bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene
Authors: Picard Véronique , Bura Alessandra , Emmerich Joseph , Alhenc‐Gelas Martine , Biron Christine , Houbouyan‐Reveillard Lucie L. , Molho Pascale , Labatide‐Alanore Agnès , Sié Pierre , Toulon Pierre , Verdy Elisabeth , Aiach Martine
Published: March 2003

We have investigated the molecular bases of familial antithrombin deficiency in eight French families. Eight mutations in the antithrombin coding exons were identified, seven of which were novel mutations. In all cases, individuals were heterozygous for the mutation. We found two small frameshift deletions in exon 3a, leading to type I deficiency. Five missense mutations in exons 3b or 5 also caused type I deficiency and their potential consequences on the antithrombin three‐dimensional structure were analysed. The last mutation in exon 4 was associated with a type II ‘reactive site’ deficiency: a dysfunctional antithrombin that is affected in its interaction with thrombin was present in circulation.
Expression of the multidrug resistance‐associated protein in myelodysplastic syndromes
Authors: Poulain Stéphanie , Lepelley Pascale , Preudhomme Claude , Cambier Nathalie , Cornillon Jérome , Wattel Eric , Cosson Alain , Fenaux Pierre
Published: March 2003

In the myelodysplastic syndromes (MDS), P‐glycoprotein (P‐gp) expression is clinically associated with drug resistance, whereas the clinical significance of multidrug resistance‐associated protein (MRP1) is uncertain. Bone marrow from 56 patients with MDS, including six with refractory anaemia (RA)/RA with ringed sideroblasts (RARS), 23 cases of RA with excess blasts/in transformation (RAEB/T), four patients with chronic myelomonocytic leukaemia (CMML) and 23 cases of MDS having progressed to acute myeloid leukaemia (MDS‐AML), were studied. MRP1 expression was investigated by immunocytochemistry (ICC) and by flow cytometry using MRPm6 monoclonal antibody. The efflux test using calcein‐AM (CAM) ± probenecid to evaluate MRP1 activity was performed in ten of the 56 patients. Twenty‐eight of the 56 cases (50%) expressed MRP1. MRP1 expression was more frequent in MDS‐AML than in MDS (70% vs. 36%). The efflux test using CAM was positive in three out of the ten patients tested. The results were in agreement with expression of MRP1 in six cases, and were discordant in four cases (1 MRP‐/CAM+, 3 MRP+/CAM–). No correlation was observed between MRP1 expression and P‐gp, lung resistance‐associated protein (LRP) or CD34 expression, although there was a trend for more frequent MRP1 expression in P‐gp‐positive cases in MDS‐AML (P = 0·08). Ten of the 26 patients treated with intensive chemotherapy achieved complete remission including six out of 16 MRP1+ and four out of ten MRP1– cases (P = NS). In conclusion, MRP1 expression was correlated with disease stage in MDS in our study. As for P‐gp, discordant expression/function of MRP1 could be found in some cases, suggesting the existence of non‐functional transport proteins in MDS. MRP1 expression did not seem to be a prognostic factor in MDS in our experience.
Chelator‐induced iron excretion in iron‐overloaded marmosets
Authors: Sergejew Thomas , Forgiarini Peter , Schnebli Hans‐Peter
Published: March 2003

In order to test new orally active iron chelators in a predictive way, a primate model has been developed. This model makes use of the marmoset monkey (Callithrix jacchus) and its overall design is similar to a previously reported monkey model. However, this new model enables a higher compound throughput and requires lower amounts of test compound because the animals are much easier to handle and have much lower body weights. The marmosets were iron‐overloaded by three intraperitoneal injections of iron (III) hydroxide polyisomaltose. For the iron‐balance studies, the animals were kept in metabolic cages and were maintained on a low‐iron diet in order to reduce faecal background. After compound administration, the excretion of iron in urine and faeces was followed for 2 d. A series of well‐known chelators was tested for validation of the model. In particular, comparison of the iron‐clearing properties of DFO, L1, CP94 and HBED in marmosets and humans demonstrated the predictive value of the model and justify our expectation that if iron chelators such as CGP65015, ICL670A and CGP75254A are active in marmosets, they will be active in humans as well.
Two novel and one recurrent missense mutation in the factor XIII A gene in two Dutch patients with factor XIII deficiency
Authors: Gómez García E. B. , Poort S. R. , Stibbe J. , Sturk A. , Schaap M. C. L. , Kappers M. , Bertina R. M.
Published: March 2003

Congenital factor XIII (FXIII) deficiency is a rare autosomal recessive disorder, usually attributed to a defect in the FXIII A subunit, whose genetic basis has been studied in a number of cases. We describe here the genetic variations found in two unrelated patients with FXIII deficiency. Both patients, under prophylactic substitution with FXIII concentrate, showed low plasma FXIII A subunit antigen levels with undetectable A subunit antigen in the platelets and normal plasma B antigen levels, which indicate that the defects are present in the A subunit of the molecule. Both probands were heterozygous for a previously reported G→A transversion in exon 8 of the FXIII A subunit gene (Arg326Gln substitution). Proband 1 was also heterozygous for a novel G→T transversion in exon 7, which predicts a Val316Phe substitution. Two of her sons were heterozygous for this mutation and showed low FXIII activity and FXIII A subunit antigen levels. Val316 is a well‐conserved amino acid among the transglutaminase family, located within the core domain, close to the Cys314 member of the catalytic triad. Proband 2 had a unique 2‐bp (TT) insertion in one of the alleles within or adjacent to the −7 to −20 T tail of intron A. This insertion was not found in 50 healthy individuals, which supports this being the second mutation in this patient.
Insertional events as well as translocations may arise during aberrant immunoglobulin switch recombination in a patient with multiple myeloma
Authors: Pratt G. , Fenton J. A. L. , Davies F. E. , Rawstron A. C. , Richards S. J. , Collins J. E. , Owen R. G. , Jack A. S. , Smith G. M. , Morgan G. J.
Published: March 2003

