bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of IL‐6 receptor expression in fresh childhood Burkitt's lymphoma cells and induction of IL‐6 receptors by Epstein‐Barr virus in vitro
Authors: Tomeczkowski Jörg , Zur Stadt Udo , Reiter Alfred , Welte Karl , Sykora Karl‐Walter
Published: November 2003

Interleukin‐6 (IL‐6) is a cytokine which is necessary for the differentiation of activated B cells and growth of early haemopoietic progenitors. It is used for ex‐vivo expansion of myeloid progenitors in the setting of high‐dose chemotherapy with autologous bone marrow transplantation (BMT). Expression of the IL‐6 receptor (IL‐6R) was examined in six fresh Burkitt’s lymphoma (BL) cell preparations and 12 BL cell lines by reverse transcriptase polymerase chain reaction (RT‐PCR) and flow cytometry (FCM). Inducibility of IL‐6R mRNA expression by Epstein‐Barr virus (EBV) was studied by comparing two uninfected cell lines with the same cell lines infected by EBV. The phenotype of the BL cells lines was analysed by FCM and by proliferation assays in the presence of anti‐IgM antibodies.

None of the fresh BL cells expressed the IL‐6 receptor. The BL cell lines expressed varying degrees of IL‐6R mRNA and protein. In vitro infection of EBV‐negative BL cell lines resulted in up‐regulation of IL‐6R mRNA. There was no proliferative response of the BL cell lines to IL‐6, including the cells that expressed the receptor. Compared to uninfected BL cell lines, EBV‐infected cell lines and lymphoblastoid cell lines (LCLs) showed a weaker or no response to anti‐IgM antibodies, indicating a more mature phenotype of these cells. We conclude that the IL‐6 receptor is not expressed in fresh childhood BLs, but only in BL cell lines. EBV infection in vitro leads to an up‐regulation of IL‐6R mRNA but not to increased proliferation. This makes growth stimulation of contaminating BL cells in the setting of autologous BMT unlikely.
The effect of cardiopulmonary bypass on circulating megakaryocytes
Authors: Wilde N. T. , Burgess R. , Keenan D. J. M. , Lucas G. S.
Published: November 2003

Megakaryocytes (Mks) are found in the lungs and the blood stream as well as in the bone marrow. We modified a whole blood filtration method for Mks by immunostaining for CD61 using biotin streptavidin, and used this technique to study Mks and their morphology in the central venous and arterial circulations before, during and after cardiopulmonary bypass (CPB) in haematologically normal patients undergoing routine cardiac surgery.

Blood samples were taken immediately after the insertion of central venous (V) and arterial (A) catheters and after thoracotomy, immediately before bypass. Further samples were taken after 60–90 min on‐CPB and 180–240 min post‐bypass. In comparison with the steady state before bypass, circulating Mk levels in blood on bypass increased dramatically, from (V) 10.93 ± 3.94/ml (mean ± SD) to 36.48 ± 11.52/ml and from (A) 8.37 ± 4.39/ml to 38.65 ± 20.68/ml. This effect was still present, to a lesser extent, 180–240 min post‐bypass. Circulating levels of Mks were consistently lower in the arterial circulation than in the venous circulation off bypass, but levels in the two circulations were comparable during CPB, confirming previous suggestions that the lungs are net removers of Mks from the circulation. Type 4 Mks, the largest and most normal morphologically, were rarely seen in arterial blood, but increased significantly during CPB, indicating that the lungs selectively remove large Mks.

The lungs appear to play an active role in the regulation of Mk levels. This is lost during CPB and despite the extracorporeal 40 μm arterial line filter, large Mks enter the systemic circulation. More effective extracorporeal filtration of large Mks might reduce the neurological impairment seen in some patients who have undergone CPB.
Quantitation of platelet‐specific autoantibodies in platelet eluates of ITP patients measured by a novel ELISA using the purified glycoprotein complexes GPIIb/IIIa and GPIb/IX as antigens
Authors: Hürlimann‐Forster Marianne , Steiner Beat , Von Felten Arthur
Published: November 2003

Immune thrombocytopenic purpura (ITP) is a disorder caused by anti‐platelet autoantibodies (Ab), most of which are directed against epitopes on platelet membrane glycoprotein complexes GPIIb/IIIa and GPIb/IX. To detect platelet Ab, reliable techniques, such as MAIPA or immunobead assay, have been developed. They all achieve their selective specificity by the use of monoclonal antibodies (MoAb) against defined glycoproteins of the platelet membrane. In order to determine the most frequent Ab‐specificities, a novel enzyme‐linked immunosorbent assay, named platelet‐glycoprotein‐ELISA (P‐GP‐ELISA), has been developed. It uses purified GPIIb/IIIa and GPIb/IX complexes, respectively, as antigens and enables determination of platelet‐associated as well as circulating Ab (IgG, IgM). MoAbs are not required and therefore there is no risk of competition between MoAb and Ab. Levels of Ab in patients with the clinical diagnosis of an idiopathic thrombocytopenic purpura were analysed. 92.7% (76/82) platelet eluates with significantly increased levels of Ab against at least one of the glycoproteins were found, whereas no sample from healthy volunteers (0/37) gave a positive result, pointing to a high sensitivity and specificity of the test system. Since its application is also easy and quick, P‐GP‐ELISA should facilitate detection of Ab against platelet membrane proteins in routine determinations.
Hypodiploidy and 22q11 rearrangements at diagnosis are associated with poor prognosis in patients with multiple myeloma
Authors: Calasanz María J. , Cigudosa Juan C. , Odero MaríA D. , García‐Foncillas JesúS , Marín Ulián , Ardanaz Mariá T. , Rocha Eduardo , Gullón Arturo
Published: November 2003

