bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatitis C virus and non‐Hodgkin's lymphomas
Authors: Mazzaro Cesare , Zagonel Vittorina , Monfardini Silvio , Tulissi Patrizia , Pussini Elisabetta , Fanni Massimiliano , Sorio Roberto , Bortolus Roberto , Crovatto Marina , Santini Gianfranco , Tiribelli Claudio , Sasso Franco , Masutti Roberto , Pozzato Gabriele
Published: October 2003

Hepatitis C virus (HCV) seems to be the aetiologic agent of mixed cryoglobulinaemia, and as this ‘benign’ lymphoproliferative disorder can frequently develop into more aggressive haematological disorders, this study was undertaken to determine the prevalence of HCV infection in non‐Hodgkin's lymphomas. 199 unselected subjects treated by three haematological centres in Northeast Italy were investigated for the presence of HCV infection. As controls, the prevalence of HCV infection was determined in a group of patients affected by other haematological malignancies (153 subjects) and in the general population of the same geographical area in the cohort study called the Dyonisos project (6917 subjects).

The presence of anti‐HCV antibodies was determined by a commercial kit and, in positive cases, by PCR amplification of the 5′ untranslated region of the virus. The HCV genotype was also obtained by PCR amplification of the Core region with type‐specific primers. The presence of serum cryoglobulins was determined in each case of NHL.

HCV infection was significantly (P<0.00000001) higher in patients with non‐Hodgkin's lymphomas (28.0%) when compared with that of the general population (2.9%), and with the group of patients affected by other malignancies (3.1%). The prevalence is particularly high in low‐grade (38.4%), as compared with intermediate (11.4%), or high‐grade (15.2%) lymphomas. The presence of the virus is significantly (P<0.000001) associated with the presence of detectable levels of cryoglobulins. On the basis of these findings, HCV seems to play an important role in the development of low‐grade non‐Hodgkin's lymphomas.
Alloreactive CD4+ T lymphocytes can exert cytotoxicity to chronic myeloid leukaemia cells processing and presenting exogenous antigen
Authors: Jiang Yin‐zheng , Mavroudis Dimitrios , Dermime Said , Hensel Nancy , Couriel Daniel , Molldrem Jeffrey , Barrett A. John
Published: October 2003

Existing evidence supports that CD4+ T lymphocytes play a role in the graft‐versus‐leukaemia (GVL) reaction after allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML), not only as initiators of the immune response but also as effectors of GVL. In BMT between HLA‐identical pairs this CD4‐mediated GVL would require CML cells to process and present antigens through MHC class II molecules. To investigate whether CML cells are capable of processing and presenting antigens, and suitable targets for CD4+ T‐cell‐mediated cytotoxicity, we generated HLA‐DR1‐restricted CD4+ cytotoxic T‐cell clones that specifically recognized tuberculous purified protein derivative (PPD). We have shown that CML cells and B lymphoblastoid cell line (B‐LCL) cells but not PHA‐blasts from patients with CML processed exogenous antigen, PPD, and induced proliferative and cytotoxic CD4+ T‐cell responses. Antigen presentation was blocked by antibodies to HLA‐DR but not to MHC class I and by treatment with chloroquine and brefeldin. This indicates that CML cells use a classic MHC class II antigen processing pathway to present PPD antigens to CD4+ T cells. Cytotoxicity to CML was shown by antibody blocking studies to be mediated mainly through fas antigen. These findings indicate that donor CD4+ T cells alone are sufficient to mediate GVL effects following allogeneic BMT for CML.
Myelodysplastic syndrome with karyotype abnormality is associated with elevated F‐cell production
Authors: Craig Jamie E. , Sampietro Maurizio , Oscier David G. , Contreras Marcela , Thein Sweelay
Published: October 2003