The majority of patients with multiple myeloma have translocations involving the immunoglobulin heavy chain switch regions on chromosome 14q32 and a promiscuous range of partner chromosomes. We describe a patient with an insertion of 132 bp of chromosome 22q12 sequence into the 5′ region flanking Sμ on chromosome 14q32. The 132 bp region from chromosome 22q12 contains the whole of exon 3 from a novel gene of unknown function in man. The significance of such insertional events remains unclear. The description of insertional events occurring as a result of abnormal switch recombination suggests that, in myeloma, dysregulation of oncogenes may occur by a mechanism other than chromosomal translocation.
Arsenic trioxide and ascorbic acid: synergy with potential implications for the treatment of acute myeloid leukaemia?
Authors: Bachleitner‐Hofmann T. , Gisslinger B. , Grumbeck E. , Gisslinger H.
Published: March 2003

Arsenic trioxide (As2O3) induces remission in a high proportion of patients with acute promyelocytic leukaemia (APL) via induction of apoptosis. Preliminary reports suggest that the apoptotic effect of As2O3 is not specific for APL but can also be observed in non‐APL acute myeloid leukaemia (AML) cells, although these are less sensitive than APL cells. Ascorbic acid has recently been demonstrated to enhance the apoptotic effect of As2O3. We have therefore evaluated combined As2O3/ascorbic acid treatment in various clinical samples of AML. Our results indicate a significant synergistic effect of As2O3 and ascorbic acid, suggesting a possible future role of As2O3/ascorbic acid combination therapy in patients with AML.
Molecular characterization of a myelodysplasia‐associated chromosome 7 inversion
Authors: Todd Roger , Bia Britta , Johnson Emma , Jones Chris , Cotter Finbarr
Published: March 2003

Chromosome 7 abnormalities are observed in a wide range of myeloid disorders, particularly myelodysplasia (MDS) and acute myeloid leukaemia (AML). Monosomy 7 and 7q deletions are the most frequent abnormalities, although translocations and inversions involving 7q also occur. The region 7q22–q34 may contain as many as four distinct minimal regions of deletion (MDRs), which are thought to contain one or more myeloid tumour‐suppressor genes. We have defined previously the proximal breakpoint of a constitutional 7q22–q34 inversion, carried in a cell line derived from a member of a family with a history of MDS. A YAC clone spanning this breakpoint was identified. Both inversion breakpoints have now been cloned and sequenced, placing the proximal breakpoint 40 kb centromeric to the TAC2 (tachykinin 2) gene and the distal breakpoint 42 kb telomeric to the SSBP (mitochondrial single‐stranded DNA‐binding protein) gene. Sequence alignments revealed small (3–4 bp) duplications at the inversion breakpoints, suggesting that the mechanism of inversion involved the creation of staggered breaks and filling in of the overhanging ends. A 190‐bp Alu–Alu deletion close to the distal breakpoint was also detected and may have contributed to the inversion.
Band 3 Cape Town (E90K) causes severe hereditary spherocytosis in combination with band 3 Prague III
Authors: Bracher Natalie A. , Lyons Cheryl A. , Wessels Glynn , Mansvelt Erna , Coetzer Thérèsa L.
Published: March 2003

Hereditary spherocytosis (HS) is an inherited haemolytic anaemia, characterized by spheroidal, osmotically fragile red blood cells. This disorder exhibits heterogeneity in terms of both clinical severity and underlying molecular defect. We have studied a South African Cape Coloured individual with severe HS owing to a band 3 deficiency caused by two mutations, occurring in trans, in the band 3 gene: a novel variant that we have designated band 3 Cape Town and a previously described mutation, band 3 Prague III. Analysis of erythrocyte membrane proteins indicated a deficiency of both band 3 and protein 4·2, as well as a decreased functional capacity of band 3 to transport anions. Band 3 Cape Town is defined by a GAG→AAG point mutation at codon 90, substituting a glutamic acid with a lysine in the cytoplasmic domain of the molecule, while band 3 Prague III is a codon 870 CGG→TGG point mutation, replacing an arginine with a tryptophan in the transmembrane region of band 3. mRNA is transcribed from both mutant alleles, implying that mutant proteins are synthesized, but are either degraded prior to membrane incorporation or insertion is impaired. We conclude that the combination of these two mutations exacerbated the clinical presentation of the proband.
Lack of CD40‐dependent B‐cell proliferation in B lymphocytes isolated from patients with persistent polyclonal B‐cell lymphocytosis
Authors: Loembé Marguerite Massinga , Lamoureux Josée , Deslauriers Noella , Darveau André , Delage Robert
Published: March 2003