Our aim was to evaluate the clinical use of cytogenetic analysis as a prognostic factor in the outcome of newly diagnosed multiple myeloma (MM) patients. The present series includes 111 newly diagnosed MM patients treated with one of three standard‐dose regimens or autologous transplantation over an 8‐year time interval. As expected, the presence of an abnormal karyotype (39% of patients) correlated with poor prognosis (progression rate 63% v 47%, P = 0.042), shorter event‐free (EFS, P = 0.014) and overall (OS, P = 0.005) survival. Two distinct cytogenetic abnormalities were the most significant variables that influenced EFS and OS in the univariate analysis. The presence of hypodiploid karyotypes or rearrangements of band 22q11 were associated with higher progression rate (P = 0.001) and shorter EFS (P < 0.024) and OS (P < 0.004). The median EFS and OS for patients with hypodiploidy was 4 and 7 months respectively. Multivariate analysis showed that absence of hypodiploidy was the most favourable prognostic variable for OS (P = 0.022) followed by stage ≤IIA, serum calcium ≤2875 μmol/l, and absence of abnormalities 22q. The data suggest that the presence of hypodiploid karyotypes and rearrangements on 22q11 band show a higher progression rate and shorter survival in MM patients.
Rarity of microsatellite genomic instability in B‐cell non‐Hodgkin's lymphomas in hepatitis C virus‐infected patients
Authors: De Vita Salvatore , Gasparotto Daniela , Pivetta Barbara , Vukosavljevic Tamara , Zagonel Vittorina , Carbone Antonino , Boiocchi Mauro
Published: November 2003

Several groups have emphasized the likely impli‐cation of the hepatitis C virus (HCV) in a fraction of B‐cell non‐Hodgkin's lymphomas. Since only a minority of patients with HCV infection and monoclonal mixed cryoglobulinaemia develop overt lymphoma, the identification of predisposing factors has relevant clinical implications. The replication error phenotype (RER+), as revealed by widespread microsatellite instability, is caused by defects in DNA mismatch repair genes, and has been frequently disclosed in subsets of B‐cell lymphomas with underlying infection and chronic inflammation. We therefore investigated the occurrence of the RER+ phenotype in a series of eight consecutive B‐cell NHLs in patients with chronic infection by HCV. A polymerase chain reaction‐based assay was used to analyse an extended panel of 15 microsatellite loci.

Microsatellite instability was not observed in six tumour samples in any locus; the two remaining cases showed instability at only one locus.

Therefore genetic instability by defects in DNA mis‐match repair genes should not represent the general mechanism predisposing to overt lymphoma in HCV‐infected patients. Although a clearer definition of HCV‐related B‐cell disorders should better address future studies on genetic instability in larger series, we recommend additional oncogenetic pathways as the target of further research.
Instability in the ATCT variable number tandem repeat locus VWF.VNTR I in intron 40 of the von Willebrand factor gene
Authors: Haddad Albert P. , Sparrow Rosemary L.
Published: November 2003

The von Willebrand factor gene intron 40 variable number tandem repeat VWF.VNTR I exhibits 10 alleles making it highly polymorphic and useful for parentage and forensic testing. 45 unrelated families (210 meiotic events) were tested for VWF.VNTR I alleles. One spontaneous mutation was observed in a family member. Haplotype analysis demonstrated that this mutation was due to a gain of one motif repeat by a paternal allele. Sequence analysis confirmed the difference in the number of motif repeats between the proband and the alleles expressed by the parents. This instability emphasizes the importance of demonstrating exclusion in at least two separate loci in parentage testing.
Detection of maternal cells in human fetal blood during the third trimester of pregnancy using allele‐specific PCR amplification
Authors: PETIT THIERRY , DOMMERGUES MARC , SOCIÉ GÉRARD , DUMEZ YVES , GLUCKMAN ELIANE , BRISON OLIVIER
Published: November 2003

Using a highly sensitive allele‐specific PCR amplification method, we have previously shown that maternal cells could be detected in all 10 cord bloods tested. This raised the question of whether maternal cells are released into cord blood during the process of delivery or whether they are already present during pregnancy. We have now used the same PCR method to detect the presence of maternal cells in nine fetal blood samples collected at different gestational ages. Maternal cells were detected in eight samples obtained between 24 and 35 weeks of gestation. They were estimated to amount between 10−4 and 10−5 of nucleated fetal blood cells. In two cases mononuclear and polymorphonuclear cell fractions were separated by Ficoll gradient centrifugation and maternal cells were detected as comparable levels in both fractions. Maternal cells could not be detected in the one fetal blood sample obtained at 20 weeks of gestation, suggesting that maternal cells could appear at detectable levels in fetal blood during the third trimester of pregnancy. These results are discussed in terms of materno‐fetal immune tolerance and of transmission of viruses (and more specifically of the human immunodeficiency virus) from mother to child.
BCL6 gene rearrangements also occur in marginal zone B‐cell lymphoma
Authors: DIERLAMM JUDITH , PITTALUGA STEFANIA , STUL MICHEL , WLODARSKA IWONA , MICHAUX LUCIENNE , THOMAS JOSÉ , VERHOEF GREGOR , VERHEST ALAIN , DEPARDIEU CLAUDE , CASSIMAN JEAN‐JACQUES , HAGEMEIJER ANNE , DE WOLF‐PEETERS CHRISTIANE , VAN DEN BERGHE HERMAN
Published: November 2003