A sensitive F‐cell assay has been used to examine the production of fetal haemoglobin (Hb F) in a group of 77 adult patients with myelodysplastic syndrome (MDS), and a control group composed of 100 normal blood donors. Although the mean F‐cell percentage in the MDS group (6.0%) is not statistically different from that in the normal blood donors (3.1%), a higher proportion of myelodysplastic patients have elevated F‐cell values and the magnitude of the increases is greater than that observed in blood donors. In order to investigate the association further, the karyotypes of the MDS patients have been examined. 13/21 (61.9%) of the MDS patients with karyotypic abnormalities have F‐cell values >5%, compared to only 6/56 (10.9%) of the MDS patients with a normal karyotype and 11/100 (11%) of the blood donors. The observed difference in the distributions of F cells between the two subgroups of patients with MDS is highly significant (P < 0.0001).
Acquired von Willebrand factor deficiency during high‐dose infusion of recombinant factor VIII
Authors: ROCK G. , Adamkiewicz T. , Blanchette V. , Poon A. , Sparling C.
Published: October 2003

Constant infusion of factor VIII (FVIII) into patients with haemophilia A after major surgery has been recommended as optimal treatment to avoid peaks and valleys in the circulating levels of FVIII and to allow the use of much lower doses of FVIII than are historically required.

One of our young patients with severe (<0.01 U/ml FVIII) haemophilia suffered a subdural haematoma for which he received treatment with 815 190 recombinant FVIII (rFVIII) units over a period of 52 d. 2 weeks after admission, because of low FVIII levels and the presence of FVIII inhibitors, the infusion rate was increased to >100 U/kg/h for 14 d. During this time the FVIII level fluctuated between 0.6 and 4.2 U/ml. For some period it was not possible to detect ristocetin co‐factor activity in this patient’s plasma and the von Willebrand factor (VWF) level and VWF multimer pattern resembled those of a patient with von Willebrand’s disease. Subsequently, when the rFVIII dose was increased 2‐fold, this was not reflected by the plasma level of FVIII although antibodies were not detected.

The data suggest that the prolonged infusion of very high levels of rFVIII which is deficient in von Willebrand factor can result in depletion of VWF from existing stores, producing a laboratory picture which is consistent with the diagnosis of von Willebrand’s disease. Further, in the absence of complexing with VWF, FVIII appears to be cleared from the circulation at an increased rate. This is expensive and potentially compromising. Therefore, when administering very high doses of FVIII concentrates devoid of VWF for prolonged periods of time, ristocetin cofactor and VWF levels should be monitored.
Aplastic anaemia and paroxysmal nocturnal haemoglobinuria: a study of the GPI‐anchored proteins on human platelets
Authors: Vu T. , Griscelli‐Bennaceur A. , Gluckman E. , Sigaux F. , Carosella E. D. , Menier C. , Scrobohaci M. L. , Socié G.
Published: October 2003

Twenty‐six consecutive patients with acquired aplastic anaemia (AA) and nine patients with de novo paroxysmal nocturnal haemoglobinuria (PNH) were included in this study. In these 35 patients a GPI‐anchored molecule defect at the platelet surface was investigated by flow‐cytometry. Platelets from eight out of the nine patients with de novo PNH were found to be deficient for the GPI‐anchored molecule CD55, CD58 and CD59. We also detected a GPI‐anchored molecule defect on monocytes, granulocytes, and erythrocytes in all patients with de novo PNH. Among the 26 AA patients, a GPI defect was detected on platelets in five patients. Interestingly, these five patients were also found to have a GPI‐anchored molecule defect on erythrocytes, whereas in 10 patients the GPI‐anchored molecule defect was only detected on monocyte and polymorphonuclear (PMN) cells.
CD30 ligand is expressed on resting normal and malignant human B lymphocytes
Authors: Younes Anas , Consoli Ugo , Zhao Shourong , Snell Virginia , Thomas Elaine , Gruss Hans J. , Cabanillas Fernando , Andreeff Michael
Published: October 2003