Persistent B‐cell lymphocytosis (PPBL) is a haematological disorder diagnosed primarily in adult female smokers that is characterized by a polyclonal increase in peripheral blood B lymphocytes and a moderate elevation of serum IgM. B lymphocyte‐associated cellular abnormalities, such as the occurrence of multi‐lobed nuclei, increased bcl2/Ig gene rearrangements and the identification of an extra long‐arm chromosome (i3)(q10) in the B‐cell population, indicate that PPBL could be part of a multi‐step process leading to the emergence of a malignant B lymphoproliferation. However, the resulting impact on cellular functional properties remains to be elucidated. Our goal was to address that aspect via the study of B‐cell activity following stimulation through CD40, a key molecule of the tumour necrosis factor receptor superfamily involved in B lymphocyte development. In contrast to normal B cells, PPBL B lymphocytes were unable to respond to the proliferative signal delivered in vitro by CD40, indicating a defect in the CD40 activation pathway. Polymerase chain reaction amplification and sequencing of the receptor as well as FACScan analysis of patient B lymphocytes dismissed the possibility of a defect in either CD40 structure or expression. Moreover, Western blot analysis of tyrosine phosphorylation, an early event in the CD40‐signalling cascade, was similar in patients and controls, leading to the conclusion that the defect affecting B lymphocytes in PPBL patients is probably located downstream of that signalling cascade.
Protein assays for diagnosis of Wiskott–Aldrich syndrome and X‐linked thrombocytopenia
Authors: Qasim Waseem , Gilmour Kimberly C. , Heath Stephanie , Ashton Eleanor , Cranston Treena , Thomas Angela , Finn Adam , Davies E. Graham , Thrasher Adrian J. , Kinnon Christine , Jones Alison , Gaspar Hubert B.
Published: March 2003

Mutations in the gene encoding the Wiskott–Aldrich syndrome protein (WASp) give rise to Wiskott–Aldrich syndrome (WAS), a condition that exhibits a wide spectrum of clinical severity. Patients may develop mild thrombocytopenia or suffer from a wide range of associated disorders including eczema, immune dysfunction, autoimmune disease and malignancy. The clinical diagnosis of Wiskott–Aldrich syndrome (WAS) can be difficult and is usually supported by the detection of WASp gene mutations using genetic analysis. Recently, protein‐based assays have been used to demonstrate the absence of WASp in patients known to have WASp gene mutations. We have now reversed this approach and report on the use of immunoblot assays to rapidly diagnose WAS in 13 patients. There was a complete absence of WASp in 10 out of 13 patients and an abnormal protein form was detected in the remaining three patients. In all cases, subsequent genetic analysis confirmed the presence of a WASp gene mutation. We believe that protein‐based assays should be employed as the first line of investigation in the diagnosis of WAS spectrum disorders.
Characterization of a partial prostaglandin endoperoxide H synthase‐1 deficiency in a patient with a bleeding disorder
Authors: Dubé Jean‐Nicolas , Drouin Jeanne , Aminian Merhdad , Plant Matthew H. , Laneuville Odette
Published: March 2003

Thromboxane A2 (TXA2), synthesized in platelets, is a powerful aggregating agent and vasoconstrictor. To induce platelet aggregation, the platelets' enzyme, prostaglandin endoperoxide H synthase‐1 (PGHS‐1), first converts arachidonic acid (AA) into prostaglandin H2 (PGH2). PGH2 is then converted by the enzyme thromboxane synthase into TXA2. Finally, TXA2 is secreted and can activate the TXA2 receptor on the platelet surface. The importance of TXA2 in haemostasis has been demonstrated by the presence of a bleeding tendency in patients showing an inherited defect in the TXA2 production pathway. We studied an 18‐year‐old woman with a lifelong bleeding disorder, moderate thrombocytopenia (55–71 × 109/l) and a prolonged bleeding time (12·5 min). Her platelets aggregated in the presence of both PGH2 and a stable TXA2 analogue, but did not aggregate in the presence of AA. The activity of PGHS‐1 in platelets, measured using thin‐layer chromatography and radioactive counting of TXA2 formation from [14C]‐AA, was reduced to 13% of the activity measured in control subjects. PGHS‐1 protein levels, measured using Western blot analysis, were also markedly reduced to 10% of control values. Such levels of PGHS‐1 enzyme were too low to sustain platelet aggregation in the patient, even if the enzyme was active. The PGHS‐1 protein level was also reduced in the patient's immortalized B lymphocytes, suggesting a systemic expression defect. Northern blot analysis was then carried out with poly (A)+ RNA extracted from the patient's immortalized B lymphocytes. PGHS‐1 mRNA was detected as a 2·8‐kb band in both the patient and control. The intensity of the band representing the patient's PGHS‐1 mRNA was similar to that of the control subject. The Northern blot result suggests a normal transcriptional rate of the PGHS‐1 gene for the patient. Therefore, the defect responsible for the reduced levels of PGHS‐1 protein is probably post‐transcriptional.
High‐dose factor VIIa increases initial thrombin generation and mediates faster platelet activation in thrombocytopenia‐like conditions in a cell‐based model system
Authors: Kjalke Marianne , Ezban Mirella , Monroe Dougald M. , Hoffman Maureane , Roberts Harold R. , Hedner Ulla
Published: March 2003

Clinical experience has shown that high doses of recombinant factor VIIa (rFVIIa) may ensure haemostasis in thrombocytopenic patients. We have used a cell‐based model system to mimic thrombocytopenia and analyse the effect of rFVIIa. Lowering the platelet density from 200 × 109/l (reflecting normal conditions) to 100, 50, 20 and 10 × 109/l revealed a platelet density‐dependent decrease in the maximal rate of thrombin generation, a prolongation in the time to maximal thrombin activity and a lower maximal level of thrombin formed. The platelet activation, measured as the time to half‐maximal P‐selectin (CD62) exposure, was not significantly dependent on the platelet density in the range of 200 × 109/l to 10 × 109/l, although there was a tendency for slower platelet activation at 20 × 109 and 10 × 109 platelets/l than at the higher platelet densities. Addition of 50–500 nmol/l rFVIIa to samples with 20 × 109 or 10 × 109 platelets/l shortened the lag phase of thrombin generation as well as the time to half‐maximal platelet activation. Our data indicate that high doses of rFVIIa may help to provide haemostasis in thrombocytopenic patients by increasing the initial thrombin generation, resulting in faster platelet activation and thereby compensating for the lower number of platelets present.
In vitro T‐cell receptor Vβ repertoire analysis may identify which T‐cell Vβ families mediate graft‐versus‐leukaemia and graft‐versus‐host responses after human leucocyte antigen‐matched sibling stem cell transplantation
Authors: Epperson Diane E. , Margolis David A. , McOlash Laura , Janczak Teresa , Barrett A. John
Published: March 2003