Marginal zone B‐cell lymphoma (MZBCL) represents a distinct subtype of B‐cell non‐Hodgkin's lymphoma (NHL) which has been recently recognized and defined as a disease entity. Cytogenetically, these lymphomas reveal a high prevalence of trisomy 3, and recent data obtained by comparative genomic hybridization indicate that the chromosomal regions 3q21‐23 and 3q25‐29 might be of particular pathogenetic significance. We identified structural chromosomal abnormalities involving the region 3q27 and rearrangements of the BCL6 proto‐oncogene in three out of 34 (9%) well‐defined cases of extranodal, nodal and splenic MZBCL using cytogenetic analysis, Southern blot, and fluorescence in situ hybridization (FISH). All three cases were characterized by a t(3;14)(q27;q32). Two of them showed additional chromosomal abnormalities including trisomy 3, which was found in one case. The patients displayed extranodal disease and did not demonstrate any striking clinical and histological differences when compared with MZBCL lacking BCL6 rearrangement. The present study for the first time demonstrates the occurrence of t(3;14)/BCL6 gene rearrangement in MZBCL, thus suggesting a role of the BCL6 proto‐oncogene in the pathogenesis of MZBCL.
Bisphosphonates induce apoptosis in human myeloma cell lines: a novel anti‐tumour activity
Authors: Shipman Claire M. , Rogers Michael J. , Apperley Jane F. , Russell R. Graham G. , Croucher Peter I.
Published: November 2003

Bisphosphonates are in widespread use to prevent bone resorption in a number of metabolic and tumour‐induced bone diseases including multiple myeloma. Recent reports suggest that bisphosphonate treatment may be associated with an increase in patient survival, raising the possibility that these compounds may have a direct effect on the tumour cells. We have investigated whether the bisphosphonates clodronate, pamidronate and YM175 can directly affect the human myeloma cell lines U266‐B1, JJN‐3 and HS‐Sultan in vitro. The effect of bisphosphonate treatment on cell number and cell cycle progression was examined using flow cytometry. The ability of bisphosphonates to induce apoptosis in human myeloma cell lines was determined on the basis of changes in nuclear morphology and of DNA fragmentation. Pamidronate and the more potent bisphosphonate, YM175, significantly decreased cell number (P < 0.001) in JJN‐3 and HS‐Sultan cells. YM175 also caused cells to arrest in the S‐phase of the cell cycle in the JJN‐3 cell line. Both pamidronate and YM175 also caused an increase in the proportion of cells with altered nuclear morphology (P < 0.05) and fragmented DNA, characteristic of apoptosis, in both JJN‐3 and HS‐Sultan cells. In contrast, clodronate had little effect on cell number and did not cause apoptosis at the concentrations examined. These data raise the possibility that some bisphosphonates could have direct anti‐tumour effects on human myeloma cells in vivo.
Effects of in vivo administration of G‐CSF on neutrophil and eosinophil adhesion
Authors: Håkansson L. , Höglund M. , Jönsson U.‐B. , Torsteinsdottir I. , Xu X. , Venge P.
Published: November 2003

The effect in vivo of G‐CSF on neutrophil and eosinophil adhesion was studied after subcutaneous administration to six healthy individuals of human recombinant glycosylated G‐CSF (lenograstim) (3 μg/kg) for 6 consecutive days.

Basal adhesion and adhesion to E‐selectin, VCAM‐1 and ICAM‐1 of neutrophil and eosinophil granulocytes were measured selectively. During G‐CSF administration neutrophil basal adhesion increased from 7.4 ± 3.9% (mean ± SD) to 55.8 ± 12.9% and 23.2 ± 4.4%, 4 and 7 d, respectively, after start of the administration. At the same time points eosinophil basal adhesion increased from 7.1 ± 2.4% to 37.7 ± 6.1% and 13.1 ± 5.3%, respectively. When adhesion was measured in the presence of Mn2+, which increases the functional activity of integrins, an even higher increase of neutrophil and eosinophil basal adhesion was noted 4 and 7 d, respectively, after start of G‐CSF administration. In parallel with the enhanced basal adhesion neutrophil adhesion to E‐selectin and ICAM‐1 and eosinophil adhesion to E‐selectin, VCAM‐1 and ICAM‐1 were significantly (P < 0.05) increased after 4 d of G‐CSF administration as was neutrophil cell surface expression of CD11b and CD18. In vitro G‐CSF induced minimal changes of granulocyte basal adhesion and inhibition of the adhesion to E‐selectin. 10 ng/ml TNFα significantly increased neutrophil and eosinophil basal adhesion and adhesion to VCAM‐1 and ICAM‐1.

In summary, administration of G‐CSF to healthy subjects induced enhanced adhesion of neutrophil and eosinophil granulocytes, probably mediated by an increase of the functional capacity of β1‐ and β2‐integrins. The induction of increased levels of TNFα might be one mechanism behind the in vivo effect of G‐CSF administration.
Rat liver biliary epithelial cells support long‐term production of haemopoietic progenitors from human CD34+ cells
Authors: Lamy Isabelle , Corlu Anne , Fardel Olivier , Gandemer Virginie , Rialland Mickael , Leberre Claudine , Le Prise Pierre‐Yves , Fauchet Renee , Coulombel Laure , Guguen‐Guillouzo Christiane
Published: November 2003