CD30, a member of the tumour necrosis receptor superfamily, is physiologically expressed on a subpopulation of T helper cells in normal individuals but is also expressed on several malignant and virally transformed cells. Its ligand (CD30L) is a pleiotropic cellular transmembrane protein that can induce cell death in several CD30+ cell lines. CD30L expression has been reported on activated human peripheral blood T lymphocytes and macrophages but not on B cells. Here we show that the CD30L is expressed on resting normal and on malignant B cells in addition to both CD4+ and CD8+ subsets of activated T cells, making it the second tumour necrosis family member, in addition to the CD27 ligand, that can be expressed on both T and B cells. These findings raise the possibility that the CD30L has a role in B‐cell/T‐cell communication and that B and T cells are likely to be involved in the growth regulation of CD30+ tumours.
Interleukin‐6 inhibits the chemotaxis of human malignant plasma cell lines
Authors: SHIBAYAMA HIROHIKO , TAGAWA SHINICHI , HATTORI HIDEKI , HARIGAYA KENICHI , TAGA TETSUYA , MACHII TAKASHI , KITANI TERUO
Published: October 2003

The chemotaxis of human malignant plasma cells is promoted by two extracellular matrix proteins (ECMs): fibronectin (FN) and laminin (LN). We examined the effect of the supernatant from a bone marrow stroma cell line, KM‐101, on the chemotaxis of human malignant plasma cell lines to assess the chemotaxis‐regulatory roles of the bone marrow microenvironment. Five human malignant plasma cell lines, FR4ds, OPM‐1ds, U266/B1, RPMI‐8226 and ARH‐77 showed different profiles of the expression of β1 integrins of FN and LN receptors. FR4ds, OPM‐1ds and U266/B1 cells showed chemotaxis promoted by FN (ChFN) and LN (ChLN). ARH‐77 cells showed ChFN but not ChLN. RPMI‐8226 cells did not show either ChFN or ChLN. The supernatant from KM‐101 cells inhibited the chemotaxis of each of these cell lines regardless of whether the chemotaxis was promoted by FN or LN. Among the cytokines produced by KM‐101 cells, it was postulated that IL‐6 mediated this inhibitory effect because anti‐IL‐6 monoclonal antibody (MoAb) and anti‐IL‐6 receptor MoAb significantly reversed the inhibition. Recombinant IL‐6 (rIL‐6) also exhibited a similar inhibitory effect. Because anti‐gp130 MoAb significantly reversed the chemotaxis inhibitory effect of rIL‐6, the inhibitory signal is probably transduced via the signal transducing receptor component, gp130. The chemotaxis‐regulatory effect is another previously unrecognized function of this pleiotropic cytokine, IL‐6. High levels of IL‐6 in the bone marrow microenvironment of patients with multiple myeloma appears to be favourable for the localization of myeloma cells there.
Composition of the intra‐erythroblastic precipitates in thalassaemia and congenital dyserythropoietic anaemia (CDA): identification of a new type of CDA with intra‐erythroblastic precipitates not reacting with monoclonal antibodies to α‐ and β‐globin chains
Authors: WICKRAMASINGHE S. N. , LEE M. J. , FURUKAWA T. , EGUCHI M. , REID C. D. L.
Published: October 2003