We studied oligoclonal T‐cell expansions of 24 T‐cell receptor (TCR) Vβ families in normal donor lymphocytes stimulated with patient's cells and in recipient blood after transplant, using a polymerase chain reaction‐based assay (spectratyping). T cells from donor blood were incubated with separated myeloid leukaemia cells or T cells from the HLA‐identical sibling recipient. In five of the six patients tested, the T‐cell Vβ skewing pattern observed in vitro was seen in vivo after transplant. After transplant, the myeloid‐specific Vβ skewing coincided with the disappearance of residual disease in three patients and in one patient skewing was lost at the time of leukaemic relapse. In functional tests, T cells generated against leukaemic cells in vitro produced interferon γ in response to the leukaemia. Removal of the leukaemia‐expanded skewed Vβ families significantly decreased cytotoxic killing of the leukaemia. However, while there was a general concordance in the Vβ family exhibiting clonal expansion in vitro and in vivo, the exact clonotype expanded in vitro and in vivo differed. These findings suggest that alloresponses involve multiple T‐cell clones within a restricted TCR Vβ repertoire that undergo different selection pressures in vitro and in vivo.
Bruton's tyrosine kinase is present in normal platelets and its absence identifies patients with X‐linked agammaglobulinaemia and carrier females
Authors: Futatani Takeshi , Watanabe Chiaki , Baba Yoshihiro , Tsukada Satoshi , Ochs Hans D.
Published: March 2003

X‐linked agammaglobulinaemia (XLA) is a primary immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk) and is characterized by an arrest of B‐cell development. We analysed Btk protein expression in platelets using flow cytometry and found that normal platelets express large amounts of Btk. Assessment of affected males from 45 unrelated XLA families revealed that platelets of the majority of the patients (37 out of 45 families) had decreased or absent Btk expression, and that platelets from carrier females of these families had both normal and mutated Btk expression, indicating that megakaryocytes in XLA carriers undergo random X‐chromosome inactivation. These observations demonstrate that Btk is not crucial for maturation of megakaryocytes and the production of platelets. No correlation between Btk expression in platelets and clinical phenotype was observed in this study. Flow cytometric evaluation using platelets is a simple and rapid method to test Btk expression. It may be used as a screening test for XLA and for carrier detection, followed, if necessary, by more expensive mutation analyses.
Fludarabine plus cyclophosphamide is an efficient treatment for advanced chronic lymphocytic leukaemia (CLL): results of a phase II study of the German CLL Study Group
Authors: Hallek Michael , Schmitt Barbara , Wilhelm Martin , Busch Raymonde , Kröber Alexander , Fostitsch Hans‐Peter , Sezer Orhan , Herold Michael , Knauf Wolfgang , Wendtner Clemens M. , Kuse Rolf , Freund Mathias , Franke Astrid , Schriever Folke , Nerl Christoph , Döhner Hartmut , Thiel Eckhard , Hiddemann Wolfgang , Brittinger Günter , Emmerich Bertold ,   the German Chronic Lymphocytic Leukaemia Study Group
Published: March 2003

The efficacy and toxicity of a combination of fludarabine and cyclophosphamide (FC) was evaluated in patients with B‐cell chronic lymphocytic leukaemia (CLL). Between April 1997 and July 1998, 36 patients with CLL (median age 59 years) received a regimen that consisted of fludarabine 30 mg/m2 in a 30‐min IV infusion, d 1–3, and cyclophosphamide 250 mg/m2 in a 30‐min IV infusion on d 1–3. Cycles were repeated every 28 d. Twenty‐one patients had received between one and three different treatment regimens prior to the study, while 15 patients had received no prior therapy. The median Eastern Cooperative Oncology Group performance score was 1. One patient was at Binet stage A, 18 were stage B and 17 patients were stage C. Objective responses, assessed according to the revised guidelines of the National Cancer Institute‐sponsored Working Group, were recorded in 29 out of 32 assessable patients (90·6%). Twenty‐four partial remissions and five complete remissions were observed. Two patients showed no change and one patient showed disease progression. At February 2000, three of the responders had relapsed. Severe neutropenia, anaemia and thrombocytopenia (Common Toxicity Criteria grade 3 and 4) were observed in 25, six and six patients (69·4%, 16·7% and 16·7%) respectively. Other side‐effects were uncommon. No treatment‐related deaths and no grade 3 or 4 infections occurred. We conclude that the combination of fludarabine and cyclophosphamide showed significant activity in patients with CLL. Myelosuppression was the major side‐effect. These results warrant further study on the FC combination in randomized trials.
Analysis of CD34+ cell subsets in stem cell harvests can more reliably predict rapidity and durability of engraftment than total CD34+ cell dose, but steady state levels do not correlate with bone marrow reserve
Authors: Pratt G. , Rawstron A. C. , English A. E. , Johnson R. J. , Jack A. S. , Morgan G. J. , Smith G. M.
Published: March 2003

In peripheral blood stem cell transplantation (PBSCT), the number of CD34+ cells transplanted has been shown to correlate well with both rapidity and durability of engraftment. However, it is clear that engraftment does not necessarily correlate with total CD34+ cell numbers in some patients. Consequently, there is increasing interest in evaluating the role of CD34+ subsets in haemopoietic recovery as a more accurate marker of harvest quality. We analysed the numbers of CD34+ cell subsets, namely Thy‐1+, L‐Selectin+ and CD38−, and correlated this with engraftment in 86 patients undergoing PBSCT. Adequate engraftment was defined as being a platelet count greater than 50 × 109/l and a neutrophil count greater than 1·0 × 109/l. CD34+L‐Selectin+ provided the best prediction of engraftment rapidity, although the improvement over total CD34+ cell dose was minor. Only the dose of CD34+Thy‐1+ cells transplanted correlated with durable engraftment. The probability of adequate 3‐month engraftment increased with the dose of CD34+ cells transplanted, but 10% of patients receiving > 5 × 106/kg still showed poor engraftment at 3 months. However, all patients receiving > 2·5 × 105/kg CD34+Thy‐1+ showed adequate engraftment at this time point. We also demonstrated that CD34+Thy‐1+ progenitors were restricted to the bone marrow under normal conditions and, during stem cell mobilization, their kinetics generally paralleled total CD34+ numbers.
Molecular analysis of transferrin receptor mRNA expression in acute myeloid leukaemia
Authors: Kollia P. , Stavroyianni N. , Stamatopoulos K. , Zoi K. , Viniou N. , Mantzourani M. , Noguchi C. T. , Paterakis G. , Abazis D. , Pangalos C. , Loukopoulos D. , Yataganas X.
Published: March 2003

Transferrin receptor (TfR, CD71) is an integral membrane glycoprotein that mediates cellular uptake of iron. In most tissues, TfR expression is correlated positively with proliferation and regulated at the post‐transcriptional level. The available data regarding the pattern of TfR gene expression in haematological malignancies are very limited. In the present study, we evaluated TfR gene expression at the molecular level in bone marrow (BM) samples of 44 patients with de novo acute myeloid leukaemia (AML) at diagnosis with BM blasts > 85%. TfR mRNA levels were determined by densitometric analysis of quantitative reverse transcription polymerase chain reaction products corresponding to TfR exons 15–17. Each sample was tested in at least two independent experiments. In 13/44 patients, TfR messages were not detected (this is probably an underestimate as some positive results may be attributed to residual normal erythroid cells present in the samples). In 17/44, TfR mRNA levels were low–intermediate, and were high in the remaining patients (14/44). TfR mRNA positivity was significantly associated with older age. No statistically significant correlations were found either with specific French–American–British (FAB) subtypes or attainment of complete remission, incidence of relapse and survival (after adjusting accordingly for age and FAB subtype). The absence of TfR mRNA transcripts in a significant minority of cases suggests that alternative mechanisms of iron uptake may function in AML blast cells.
Ex vivo expansion of umbilical cord blood (UCB) CD34+ cells alters the expression and function of α4β1 and α5β1 integrins
Authors: Ramírez Manuel , Segovia José Carlos , Benet Isana , Arbona Cristina , Güenechea Guillermo , Blaya Carolina , García‐Conde Javier , Bueren Juan A. , Prosper Felipe
Published: March 2003

We have investigated the influence of ex vivo expansion of human CD34+ cord blood cells on the expression and function of adhesion molecules involved in the homing and engraftment of haematopoietic progenitors. Ex vivo expansion of umbilical cord blood CD34+ cells for 6 d in the presence of interleukin 3 (IL‐3), IL‐6 and stem cell factor (SCF) or IL‐11, SCF and Flt‐3L resulted in increased expression of α4, α5, β1, αΜM and β2 integrins. However, a significant decrease in the adhesion of progenitor cells to fibronectin was observed after the ex vivo culture (adhesion of granulocyte‐macrophage colony‐forming units (CFU‐GM) was 22 ± 4% in fresh cells versus 5 ± 2% and 2 ± 2% in each combination of cytokines). Incubation with the β1 integrin‐activating antibody TS2/16 restored adhesion to fibronectin. Transplantation of ex vivo expanded umbilical cord blood CD34+ cells was associated with an early delayed engraftment in non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Incubation of cells with the monoclonal antibody TS2/16 before transplantation almost completely abrogated NOD/SCID repopulating ability of both fresh and expanded CD34+ cells. The seeding efficiency of fresh and expanded CD34+ cells was similar, but markedly reduced after incubation with the TS2/16 monoclonal antibody. Our results show that functional activation of β1 integrins could overcome the decreased very late antigen (VLA)‐4‐ and VLA‐5‐mediated adhesion observed after ex vivo expansion of haematopoietic progenitors. However, in vivo, these effects induced an almost complete abrogation of the homing and repopulating ability of CD34+ UCB cells.
Deregulation of immunoglobulin gene transcription in the Hodgkin–Reed Sternberg cell line L1236
Authors: Re Daniel , Staratschek‐Jox Andrea , Holtick Udo , Diehl Volker , Wolf Jürgen
Published: March 2003

Hodgkin–Reed Sternberg (H‐RS) cells harbour clonal immunoglobulin gene (Ig) rearrangements in almost all cases of classical Hodgkin's disease but lack Ig gene expression. In the H‐RS cell line L1236, a somatic mutation of the Ig heavy‐chain gene promoter octamer motif has been described as a putative reason for absence of Ig gene expression. We addressed transcriptional activity of this mutated promoter by performing reporter gene studies and gel retardation assays. The results showed that the mutation outside the coding region of the rearranged Ig gene in L1236 cells leads to downregulation of Ig gene expression in H‐RS cells.
Effect of intermediate‐purity factor VIII (FVIII) concentrate on lymphocyte proliferation and apoptosis: transforming growth factor‐β is a significant immunomodulatory component of FVIII
Authors: Hodge G. , Han P.
Published: March 2003