In this study we report the supportive activity of rat liver epithelial cells (RLEC) on human haemopoiesis in the absence of exogeneously supplied growth factors. RLEC is a rat cell line derived from primitive biliary cells with epithelial characteristics which induce the long‐term differentiation of hepatocytes through cell–cell contacts. We have established the ability of these cells to sustain long‐term survival and multilineage differentiation of human haemopoietic progenitors from unfractionated bone marrow and growth‐factor mobilized peripheral blood cells, and from human CD34+ and CD34+ CD38− haemopoietic cells, with a higher efficiency than the murine MS‐5 stromal cell line: the numbers of committed progenitors recovered from RLEC cocultures after 8 weeks were 3‐fold higher than fromMS‐5 cocultures, with an unusually high BFU‐E production. Furthermore, using diffusible insert cultures, we demonstrated that, despite the lack of strong adhesive interaction between haemopoietic cells and RLEC, physical proximity was absolutely required for optimum stimulation of LTC‐IC by RLEC. Taken together, these results show that biliary epithelial cells support human haemopoiesis and cause speculation that common mechanisms might be used by RLEC to regulate both the hepatocyte and the haemopoietic progenitors differentiation.
High‐resolution cell division tracking demonstrates the Flt3‐ligand‐dependence of human marrow CD34+CD38− cell production in vitro
Authors: Nordon Robert E. , Ginsberg Steven S. , Eaves Connie J.
Published: November 2003

Investigation of primitive human haemopoietic cell behaviour requires methodologies for monitoring asynchronously activated cells over several generations. We describe a high‐resolution procedure for tracking 5‐ (and 6‐) carboxyfluorescein diacetate succinimidyl ester (CFSE)‐labelled human haemopoietic cells through six cell cycles based on the precise halving of their CFSE‐fluorescence at each mitosis. Using this approach in combination with DNA or surface antigen staining, we show that the addition of Flt3‐ligand (FL) to a cytokine cocktail consisting of Steel factor, IL‐3, IL‐6 and G‐CSF increased the proportion of CD34+ (CD45RA/CD71)−, but not CD34+(CD45RA/CD71)+, human marrow cells initially recruited into division in vitro, shortened the overall cycle time of their progeny, and enhanced the production of a derivative CD34+CD38− population through several (up to four) cell generations. These studies also showed that during the first 4 d there was no detectable apoptosis among the progeny of the CD34+(CD45RA/CD71)− cells generated in the presence of this four‐cytokine cocktail, regardless of the presence of FL. The availability of a technique for monitoring changes in the properties of individual cells as a function of their mitotic history and under conditions where they are asynchronously recruited to divide provides a new and powerful approach for studies of the regulation of primitive human haemopoietic cell proliferation and differentiation.
Cyclophosphamide therapy for pure red cell aplasia associated with granular lymphocyte‐proliferative disorders
Authors: YAMADA OSAMU , MIZOGUCHI HIDEAKI , OSHIMI KAZUO
Published: November 2003

Granular lymphocytes have been characterized as cells with azurophilic granules in the cytoplasm. Patients with increased numbers of granular lymphocytes are designated as granular lymphocyte‐proliferative disorders (GLPDs). A variety of haematological abnormalities are associated with T‐cell‐lineage GLPD. Among these, pure red cell aplasia is frequent, and adequate therapy is required. Seven patients with pure red cell aplasia, or a related condition complicating T‐cell‐lineage GLPD, were entered into this study. Cyclophosphamide was initiated at a daily oral dose of 100 mg. After 2 weeks the dose was reduced to 50 mg/d, and maintained at that dose. Cyclophosphamide was administered until the lymphocyte count was <1 ×109 l and T‐cell receptor‐β gene analysis was used to monitor the response to treatment. All the patients were successfully treated, irrespective of their former treatment. Clinical remission was associated with the disappearance of the abnormal granular lymphocyte clone, as detected by Southern blot hybridization analysis. Therapeutic responses began after 8 weeks, and clinical complete remissions were obtained after 6 months. Oral cyclophosphamide monotherapy can successfully treat the pure red cell aplasia associated with T‐cell‐lineage GLPD.
Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis
Authors: MARTYRÉ M.‐C. , LE BOUSSE‐KERDILES M.‐C. , ROMQUIN N. , CHEVILLARD S. , PRALORAN V. , DEMORY J.‐L. , DUPRIEZ B.
Published: November 2003

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte‐derived growth factors in its pathogenesis has been documented.

We previously reported increased expression of TGF‐β in patients with idiopathic myelofibrosis. In the present study we show that circulating megakaryocytic cells from such patients expressed high levels of basic fibroblast growth factor (bFGF). An increased expression of bFGF was also detected in patients’ platelets. Under culture conditions, bFGF present in megakaryocytic cells was not exported into the medium, consistent with the fact that bFGF is devoid of a secretion peptide signal. Interestingly, this lack of bFGF secretion was observed in all patients but one, who was in an accelerated phase of the disease and presented an important percentage of circulating megakaryoblasts.
Collection of peripheral blood stem cells from normal donors 60 years of age or older
Authors: Anderlini Paolo , Przepiorka Donna , Lauppe Jo , Seong David , Giralt Sergio , Champlin Richard , Körbling Martin
Published: November 2003

We report 14 normal peripheral blood stem cell (PBSC) donors ≥ 60 years of age who had cytokine mobilization followed by PBSC apheresis for allogeneic transplantation. Mobilization was achieved with filgrastim (6 μg/kg twice daily). Their median age was 63.5 years (range 60–77), and 43% had a positive medical history, mainly hypertension and/or cardiac problems. Their median pre‐apheresis leucocyte count (×109/l) was 38.6 (range 29.6–63.4). The median apheresis yield (×106 CD34+ cells/litre blood processed, first apheresis) was 27.9 (range 1.6–54.8). The target cell dose (≥4& times; 106 CD34+ cells/kg recipient) was reached with one procedure in eight (57%) donors. Filgrastim‐related adverse events were acceptable and apheresis was well tolerated. When compared to younger donors (<60 years of age), a trend to a lower CD34+ apheresis yield and a requirement for more than one apheresis to achieve the collection target (≥4 ×106 CD34+ cells/kg) was evident. Although older (≥60 years) donors seem to mobilize less effectively, these data suggest that PBSC collection from them is feasible and has an acceptable short‐term safety profile.
AMYLOIDOSIS: A REVIEW OF RECENT DIAGNOSTIC AND THERAPEUTIC DEVELOPMENTS
Authors: Gillmore Julian D. , Hawkins Philip N. , Pepys Mark B.
Published: November 2003

Amyloid deposition is associated with a diverse range of disorders that includes Alzheimer's disease, type II diabetes mellitus and dialysis arthropathy. Although less common, systemic AA and AL amyloidosis remain important because effective treatments have increasingly become available.