Ultrathin sections of bone marrow cells from two patients with homozygous β‐thalassaemia, two patients with haemoglobin H (HbH) disease, a patient with congenital dyserythropoietic anaemia (CDA) type III and two patients with severe congenital dyserythropoietic anaemia of an unusual type were reacted with mouse monoclonal antibodies against various globin chains and the reaction visualized using a gold‐labelled goat antibody against mouse IgG. The multiple rounded intra‐erythroblastic inclusions found in homozygous β‐thalassaemia reacted with the monoclonal antibody against α‐globin chains but not β‐globin chains, thus confirming that they consisted of precipitated α‐globin chains. The branching intra‐erythroblastic inclusions found in HbH disease and CDA type III reacted with the monoclonal antibody against β‐globin chains but not α‐globin chains, indicating that they consisted of precipitated β‐globin chains. The two patients with severe CDA had been transfusion‐dependent since infancy, had a normal α:β globin chain synthesis ratio or parents with normal red cell indices, displayed prominent dysplastic changes in their erythroblasts, and had intra‐erythroblastic inclusions resembling those seen in homozygous β‐thalassaemia. However, unlike those in β‐thalassaemia, the inclusions in these two patients did not react with the monoclonal antibody against either α‐ or β‐globin chains. The inclusions reacted with antibody against ζ‐globin chains, but detailed studies in one of the patients indicated that the antigen involved was not ζ‐globin. These patients have features not reported in the condition known as dominantly inherited inclusion body β‐thalassaemia and appear to suffer from a novel type of CDA in which the intra‐erythroblastic inclusions may consist of some non‐globin protein or structurally‐abnormal α‐globin chains.
Characterization of TGF‐β1‐binding proteins in human bone marrow stromal cells
Authors: Robledo Mar M. , Hidalgo Andrés , Lastres Pedro , Arroyo Alicia G. , Bernabeu Carmelo , Sánchez‐Madrid Francisco , Teixidó Joaquín
Published: October 2003

The proliferation and differentiation of haemopoietic progenitor cells is dependent on their close relation with bone marrow stromal cells, which constitute a source of cytokines as well as expressing receptors for both the cytokines and progenitor cell adhesion molecules necessary for regulated haemopoiesis. We have generated human bone marrow stromal cell cultures and analysed the TGF‐β1 receptor components expressed by these cells. [125I]TGF‐β1‐affinity labelling experiments showed the involvement of type I and II receptors in the binding of TGF‐β1, as demonstrated by specific immunoprecipitation of [125I]TGF‐β1–receptor complexes. In addition, large TGF‐β1‐labelled complexes displaying an electrophoretic mobility similar to betaglycan were also observed in these experiments. Endoglin, another component of the TGF‐βreceptor system, was detected by flow cytometry on the surface of cultured marrow stromal cells, and in the human bone marrow stromal cell line Str‐5, and was immunoprecipitated from surface‐iodinated cells. Endoglin on the stromal cells was able to bind TGF‐β1, as demonstrated by specific immunoprecipitation of [125I]TGF‐β1–endoglin complexes using anti‐endoglin antibodies. The results presented provide evidence that bone marrow stromal cells are fully capable of responding to TGF‐β1. Given the important role of TGF‐βas a regulator of the synthesis of cytokines and cytokine receptors, as well as cell adhesion molecules, these data indicate that the binding of TGF‐β1 by stromal cells might represent an important step in the regulation of the proliferation and differentiation of haemopoietic progenitor cells.
Matched and mismatched unrelated donor bone marrow transplantation for juvenile chronic myeloid leukaemia
Authors: Chown S.R. , Potter M.N. , Cornish J. , Goulden P. , Goulden N. , Pamphilon D. , Steward C.G. , Oakhill A.
Published: October 2003

Juvenile chronic myeloid leukaemia (JCML) is a rare haematological condition of childhood curable only by bone marrow transplantation (BMT). We report our experience using matched and mismatched unrelated donor BMT for JCML in five patients. Although the procedure is hazardous in terms of toxicity and relapse, two patients are alive and disease‐free 28 and 49 months post BMT.
Identification and characterization of an inherited mutation of PIG‐A in a patient with paroxysmal nocturnal haemoglobinuria
Authors: Endo Morito , Ware Russell E. , Vreeke Teresa M. , Howard Thad A. , Parker Charles J.
Published: October 2003

Analysis of the X‐linked gene PIG‐A from haemopoietic cells of a female PNH patient showed a homozygous C‐55‐T substitution that caused replacement of arginine with tryptophan at codon 19. AvaI restriction analysis of PIG‐A cDNA demonstrated that the patient was homozygous for this mutation, whereas her mother was heterozygous and her father was hemizygous. Flow cytometry, however, showed normal expression of glycosyl phosphatidylinositol anchored proteins on blood cells of the patient’s mother and father. Therefore the C‐55‐T mutation is an inherited sequence variant that does not account for the PNH phenotype of this patient.
Circulating thrombopoietin level in chronic immune thrombocytopenic purpura
Authors: KOSUGI SATORU , KURATA YOSHIYUKI , T OMIYAMA YOSHIAKI , T AHARA TOMOYUKI , KATO TAKASHI , TADOKORO SEIJI , SHIRAGA MASAMICHI , H ONDA SHIGENORI , KANAKURA YUZURU , MATSUZAWA YUJI
Published: October 2003

The circulating thrombopoietin (TPO) level in 43 patients with chronic immune thrombocytopenic purpura (ITP) was examined by an ELISA system. The TPO level (mean±SD) in ITP patients was mildly elevated (1.86±1.17 fmol/ml) compared to that in normal subjects (0.76±0.21), and was within the normal range in 30% of ITP patients. In contrast, the TPO level in patients with aplastic anaemia was very high, 12.35±6.42 fmol/ml. There was no correlation between TPO level and platelet count in ITP patients. Splenectomy was performed in two ITP patients, after which platelet counts increased to normal levels and TPO levels showed a transient increase. These data suggest that reactive TPO production against thrombocytopenia in ITP is small when compared to that in aplastic anaemia. Relative endogenous TPO deficiency may play some role in the pathophysiology of thrombocytopenia in ITP patients.
Activated protein C resistance: a comparison between two clotting assays and their relationship to the presence of the factor V Leiden mutation
Authors: Legnani C. , Palareti G. , Biagi R. , Coccheri S. , Bernardi F. , Rosendaal F. R. , Reitsma P. H. , De Ronde H. , Bertina R. M.
Published: October 2003

Resistance to the anticoagulant effect of activated protein C (APC resistance), a frequent abnormality in patients with a history of venous thrombosis, is known to be due, in the large majority of cases, to the presence of an abnormal factor V: the factor V Leiden. It is reasonable to surmise that screening for this abnormality should be performed with a clotting method for APC resistance, before submitting the patients with abnormal results to DNA analysis. The present study was performed on 216 individuals enrolled at the Bologna centre, of which 189 were unrelated patients with a history of juvenile venous thromboembolism and 27 were relatives with or without thrombosis. APC resistance was first measured in Bologna by a standard commercial method and then, in Leiden, by an in‐house method; DNA analysis was performed in those cases in which at least one of the clotting methods was abnormal. The data obtained confirm the good performance and the optimal positive predictive value for the Leiden mutation (100%) of the Leiden in‐house clotting method. Performance of the commercial method was less satisfactory but markedly improved by expressing the data in relation to the values simultaneously obtained with a normal plasma pool. Even with optimal data expression, however, the positive predictive value of the commercial method, versus DNA analysis, did not exceed 88%.

It is concluded that further standardization of the commercial method here evaluated is necessary before it can be widely adopted for the screening of APC resistance and prediction of the presence of factor V Leiden.
The genetic basis of a new partial D antigen: DDBT
Authors: BECKERS E. A. M. , FAAS B. H. W. , SIMSEK S. , OVERBEEKE M. A. M. , VAN RHENEN D. J. , WALLACE M. , VON DEM BORNE A. E. G. KR , VAN DER SCHOOT C. E.
Published: October 2003

The Rh system, the most polymorphic system on red cells, is genetically controlled by two different but highly homologous genes on chromosome 1. The RHCE gene encodes different RhCcEe polypeptides and the RHD gene encodes D antigens. It is well established that in D negative individuals the RHD gene is either absent or grossly deleted. The D antigen comprises at least nine serologically defined D epitopes. The D antigen can be divided into different partial D categories, reflecting a different pattern of specific D epitopes.

In this study a newly defined partial D antigen, DDBT, was studied. D epitope mapping revealed the presence of D epitopes 6/7 and 8 and the absence of the other D epitopes. The molecular basis of this phenotype was studied by Southern blotting, by RHD typing using the polymerase chain reaction (RHD‐PCR) and by sequence analysis of Rh transcripts. The DBT phenotype appeared to be encoded by a hybrid RHD gene, in which exons 5, 6 and 7 (and possibly the identical exon 8) were replaced by the corresponding exons of the RHCE gene.

From this study it may be concluded that D epitopes 1, 2, 3, 4, 5 and 9 are dependent on the presence of RHD exons 5, 6, and 7.
Splenic lymphoma with villous lymphocytes: clinical presentation, biology and prognostic factors in a series of 100 patients
Authors: TROUSSARD X. , VALENSI F. , DUCHAYNE E. , GARAND R. , FELMAN P. , TULLIEZ M. , HENRY‐AMAR M. , BRYON P. A. , FLANDRIN G.
Published: October 2003

The diagnosis of splenic lymphoma with villous lymphocytes (SLVL) was assessed by a panel of cytologists in a series of 100 patients. Clinical and biological characteristics were analysed in relation to prognosis.

SLVL is a chronic B‐cell lymphoproliferative disorder characterized by splenomegaly and the presence, in peripheral blood, of lymphocytes with ‘villous’ projections. The cytological diagnosis can be difficult in patients without an absolute lymphocytosis which was observed in 24% of cases. B‐cells expressed CD19+, CD20+, CD22+, CD24+ and DBA44+, whereas the expression of CD5, CD10 and CD25 was usually negative.

SLVL is a disease of the elderly with a relatively benign clinical course. In the present series the 5‐year overall survival was 78%. Deaths in 15 patients were related to disease progression or treatment (nine cases). Patients with a leucocyte count >30 × 109/l or lymphocyte count <4 × 109/l or initially treated with chemotherapy had significantly (P < 0.001) lower overall survival than other patients. From these findings, treatment abstention should be considered in patients with favourable prognostic factors; on the other hand, the efficacy of conventional chemotherapy remains to be evaluated in patients with unfavourable prognostic factors.
PLATINUM AGENTS AND SECONDARY MYELOID LEUKAEMIA: TWO CASES TREATED ONLY WITH PLATINUM‐BASED DRUGS
Authors: Philpott N. J. , Elebute M. O. , Powles R. , Treleaven J. G. , Gore M. , Dainton M. G. , Min T. , Swansbury G. J. , Catovsky D.
Published: October 2003

With the increasing use of chemotherapy for many different primary malignancies, secondary or therapy‐related acute myeloid leukaemias (AML) and myelodysplastic syndromes (MDS) are becoming more common. The risk of developing sAML has been estimated to be between 2% and 10%, depending upon the type, duration and dosage of previous therapy (Michels et al, 1985; Shulman, 1993; Robinson & Mertens, 1993; Ballen & Antin, 1993). It is therefore one of the most serious long‐term complications of current cancer treatment and is likely to increase as longer survival rates for the primary tumour are achieved. An increasing range of drugs have been reported to cause sAML, including the alkylating agents, the epipodophyllotoxins and the anthracyclines, both as single agents and in combination (Pedersen‐Bjergaard & Philip, 1991; Pedersen‐Bjergaard & Rowley, 1994). We report two cases of secondary AML in which platinum compounds were the sole prior chemotherapy.
The dominant β‐thalassaemia in a Spanish family is due to a frameshift that introduces an extra CGG codon (=arginine) at the 5′ end of the second exon
Authors: Negri Arjona S. , Maldonado Eloy‐Garcia J. , Gu L‐H. , Smetanina N. S. , Huisman T. H. J.
Published: October 2003

We have discovered a Spanish family with a dominant type of β‐thalassaemia. Carriers are characterized by mild anaemia, hypochromia, microcytosis, elevated Hb A2 and Hb F levels, reticulocytosis, and splenomegaly. The molecular basis of this condition is the introduction of a CGG triplet between codons 30 and 31 of the β gene; this was determined by sequencing of amplified DNA and confirmed by dot‐blot analysis. The abnormal mRNA (βTh‐mRNA) is stable and present in quantities similar to that of normal βA‐mRNA. cDNA fragments derived from βTh‐ and βA‐mRNAs can be separated on a denaturing polyacrylamide gel electrophoresis because the βTh fragment is three nucleotides (nts) longer than the βA fragment. The βTh‐mRNA translates into a β chain that is 147 amino acid residues long and carries an extra arginine residue between residues 30 and 31. This βX chain has not been detected. It may be unstable and does not bind to the α chain. It probably is continuously digested by proteolytic enzymes in red cell precursors in the bone marrow. The abnormal chain probably binds haem that is excreted after proteolysis causing a darkening of the urine.
Rearrangement of the BCL6 gene in B‐cell lymphoid neoplasms: comparison with lymphomas associated with BCL2 rearrangement
Authors: Muramatsu M. , Akasaka T. , Kadowaki N. , OHNO H. , YAMABE H. , EDAMURA S. , DOI S. , MORI T. , Okuma M. , FUKUHARA S.
Published: October 2003

We report a series of B‐cell neoplasms with regard to rearrangement of the BCL6 gene on chromosome band 3q27. Southern blot analysis using probes from the major translocation cluster (MTC) region of the BCL6 revealed rearrangement in 21/197 patients (10.7%) with B‐cell neoplasms studied at presentation, and 11/25 patients (44%) first studied at relapse. In non‐Hodgkin's lymphoma (NHL) studied at diagnosis, rearrangements of the BCL6 gene were not closely associated with a specific histopathologic subtype but distributed in subcategories in the Working Formulation. The incidence in follicular lymphoma was 12.1%, with significantly higher frequency in mixed and large cell subtypes, and that in diffuse aggressive lymphoma was 14.1%. Comigration analysis using probes from the immunoglobulin genes revealed association of the BCL6 gene with one of the three immunoglobulin loci in 9/25 cases analysed. A comparative study between NHL associated either with BCL2 or BCL6 rearrangement showed that advanced disease and bone marrow involvement were more frequent in BCL2(+) NHL. In contrast, extranodal involvement was more frequently observed in the BCL6(+) NHL. The survival curve of BCL6(+) NHL was characterized by a rapid decline followed by a plateau. Of the total of 32 BCL6(+) patients, six carried both BCL2 and BCL6 rearrangements; five of these six showed clinicopathological properties characteristic of follicular lymphoma, suggesting that the presence of the two genetic abnormalities does not necessarily have synergistic effects on malignant phenotypes. The high level of BCL6 expression in follicular lymphoma cell lines carrying a BCL2 rearrangement suggests that the deregulated BCL2 gene may have an effect on the development of genetic abnormalities of the BCL6 gene. The present study suggests that BCL6 gene rearrangement is primarily involved in large cell lymphoma irrespective of growth pattern of neoplastic cells, and that BCL6(+)BCL2(−) NHL could be curable with modern intensive chemotherapy.
Pretransplant cytotoxic donor T‐cell activity specific to patient HLA class I antigens correlating with mortality after unrelated BMT
Authors: Speiser D. E. , Löliger C.‐C. , Siren M.‐K. , Jeannet M.
Published: October 2003

Unrelated bone marrow transplantation (BMT) is associated with increased post‐transplant complication rates, partly because more transplantation antigens are mismatched than in HLA‐identical related BMT. We have shown previously that the cytotoxic T‐lymphocyte precursor (CTLp) test performed before transplantation specifically detects HLA class I mismatches demonstrating its usefulness for the identification of new HLA class I alleles. In this study we analysed the clinical relevance of the CTLp test in 41 patients who underwent unrelated BMT between 1990 and 1994. All patient–donor pairs were HLA‐A, ‐B, ‐DR compatible as defined by AB‐serology and oligotyping for DR1‐14. The host‐reactive CTLp test was performed using previously frozen peripheral blood mononuclear cells (PBMC) as stimulators and PHA blasts as target cells. We found 10 CTLp‐positive and 31 CTLp‐negative patient–donor pairs. Between the two groups there were no significant differences for age, diagnosis, sex, preconditioning and GvHD prophylaxis. The clinical results for the CTLp positive and the CTLp negative transplants were: severe acute GvHD III–IV 67% and 26% (P = 0.0315), transplant‐related mortality 60% and 26% (P = 0.0085), and patient survival at 3.5 years 10% and 54% (P = 0.0006).

Seven patient–donor pairs were mismatched for HLA‐DR and/or ‐DQ subtypes. Only one of these seven class II mismatched pairs had a positive CTLp test. In the remaining nine CTLp positive pairs the CTL reactivity was directed against HLA‐A, ‐B or ‐C antigens, revealing a statistically significant (P < 0.005) correlation between the CTLp frequency and HLA class I matching. In conclusion, the CTLp test helped to select optimally matched bone marrow donors and was particularly useful in association with high resolution oligotyping for DR‐ and DQ‐subtypes for precise matching of both classes of HLA antigens.
RELATION OF BLAST CELL SURVIVAL AND PROLIFERATION TO CHEMOTHERAPY RESISTANCE IN AML
Authors: NØrgaard Jan M. , Langkjer Sven T. , Palshof Torben , Clausen Niels , Pedersen Bent , Hokland Peter
Published: October 2003

We have investigated the in vitro blast cell survival (viability) and drug resistance to cytosine arabinoside (Ara‐C), daunorubicin (Dau), mitoxantrone (Mitox) and aclarubicin (Acla) of fresh leukaemic blast cells from 80 patients with newly diagnosed acute myeloid leukaemia (AML) employing the semiautomated colourimetric MTT(3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide)‐assay. In 15 cases we concurrently investigated the relation between in vitro blast cell survival (MTT assay) and blast cell proliferation (3H‐thymidine incorporation) in the presence and absence of myeloid growth factors (GFs) G‐CSF, GM‐CSF and IL‐3 (individually and in combination). A highly significant correlation was found between blast cell survival and blast cell proliferation (r = 0.87, P < 1 × 10−4). Furthermore, in 40 evaluable adult patients who completed intravenous induction chemotherapy and were evaluable for response to chemotherapy we found a positive correlation between in vitro blast survival (MTT assay) and response to chemotherapy with high blast survival being associated with poor response to chemotherapy (P = 0.05). Moreover, in a multivariate analysis, high blast cell survival was significantly associated with high CD13 expression and monocytic phenotype (P = 0.0003 and P = 0.02, respectively). Furthermore, we found an inverse relationship between the baseline proliferation of the blasts and the magnitude of response to the GFs (P < 0.02), indicating that cells with low baseline proliferation were more readily stimulated by growth factors. Finally, we found a significant correlation between leukaemic cell survival and cellular drug resistance towards Dau (P = 0.001) and Mitox (P = 0.03), but not towards Ara‐C (P = 0.68) and Acla (P = 0.13). We conclude that high in vitro leukaemic cell survival is associated with drug resistance in vivo and in vitro, and furthermore is correlated with high blast cell proliferation and some adverse prognostic factors previously identified in AML.
Effect of basic (FGF‐2) and acidic (FGF‐1) fibroblast growth factors on early haemopoietic cell development
Authors: Ratajczak Mariusz Z. , Ratajczak Janina , Skorska Margaret , Marlicz Wojtek , Calabretta Bruno , Pletcher, Jr Charles H. , Moore Jonni , Gewirtz Alan M.
Published: October 2003

Basic fibroblast growth factor (FGF‐2) and acidic fibroblast growth factor (FGF‐1) are mitogens for a variety of cell types. Many reports suggest that haemopoietic cells are among these. Nevertheless, when we examined the effect of recombinant human FGF‐1 or 2 on normal human marrow cell proliferation in vitro, only minimal stimulatory activity could be detected. In this regard, the addition of either growth factor to cultures of ancillary cell depleted marrow mononuclear cells (MNC), or to highly enriched CD34+ MNC, failed to enhance haemopoietic colony number and induced only a slight increase in colony size. