. , PURI S. , WONKE B. , HOFFBRAND A.V.
Published: October 2003

Fifteen iron‐overloaded thalassaemia major (TM) patients and two homozygous sickle cell patients (SCD) were treated continuously for 7d each week with the novel 48h continuous subcutaneous (s.c.) desferrioxamine (DFX) delivery device (code C1083, Baxter) and 10 TM patients received the 24h continuous intravenous (i.v.) DFX delivery device (code C1071). The 27 patients had previously received conventional s.c. DFX for 8–10h on 5 or more days each week. The serum non‐transferrin bound iron (NTBI) levels fell significantly in both groups within 12h of commencing the continuous infusion. In the s.c. group the mean level fell from 4.2 to 2.0μmol/l (P=0.001), whereas in the i.v. group the mean level fell from 3.6 to 0.1μmol/l (P=0.006) the initial levels being measured 12h after stopping conventional s.c. DFX. After 4 weeks there was a significant fall in serum ferritin in both groups (P=0.009). The new DFX delivery device is effective at removing toxic‐free iron from plasma and reducing body iron. Moreover, it is preferred by patients with much improved compliance compared to the conventional s.c. DFX pump.
Inhibition of growth and induction of apoptosis by all‐trans retinoic acid in lymphoid cell lines transfected with the PML/RARα fusion gene
Authors: Maeda Yasuhiro , Horiuchi Fusanari , Miyatake Jun‐ichi , Sono Hiroshi , Tatsumi Yoichi , Urase Fumiaki , Irimajiri Kiyohiro , Horiuchi Atsushi
Published: October 2003

The interaction of an exogenous PML/RARα fusion gene associated with acute promyelocytic leukaemia, with all‐trans retinoic acid (ATRA) was examined in two lymphoid cell lines. L1210 and MOLT‐4 cells were transfected with PML/RARα cDNA in the expression vector pGD and stable transformants (L1210PML/RARα and MOLT‐4PML/RARα) were selected with G418. ATRA inhibited the growth of these stable transformants, as assessed by [3H]thymidine incorporation, in a dose‐dependent manner, but had no effect on the growth of control cells stably transformed with neomycin resistant gene alone. ATRA also induced apoptosis, as assessed by fragmentation of genomic DNA, in L1210PML/RARα and MOLT‐4PML/RARα cells but not in control cells. The exogenous PML/RARα fusion gene therefore probably mediates the effects of ATRA on cell growth and apoptosis in these cell lines.
Granulocyte colony‐stimulating factor (G‐CSF) increases neutrophil migration across vascular endothelium independent of an effect on adhesion: comparison with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)
Authors: Yong Kwee
Published: October 2003

Granulocyte colony‐stimulating factor (G‐CSF) increases neutrophil counts, and enhances and primes many neutrophil functions, implicating a role for this growth factor in host defence. This study investigated whether G‐CSF is able to directly influence the transendothelial migration of neutrophils, and how such effects might be related to other effects on neutrophil adhesive properties. G‐CSF, like GM‐CSF, increased surface levels of the adhesive receptor, CD11b/CD18, but down‐regulated L‐selectin expression on neutrophils. Unlike GM‐CSF, however, G‐CSF had no effect on neutrophil adhesion to endothelium. Despite the lack of effect on neutrophil adhesion, G‐CSF was able to produce significant enhancement of neutrophil transmigration across unstimulated endothelium in vitro. When used at an optimal concentration of 100 ng/ml, G‐CSF increased neutrophil migration to 217 ± 19% of baseline levels (P<0.001, n = 10). This effect was similar to that previously demonstrated for GM‐CSF (which increased migration to 271 ± 40%, P<0.001, n = 12). G‐CSF‐induced transmigration, like GM‐CSF induced migration, was independent of concentration gradients, suggesting that these are not simply chemotactic effects. G‐CSF differs from GM‐CSF, however, in that although GM‐CSF inhibited neutrophil migration across IL‐1‐activated endothelium (33 ± 8% inhibition, n = 6, P<0.01), G‐CSF had no effect on neutrophil migration across IL‐1 activated endothelium. Hence G‐CSF, despite having no effect on neutrophil adhesion to endothelium, is a powerful stimulator of transmigration, and, unlike GM‐CSF, does not inhibit cell movement across inflamed endothelium. These results suggest that G‐CSF is able to influence neutrophil recruitment into local infective sites, and, further, that G‐CSF mobilized cells would be competent to migrate into tissues in response to inflammatory stimuli.
Coexistence of familial antiphospholipid syndrome and factor V Leiden: impact on thrombotic diathesis
Authors: Brenner B. , Vulfsons S. L. , Lanir N. , Nahir M.
Published: October 2003

Two young siblings who presented with an unusual recurrent severe thromboembolic phenomenon were found to have familial anti‐phospholipid syndrome and were heterozygous for the factor V R506Q mutation. The coexistence of hereditary and acquired APC‐resistance may explain the severity of thromboembolism.