The pathology in all forms of amyloidosis involves the extracellular deposition of protein as characteristic fibrillar aggregates which interfere with tissue structure and function. Amyloid fibrils are derived from different unrelated proteins in the different forms of the disease but share many common properties, including the capacity to bind the normal plasma protein serum amyloid P component (SAP). This is the basis for our development of radiolabelled SAP as a nuclear medicine tracer for the diagnosis and quantitative monitoring of amyloid. Serial studies have shown that the deposits are far from inert but are actually turned over quite rapidly in many patients.

The treatment of amyloidosis involves supportive measures whilst every effort is made to reduce the supply of the respective fibril precursor protein. Under favourable circumstances further amyloid deposition will be prevented, existing deposits will regress and improvement of organ function will occur. Since this strategy is not always possible or may fail, new approaches to inhibit fibril formation and promote regression of amyloid are being pursued.
Outcome and lineage involvement in t(12;21) childhood acute lymphoblastic leukaemia
Authors: Lanza Carlo , Volpe Gisella , Basso Giuseppe , Gottardi Enrico , Barisone Elena , Spinelli Monica , Ricotti Emanuela , Cilli Vania , Perfetto Fatima , Madon Enrico , Saglio Giuseppe
Published: November 2003

The t(12;21)(p13;q22) translocation has been described recently as the most recurrent genetic lesion in paediatric acute lymphoblastic leukaemias (ALLs). It has also been associated with B‐precursor lineage involvement and good outcome.

We tested 51 diagnostic paediatric ALLs and found 11 cases with molecular evidence of the t(12;21). Interestingly, amongst t(12;21) positive patients, we report three cases with hybrid phenotype, and two cases showing an aggressive and fatal disease. Our data show that the t(12;21) does not represent an independent good‐risk indicator. Long follow‐ups and additional molecular investigations are needed to assess the prognostic and pathogenetic relevance of t(12;21) in childhood ALLs.
Automated red cell exchange in sickle cell disease
Authors: Janes Sarah L. , Pocock Michaela , Bishop Elizabeth , Bevan David H.
Published: November 2003

Red cell exchange is important in the care of acutely ill sickle‐cell patients, and may be life‐saving. An automated red cell exchange technique has been developed using a Baxter blood cell separator, enabling an isovolaemic exchange to be performed within 2..5 h. A total of 20 procedures have been performed in 15 patients, including one woman in the third trimester of pregnancy, with a mean decrease of 72% in the circulating sickle haemoglobin (HbS) level. This method enables almost all adult patients with sickle cell anaemia to have their HbS reduced to safe levels by only one procedure. The procedure was well tolerated by all patients, including those who were acutely ill. This technique provides an effective procedure for reducing the percentage of circulating HbS rapidly in acutely ill patients with complications of sickle cell anaemia.
Molecular response of CML patients treated with interferon‐α monitored by quantitative Southern blot analysis
Authors: Reiter Andreas , Skladny Heyko , Hochhaus Andreas , Seifarth Wolfgang , Heimpel Hermann , Bartram Claus R. , Cross Nicholas C. P. , Hehlmann Rüdiger
Published: November 2003

We analysed 459 samples from 206 chronic myeloid leukaemia (CML) patients at diagnosis and during or after treatment with interferon‐α (IFN‐α) by quantitative Southern blot analysis for BCR rearrangement. In a minority (2%) of Ph‐positive patients, no BCR rearrangement was detectable due to breakpoints outside the major breakpoint cluster region (M‐bcr) or possibly due to M‐bcr deletions. Results from 235 samples were compared with the proportion of Ph‐positive metaphases found in contemporaneous bone marrow specimens analysed by conventional cytogenetics. The rank correlation between both methods was 0.82 (P<0.001). The proportion of CML cells in samples determined by Southern blot analysis (BCR ratio) was significantly different between cytogenetically‐defined minor, partial, and complete response groups (P<0.001). Empirically‐derived cut‐off points in the BCR ratio were introduced in order to define molecular response groups for comparison to standard cytogenetic response groups: a BCR ratio of 0% was defined as complete molecular response and ratios of 1–24%, 25–50%, and >50% were defined as partial, minor, and no molecular response, respectively. Using these cut‐off points the concordance between both methods was 67% (P<0.0001), a major cytogenetic response could be predicted or excluded in more than 90% of cases (P<0.0001). Our findings demonstrated that quantitative Southern blot was as sensitive as cytogenetics and as peripheral blood samples are suitable for this technique it should be considered as the method of choice for routine monitoring IFN‐α therapy in CML patients.
Oral squamous cell carcinoma after allogeneic bone marrow transplantation for Fanconi anaemia
Authors: Millen F. J. , Rainey M. G. , Hows J. M. , Burton P. A. , Irvine G. H. , Swirsky D.
Published: November 2003

We report a case of oral squamous cell carcinoma (SCC) originating in the buccal mucosa of an 18‐year‐old female patient with chronic graft‐versus‐host disease (GVHD) 9 years after HLA‐identical sibling bone marrow transplantation (BMT) for Fanconi anaemia (FA). The case highlights the problems of malignant change in FA and also the increased risk of second malignancy after BMT. The literature is reviewed with regard to previous cases and the possible aetiology of tumour formation. A high index of suspicion to any epithelial lesion in FA is appropriate so that early diagnosis may lead to improved prognosis.
Monitoring minimal residual disease in peripheral blood in B‐lineage acute lymphoblastic leukaemia
Authors: Brisco M. J. , Sykes P. J. , Hughes E. , Dolman G. , Neoh S‐H. , Peng L‐M. , Toogood I. , Morley A. A.
Published: November 2003

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.1 × 10−2 to < 9.4 × 10−7 leukaemic cells/total cells, corresponding to 1.3 × 107 to < 2 × 103 leukaemic cells/l. In 15 paired samples, leukaemia could be quantified in both tissues and in 20 paired samples, leukaemia was not detected in one or both tissues so that only upper level limits could be set. In the former 15 pairs, the level of leukaemia in peripheral blood was directly proportional to that in marrow but was a mean of 11.7‐fold lower. Leukaemia in blood was detected in 10/12 pairs in which the level in marrow was > 10−4, but in only two of 13 pairs in which the level in marrow was < 10−5. Patients studied at multiple time‐points showed parallel declines in the number of leukaemic cells in both tissues. The results showed that leukaemia could be monitored in peripheral blood during induction therapy, and quantitative considerations based on the results suggest that monitoring of blood during post‐induction therapy may be of value in detecting molecular relapse.
Hepatitis G virus RNA and its relation to hepatitis C infection in adult haemophilic patients
Authors: Tong C. Y. W. , Sallam T. A. , Williams H. , Mutton K. J. , Gilmore I. T. , Toh C. H.
Published: November 2003

The prevalence of hepatitis C virus (HCV) infection and hepatitis G virus (HGV) RNA were studied in 50 adult haemophilic patients who had received commercial clotting factors prior to 1980. HGV RNA was detectable in 6/50 patients (12%); 49/50 (98%) had antibody to HCV and 40/49 (82%) of these were viraemic with detectable HCV RNA; 5/6 patients with detectable HGV RNA had co‐existing HCV infection and viraemia. The HGV PCR products from all six patients were directly sequenced and all were shown to be similar to that of HGV but more diverse from that of GB virus C. One patient who had persistent abnormal liver function tests had detectable HGV RNA but no evidence of hepatitis B or C. The presence of HGV RNA in the absence of hepatitis B and C infection indicates that this virus is capable of independent transmission. Independent response to interferon was demonstrated in one patient with co‐infection who lost HGV but not HCV after interferon therapy.
BEAM chemotherapy and autologous haemopoietic progenitor cell transplantation as front‐line therapy for high‐risk patients with diffuse large cell lymphoma
Authors: Cortelazzo Sergio , Rossi Andrea , Viero Piera , Bellavita Piero , Marchioli Roberto , Marfisi Rosa‐Maria , Rambaldi Alessandro , Barbui Tiziano
Published: November 2003

In two consecutive and unselected cohorts of diffuse large cell lymphoma (DLCL) patients with advanced stage disease (IIB or bulk or more) and aged < 60 years, we compared a standard (MACOP‐B for 12 weeks, 60 patients) versus a high‐dose chemotherapy programme (8 weeks of MACOP‐B plus one or two cycles of intensification with mitoxanthrone, dexamethasone, high‐dose Ara‐C, and finally BEAM chemotherapy with autologous haemopoietic progenitor cell transplantation, 61 patients). 41 patients (68%) in the standard group and 51 (84%) in the high‐dose chemotherapy group, achieved a complete remission (CR) or an uncertain complete remission (CRu) (P = 0.0491). With a median follow‐up time of 28 months for the high‐dose group and 63.5 months for the standard group, the actuarial estimate of event‐free survival (EFS) at 2 years demonstrates a significant benefit (70% v 50%, P = 0.03) for patients treated with the intensive regimen. The analysis of subgroups of patients showed that only high‐risk patients (two or three risk factors) benefitted from the high‐dose chemotherapy programme. Nevertheless, the overall survival does not show a significant difference between the two treatment modalities. The treatment‐related morbidity was similar and the mortality rate was 8% in the standard (MACOP‐B) group and 3% in the high‐dose chemotherapy programme. In conclusion, our results show that high‐dose chemotherapy and autologous stem cell transplantation is a safe procedure which should be considered for the front‐line treatment of non‐Hodgkin lymphoma patients with poor prognostic features.
Investigating severe neutropenia with a simple bone marrow immunofluorescence test
Authors: Johnson Tamzine , Minchinton Robyn
Published: November 2003

Severely neutropenic patients who are suspected of having circulating autoantibodies to neutrophils and/or myeloid precursors present a diagnostic problem. This report describes the use of a simple bone marrow immunofluorescence test (BMIFT) to demonstrate autoantibodies reactive with the patient's own bands, neutrophils and myeloid precursors represented on fresh‐frozen bone marrow aspirate smears. Positive results were seen in two of 13 evaluable marrows from children with primary neutropenia and in eight of 44 evaluable marrows from adults with primary or secondary neutropenia. The BMIFT is simple enough to perform in most laboratory environments and provides serological information to support a diagnosis of immune neutropenia when other means of investigation are precluded.
Relationship of the white blood cell count to body fat: role of leptin
Authors: Wilson Charlton A. , Bekele Genene , Nicolson Margery , Ravussin Eric , Pratley Richard E.
Published: November 2003

The white blood cell (WBC) count is correlated to the amount of body fat in humans, but the mechanism for this association is unknown. Leptin, a 16 kD protein produced in adipocytes, circulates in humans in direct proportion to the amount and percentage of body fat. Recent evidence suggests that leptin and the leptin receptor are part of a novel pathway which stimulates haemopoiesis. This study was designed to test whether fasting plasma leptin concentrations contribute to the relationship between the WBC count and body fat. 117 Pima Indians with a wide range in body composition were studied. The WBC count was positively correlated with percentage of body fat (r = 0.44, P = 0.0001) and fasting plasma leptin concentration (r = 0.38, P = 0.0001). In multiple regression analyses, age, gender and percent body fat were significant independent determinants of the WBC count. After controlling for age and gender, percent body fat accounted for 23% of the variance in the WBC count (partial r = 0.48, P = 0.0001). In similar models which also included plasma leptin concentration, percent body fat remained significantly related to the WBC count, but only accounted for 7% of its variance (partial r = 0.27, P = 0.003). Based on these results, and the demonstration that leptin directly effects the stimulation of proliferation of haemopoietic stem cells in vitro, we hypothesize that the relation of the WBC count to percent body fat may be mediated, in part, through the effect of leptin.
A hitherto unknown splice site defect in the protein S gene (PROS1): the mutation results in allelic exclusion and causes type I and type III protein S deficiency
Authors: Mustafa Stefan , Pabinger Ingrid , Vàradi Katalin , Halbmayer Walter‐Michael , Lechner Klaus , Schwarz Hans Peter , Fischer Michael , Mannhalter Christine
Published: November 2003

A hitherto unknown splice site mutation, in the splice acceptor of intron B (tctag to tctgg), was identified in a symptomatic patient with type III protein S deficiency. The mutation co‐segregated with type I/III protein S deficiency in the patient's family. RNA analysis showed allelic exclusion of the mutant transcript in affected individuals. The apparent type III deficiency in the propositus was not associated with the protein S Heerlen variant.
mRNA expression of Fas receptor (CD95)‐associated proteins (Fas‐associated phosphatase‐1/FAP‐1, Fas‐associating protein with death domain/FADD, and receptor‐interacting protein/RIP) in human leukaemia/lymphoma cell lines
Authors: Komada Yoshihiro , Inaba Hiroto , Zhou Yan‐Wen , Zhang Xao‐Li , Tanaka Shigeki , Azuma Eiichi , Sakurai Minoru
Published: November 2003

mRNA expression of Fas (CD95)‐associated proteins [Fas‐associating protein with death domain (FADD), receptor‐interacting protein (RIP), and Fas‐associated phosphatase‐1 (FAP‐1)] has been investigated in 26 Fas‐positive human leukaemia/lymphoma cell lines. Reverse transcriptase‐polymerase chain reaction analysis revealed that FADD and RIP mRNA were invariably expressed in both Fas‐sensitive and Fas‐insensitive cell lines. However, FAP‐1 mRNA was detected in only 11 of 26 cell lines. Interestingly 7/14 cell lines in the Fas‐sensitive group were positive for FAP‐1 mRNA expression. 8/12 cell lines in the Fas‐refractory group did not express FAP‐1 mRNA, but half of these cell lines were susceptible to tumour necrosis factor α‐induced growth inhibition. These findings suggest that the presence or absence of FAP‐1 mRNA expression did not always correlate with relative sensitivity of Fas‐mediated growth inhibition. Furthermore, it is assumed that leukaemia/lymphoma cells could possess structural or functional defects of Fas or Fas‐associated proteins resulting in the failure to trigger apoptotic cell death.
The increase of plasminogen activator inhibitor activity is associated with graft occlusion in patients undergoing aorto‐coronary bypass surgery
Authors: Rifón J. , Páramo J. A. , Panizo C. , Montes R. , Rocha E.
Published: November 2003

Early graft occlusion is a common complication in patients undergoing aorto‐coronary bypass surgery. Both mechanical and haemostatic factors play a role in the pathogenesis of thrombotic occlusion. Several studies have demonstrated a relationship between fibrinolytic activity and venous or arterial thrombosis. We undertook this study to evaluate the possible contribution of the fibrinolytic system to postoperative occlusion in patients undergoing aorto‐coronary bypass graft (CABG).

A venous occlusion (VO) test was performed preoperatively in 82 patients undergoing revascularization procedures. Before and after VO the euglobulin fibrinolytic activity and tissue type plasminogen activator (t‐PA) activity and antigen were measured. Plasminogen activator inhibitor (PAI) activity and antigen and fibrinogen were also assessed in the preocclusion sample. An angiography performed 10 d postoperatively showed graft occlusion in 23% of patients. Patients with graft occlusion had significantly higher preoperative PAI activity than patients without occlusion (P < 0.001). Reduced fibrinolytic response and t‐PA capacity was also observed in the group of patients with graft occlusion (P < 0.03 and P < 0.02 respectively).

We found a reduced preoperative fibrinolytic response, mainly related to high plasma PAI activity in patients with postoperative graft occlusion. These results suggest that increased PAI activity might have a predictive value for early thrombosis in patients undergoing CABG.
Thrombopoietin‐responsive essential thrombocythaemia with myelofibrosis
Authors: Ozaki Shuji , Kosaka Masaaki , Ozaki Keiji , Wakatsuki Shingo , Hashimoto Toshihiro , Miyazaki Hiroshi
Published: November 2003

Platelet‐derived growth factor (PDGF) and other cytokines released from megakaryocytes are thought to play a crucial role in the pathogenesis of myelofibrosis. We describe a patient with essential thrombocythaemia (ET) who developed myelofibrosis with an increased level of serum thrombopoietin (TPO). Recombinant human (rh) TPO stimulated the proliferation and spontaneous megakaryocyte colony formation of the neoplastic cells in the peripheral blood. Moreover, serum concentrations of PDGF, platelet factor 4, and β‐thromboglobulin were elevated and the production of these growth factors from the megakaryocyte progenitors was augmented with the addition of rhTPO in vitro. These results indicate that TPO may contribute to the development of myelofibrosis in ET.
Successful collection of peripheral blood progenitor cells in patients with acute myeloid leukaemia following early consolidation therapy with granulocyte colony‐stimulating factor‐supported high‐dose cytarabine and mitoxantrone
Authors: Schlenk R. F. , Döhner H. , Pförsich M. , Benner A. , Fischer K. , Hartmann F. , Fischer J. Th. , Weber W. , Gunzer U. , Pralle H. , Haas R.
Published: November 2003

We evaluated the feasibility of collecting peripheral blood progenitor cells (PBPC) in patients with acute myeloid leukaemia (AML) following two cycles of induction chemotherapy with idarubicin, cytarabine and etoposide (ICE), and one cycle of consolidation therapy with high‐dose cytarabine and mitoxantrone (HAM). Thirty‐six patients of the multicentre treatment trial AML HD93 were enrolled in this study, and a sufficient number of PBPC was harvested in 30 (83%). Individual peak concentrations of CD34+ cells in the blood varied (range 13.1–291.5/μl; median 20.0/μl). To reach the target quantity of 2.5 × 106 CD34+ cells/kg, between one and six (median two) leukaphereses (LP) were performed. The LP products contained between 0.2 × 106 and 18.9 × 106 CD34+cells/kg (median 1.2 × 106/kg). Multivariate analysis showed that the white blood cell count prior to HAM and the time interval from the start of HAM therapy to reach an unsupported platelet count > 20 × 109/l were predictive for the peak value of CD34+ cells in the blood during the G‐CSF stimulated haematological recovery. In 16 patients an intraindividual comparison was made between bone marrow (BM) and PBPC grafts. Compared to BM grafts, PBPC grafts contained 14‐fold more MNC, 5‐fold more CD34+ cells and 36‐fold more CFU‐GM. A CD34+ subset analysis showed that blood‐derived CD34+ cells had a more immature phenotype as indicated by a lower mean fluorescence intensity for HLA‐DR and CD38. In addition, the proportion of CD34+/Thy‐1+ cells tended to be greater in the PBPC grafts. The data indicate that sufficient PBPC can be collected in the majority of patients with AML following intensive double induction and first consolidation therapy with high‐dose cytarabine and mitoxantrone.
Both cycling and noncycling primitive progenitors continue to be mobilized into the circulation during the leukapheresis of patients pretreated with chemotherapy and G‐CSF
Authors: Ponchio Luisa , Eaves Connie , Hogge Donna , Perotti Cesare , Torretta Lorella , Cerani Paola , Salvaneschi Laura , Ascari Edoardo , Cazzola Mario
Published: November 2003

Colony‐forming cells (CFC) and long‐term culture‐initiating cells (LTC‐IC) include a spectrum of progenitor types whose potential contributions to the haemopoietic recovery seen in patients transplanted with mobilized peripheral blood progenitor cells (PBPC) remains unclear. We evaluated both the number and cycling status of the circulating LTC‐IC and CFC harvested from 12 patients treated with chemotherapy and G‐CSF using a modified 6‐week LTC‐IC assay. The frequency of the LTC‐IC and CFC in the mobilized PB samples were increased 45‐ and 750‐fold, respectively. Interestingly, comparison of these values for PB samples, taken just prior to the start of the leukapheresis, with the progenitor content of the 3 h harvest, showed that, on average, the leukapheresis product contained 19 times more LTC‐IC (P < 0.01) than had been detectable in the entire blood volume of the patients at the start of the collection, whereas the number of CFC collected was approximately the same as the number in the initial circulating pool of PBPC. Cycling studies showed many of the LTC‐IC in the apheresis collections to be proliferating although not more so than in the steady‐state marrow LTC‐IC compartment (i.e. per cent kill of mobilized LTC‐IC after 16 h in 3H‐Tdr = 70 ± 8%, n = 9). On the other hand, the majority of the CFC in the apheresis collections were initially quiescent (per cent kill after 16 h in 3H‐Tdr = 37 ± 6%, n = 12). These findings demonstrate the rapidity with which a primitive subset of LTC‐IC may enter the circulation during the early phase of rebound haemopoiesis induced by chemotherapy plus G‐CSF and provide evidence of differences in the mechanisms regulating LTC‐IC and CFC mobilization.
Phenotypic homozygous activated protein C resistance associated with compound heterozygosity for Arg506Gln (factor V Leiden) and His1299Arg substitutions in factor V
Authors: Castaman Giancarlo , Lunghi Barbara , Missiaglia Edoardo , Bernardi Francesco , Rodeghiero Francesco
Published: November 2003

Two patients from two unrelated families with a history of thrombosis showed severe plasma activated protein C (APC) resistance. However, genotypic analysis demonstrated that the patients were heterozygous for factor V (FV) Leiden mutation. Coagulation studies revealed that FV clotting activity and antigen were similarly reduced at about 50% of normal in the patients. One brother of propositus A also showed the same abnormalities. Genetic analysis showed that, in addition to FV Leiden mutation in exon 10 of the FV gene (G1691A), these patients had a transition in exon 13 of the FV gene (A4070G; R2 allele) predicting His1299Arg substitution in the mature FV. Study by RT‐PCR of platelet FV mRNA indicated that the mRNA produced by the FV gene, marked by the R2 allele, was reduced in amount in both pseudohomozygous patients of family A. The R2 allele has previously been demonstrated to be significantly associated with plasma FV deficiency in the Italian population. The presence of FV deficiency did not protect the propositi from thrombosis. These data confirm that genotypic analysis is mandatory in patients with phenotypic severe APC resistance before these patients are definitely classified as homozygotes for FV Leiden and that further genotypic analysis is advisable.