Factor concentrates have been shown to have a variety of immunomodulatory effects in vitro. The presence of plasma‐derived factor VIII (pdFVIII) has been shown to diminish lymphocyte proliferative response to mitogens. Recently, we have shown the presence of transforming growth factor‐β (TGF‐β) as an immunomodulatory component present in plasma‐derived FVIII concentrate. However, the addition of neutralizing antibody to TGF‐β did not abrogate the inhibitory effect of pdFVIII on monocyte cytokine production, suggesting the presence of other, as yet undetermined, immunomodulatory agent/s in pdFVIII. To further characterize the immunomodulatory effects of pdFVIII, the in vitro effect of pdFVIII concentrate on proliferation and apoptosis of mitogen‐stimulated T cells was studied using whole blood and purified T cells. The presence of pdFVIII increased the apoptosis of phytohaemagglutinn (PHA) ‐stimulated CD4 and CD8 T‐cell subsets as determined by Annexin V binding and DNA fragmentation. T‐cell subsets showed a pdFVIII dose‐dependent inhibition of entry into S‐phase and G1 arrest. Addition of neutralizing anti‐TGF‐β reduced some of these changes. To determine the physiological relevance of these findings, blood samples from five patients receiving FVIII prophylaxis were similarly studied ex vivo and showed significantly increased apoptosis of T‐cell subsets as determined by Annexin V staining. TGF‐β has been reported to be a potent inhibitor of T‐cell proliferation, arresting the cell cycle in G1 phase and causing apoptosis. Together, these findings suggest that TGF‐β is a significant immunomodulatory component of pdFVIII concentrates.
An in vitro system for testing leucocyte and leukaemic cell line adhesion to synthetic fibres
Authors: Barbe Laurent L. , Boval Bernadette M. , Wautier Marie‐Paule S. , Wautier Jean‐Luc T.
Published: March 2003

Leucocyte adhesion is an important phenomenon in antimicrobial defence, inflammation and immunological mechanisms and has been shown to be dependent upon specialized adhesion molecules. To prevent side‐effects related to blood transfusion (e.g. anti‐human leucocyte antigen immunization and transmission of infectious agents) leucocyte reduction of blood products is now systematically performed in various countries. The most common system used for leucoreduction is blood filtration. For further understanding of the mechanisms responsible for the interaction between leucocytes and the fibres present in filters we used a flow chamber to study the adhesion of leucocytes and leukaemic cell lines to different types of fibre. Adhesion was quantified using video‐microscopy and computer image analysis. Our results demonstrate that adhesion to filter fibres was dependent on the expression of β2‐integrins CD11–‐CD18 and was inhibited by anti‐CD18. The amount of fibres present, their spatial arrangement and the physicochemical characteristics of the fibres were important factors in leucocyte adhesion. Leucocyte adhesion was the highest to polyethylene terephthalate (PET) and polyimide fibres. Lymphocytes or lymphocytic cell lines were poorly adherent to PET fibres. The retaining capacity of leucocyte filters can be improved by taking into account the different parameters for the design of new filters
MCP‐1 modulates chemotaxis by follicular lymphoma cells
Authors: Husson Hervé , Carideo Elizabeth G. , Cardoso Angelo A. , Lugli Serena M. , Neuberg Donna , Munoz Olivier , De Leval Laurence , Schultze Joachim , Freedman Arnold S.
Published: March 2003

The localization and establishment of follicular lymphoma (FL) cells in distinct anatomic sites probably involves chemokine and adhesion receptors on the neoplastic cells and appropriate chemokines and adhesion receptor ligands in the microenvironment. Several chemokines play an important role in normal B‐cell trafficking and differentiation. Monocyte chemoattractant protein‐1 (MCP‐1) is a C‐C chemokine that induces chemotaxis of a variety of lymphoid cells through its receptor CCR2. CCR2 is also expressed on B cells, and MCP‐1 induces chemotaxis of normal B cells. In this report, we investigated expression and function of CCR2 on FL cells. We found FL cells as well as the t(14; 18)+ B‐cell lymphoma line H2 expressed CCR2. MCP‐1 potentiated SDF‐1‐induced chemotaxis of FL cells and H2 cells, but MCP‐1 alone did not induce chemotaxis. The specificity of the effects of MCP‐1 and SDF‐1 was demonstrated by antibody blocking studies. Because FL cells are generally associated with follicular dendritic cells (FDCs), FDCs may be an important source of chemokines. We found that cultured FDCs produced MCP‐1, and this production was enhanced by tumour necrosis factor. These data implicate MCP‐1 in the migration and localization of FL cells.
Philadelphia chromosome‐positive mature B‐cell (Burkitt cell) leukaemia
Authors: Mann Georg , Trebo Monika M. , Haas Oskar A. , Grümayer‐Panzer Eva R. , Dworzak Michael N. , Lion Thomas , Gadner Helmut
Published: March 2003

Summary. Philadelphia chromosome‐positive (Ph+) acute leukaemia usually shows lymphoblastic morphology and a B‐precursor phenotype. The bone marrow aspirate of a 9‐year‐old boy showed a L3 blast cell morphology in 90% of cells; immunophenotyping revealed a mature B‐blast population. The translocation t(9;22) (q34;q11) was seen in 45 out of 50 metaphases, and expression of the corresponding bcr1/abl fusion transcripts, but no IgH/myc co‐localization or splitting of c‐myc, was demonstrated. Chemotherapy according to the Berlin–Frankfurt–Munster non‐Hodgkin's lymphoma (NHL‐BFM 95) protocol with maintenance according to the BFM acute lymphoblastic leukaemia (ALL‐BFM 90) protocol resulted in continuing complete remission of 54 months. The occurrence of Ph+ Burkitt's leukaemia might reflect multiple‐step cancer development.
Lamivudine for the treatment of hepatitis B virus reactivation following chemotherapy for non‐Hodgkin's lymphoma
Authors: Liao Chun‐Ann , Lee Chuan‐Mo , Wu Hong‐Cheng , Wang Ming‐Chung , Lu Sheng‐Nan , Eng Hock‐Liew
Published: March 2003

Summary. Chemotherapy for non‐Hodgkin's lymphoma (NHL) patients with chronic hepatitis B virus (HBV) infection may be accompanied by severe hepatitis. Of 86 consecutive NHL patients, 11 (12·8%) exhibited a positive serum HBsAg. Six of these patients (54·5%) developed acute exacerbation of chronic HBV infection following chemotherapy and received lamivudine. Five of the six patients demonstrated a clinical improvement, one patient died from fulminant hepatic failure owing to delayed lamivudine therapy and poor compliance. These data suggest that HBsAg screening is necessary before commencing chemotherapy for NHL patients in a hyperendemic area and that lamivudine is effective in treating hepatitis B reactivation during chemotherapy.
t(7;11)(p15;p15) chronic myeloid leukaemia  developed into blastic transformation showing a novel  NUP98/HOXA11 fusion
Authors: Suzuki Akitaka , Ito Yoshikazu , Sashida Goro , Honda Seiko , Katagiri Tomoko , Fujino Takashi , Nakamura Takuro , Ohyashiki Kazuma
Published: March 2003

Summary. We encountered a patient with Philadelphia‐negative chronic myeloid leukaemia, witht(7;11)(p15;p15), in whom acute leukaemia phase (acute myeloid leukaemia‐M2 morphology) developed within a short period. We detected a novel gene fusion between NUP98 and HOXA11 both in the chronic phase and in the acute leukaemia phase in this case. Although it is well known that a fusion of NUP98–HOXA9 in myeloid malignancies is created by the t(7;11)(p15;p15), this case suggests the possibility that HOXA11 might be another partner gene for NUP98 in t(7;11)(p15;p15) leukaemia.
Immunochemical studies of antigenic  lymphoma‐associated proteins
Authors: Pulford Karen , Roberton Helen , Banham Alison H. , Hatton Christian S. R. , Mason David Y.
Published: March 2003

Summary. We have previously reported that plasma from patients with anaplastic lymphoma kinase (ALK)‐positive lymphoma contains antibodies against the oncogenic kinase NPM‐ALK protein characteristic of this disease. We investigated whether this reactivity represents a phenomenon unique to ALK‐positive lymphoma by screening plasma from patients with follicular lymphoma for antibodies to BCL‐2 protein. Eight out of 10 samples showed such reactivity (and in six cases gave specific staining of BCL‐2‐transfected cells). As these findings suggest a new biochemical approach to the identification of oncogenic proteins in lymphoma, we investigated whether antibodies present in patients with ALK‐positive lymphoma can precipitate NPM‐ALK in quantities which should be sufficient for further analysis. We found that plasma samples from all10 patients studied immunoprecipitated NPM‐ALK asaprotein visible in silver‐stained sodium dodecyl sulphatepolyacrylamide gels. Finally we demonstrated that NPM‐ALK could be visualized more clearly if it were immunoprecipitated from extracts of cells in which newly synthesized proteins had been labelled with 35S and then identified by autoradiography. These results suggest a strategy for using patients' autoantibodies to screen for antibodies to other tumour‐associated proteins.
Epstein–Barr virus‐associated T‐cell acute lymphoblastic leukaemia
Authors: Sakajiri Sakura , Mori Kiyoshi , Isobe Yasushi , Kawamata Norihiko , Oshimi Kazuo
Published: March 2003

Summary.  Epstein–Barr virus (EBV) is frequently detected in haematological malignancies, including Burkitt's lymphomas/leukaemias, Hodgkin's diseases and non‐Hodgkin's lymphomas. However, immature T‐cell malignancies associated with EBV have not been reported previously. We report a patient with T‐cell acute lymphoblastic leukaemia (T‐ALL), whose leukaemic cells had EBV, confirmed by Southern blotting and in situ hybridization. The EBV existed in episomal form and was detected in most leukaemic cells, but not in bystander normal B‐cells. The leukaemic cells, massively infiltrated into the liver and spleen, were resistant to chemotherapy. EBV might be associated with tumorigenesis of T‐ALL, and characteristic clinical features of the patient.
Rearrangement of the BCL6 locus at 3q27 is an independent poor prognostic factor in nodal diffuse large B‐cell lymphoma
Authors: Barrans Sharon L. , O'Connor Sheila J. M. , Evans Paul A. S. , Davies Faith E. , Owen Roger G. , Haynes Andrew P. , Morgan Gareth J. , Jack Andrew S.
Published: March 2003

Summary. Diffuse large B‐cell lymphomas (DLBCL) are a heterogeneous group of tumours, varying in clinical features, immunophenotype and cytogenetics. The aim of this study was to investigate the prognostic significance of BCL6 gene rearrangement at the 3q27 locus in patients with primary nodal disease, and to examine interrelationships with immunophenotype and International Prognostic Index (IPI). We have developed a fluorescent in situ hybridization (FISH)‐based technique for the retrospective analysis of the effect of BCL6 gene rearrangements on survival, using nuclei extracted from paraffin‐embedded tissue. FISH results were obtained in 111 presentation cases of nodal DLBCL. The IPI was calculated and each case was stained immunocytochemically for BCL6, BCL2 and CD10. 3q27 rearrangements were detected in 25% of cases. BCL2 protein and a germinal centre (GC) phenotype (defined as CD10+, BCL6+) were expressed in 56% and 41% of cases respectively. In multivariate analysis, rearrangement of 3q27 and BCL2 expression and the absence of a GC phenotype were associated with a poor prognosis. These factors can be used in conjunction with the IPI to improve risk stratification in nodal DLBCL.
Expression pattern of MUM1/IRF4 in the spectrum of pathology of Hodgkin's disease
Authors: Carbone Antonino , Gloghini Annunziata , Aldinucci Donatella , Gattei Valter , Dalla‐Favera Riccardo , Gaidano Gianluca
Published: March 2003

Summary.  Biological and clinical studies have shown that Hodgkin's disease (HD) can be divided into two major categories, termed nodular lymphocyte predominance HD (NLP HD) and classic HD (CHD). Within CHD four subtypes have been distinguished: nodular sclerosis, mixed cellularity, lymphocyte rich and lymphocyte depletion. To refine the histogenesis of the pathological spectrum of HD, 75 CHD and 13 NLP HD were analysed for the expression pattern of MUM1/IRF4 (Multiple Myeloma‐1/Interferon Regulatory Factor‐4), a lymphocyte‐specific member of the IRF family, that is expressed by late centrocytes and post‐germinal centre (GC) B cells. MUM1 reacted with Hodgkin's and Reed–Sternberg (HRS) cells of all CHD cases (75/75 cases), with a moderate to strong staining intensity. Conversely, lymphocyte and histiocyte (L & H) cells, the putative tumour cells of NLP HD, were negative for MUM‐1 expression (9/13 cases) or displayed a weak reactivity for the antigen in < 10% neoplastic cells (4/13 cases). With respect to HD microenvironment, NLP HD displayed numerous MUM1‐positive T lymphocytes located in close proximity to L & H cells whereas, in CHD, MUM1‐positive T lymphocytes appeared to be distributed randomly with no specific relationship with HRS cells. Overall, this study shows that MUM1 expression differs in L & H cells versus HRS cells, corroborating the notion that NLP HD and CHD represent different stages of B‐cell differentiation. As MUM1‐positive T lymphocytes form rosettes around tumour cells of NLP HD, but not of CHD, these data point also to differences in the microenvironment of NLP HD and CHD, and postulate an interactive role of MUM1‐positive T lymphocytes with L & H cells.
Early and late invasive pneumococcal infection following stem cell transplantation: a European Bone Marrow Transplantation survey
Authors: Engelhard Dan , Cordonnier Catherine , Shaw Peter J. , Parkalli Terttu , Guenther Christine , Martino Rodrigo , Dekker Adriaan W. , Prentice H. Grant , Gustavsson Anita , Nurnberger Wenzel , Ljungman Per ,   the Infectious Disease Working Party of the European Bone Marrow Transplantation (IDWP‐EBMT)
Published: March 2003

Summary.  Streptococcus pneumoniae (S. pneumoniae) may cause severe and lethal infections months and years following stem cell transplantation (SCT). In a prospective survey over a 3·5‐year period, we assessed the incidence, risk factors and outcome for invasive pneumococcal infection (IPI) following SCT. Fifty‐one episodes of IPI were reported: 43 episodes after bone marrow transplantation (BMT) and 8 after peripheral blood stem cell transplantation (PBSCT); 35 after allogeneic SCT and 16 after autologous SCT. Seven IPI episodes, all bacteraemias, were defined as early, occurring 1–35 d (median 3 d) post transplantation. Forty‐four episodes were defined as late (≥ 100 d post SCT), occurring 4 months to 10 years (median 17 months) post transplantation. The incidences of early and late IPI were 2·03/1000 and 8·63/1000 transplantations respectively (P = 0·001). A higher incidence of late IPI was observed after BMT than after PBSCT (10·99 versus 3·23/1000; P < 0·01) and after allogeneic versus autologous SCT (12·20 versus 4·60/1000; P < 0·01). There was a higher estimated incidence of IPI in allogeneic patients with than in those without graft‐versus‐host disease (GVHD) (18·85 versus 8·25/1000; P = 0·015). The mortality rate was 20%, including 2/7 of early and 8/44 of late IPI. S. pneumoniae is a rare but important complication during the aplastic phase after SCT. In conclusion, S. pneumoniae is a significant cause of morbidity late post‐transplantation, especially in allogeneic patients, and particularly those with GVHD. The high IPI mortality rate, both early and late post‐transplantation, requires preventive approaches, mainly effective immunization.
Epstein–Barr virus (EBV)‐positive pyothorax‐associated lymphoma (PAL): chromosomal integration of EBV in a novel CD2‐positive PAL B‐cell line
Authors: Daibata Masanori , Taguchi Takahiro , Nemoto Yuiko , Saito Tsuyako , Machida Hisanori , Imai Shosuke , Miyoshi Isao , Taguchi Hirokuni
Published: March 2003

Summary.  Pyothorax‐associated lymphoma (PAL) is a clinico‐pathological entity arising in the pleural cavity of patients with long‐standing inflammatory pyothorax. PAL is closely associated with Epstein–Barr virus (EBV), but how this virus contributes to the development of the lymphoma is unknown. We have successfully obtained a novel EBV‐infected PAL cell line, designated Pal‐1. The cell line and its source coexpressed CD2 and CD20 molecules, but other representative B‐ and T‐cell markers such as CD1, CD3, CD5, CD7, CD10 and CD19 were not found. The B‐cell origin of Pal‐1 cells was proven by rearrangement of the immunoglobulin heavy‐ and light‐chain genes without rearranged T‐cell receptor genes. Both the cell line and primary tumour cells carried monoclonal EBV genome. Although EBV genome is known to be maintained as circular extrachromosomal DNA, neither circular nor linear extrachromosomal EBV DNA was detectable in Pal‐1 cells by in situ lysis gel analysis. Fluorescence in situ hybridization demonstrated viral integration at a marker chromosome mostly consisting of the centromere region of chromosome 1. The viral integration event may enhance a chromosomal instability at the insertion site. This cell line represents the first example of EBV integration in PAL and could enable the study of the potential role of integrated viral infection in the development of PAL as well as mechanism of the aberrant phenotype expression.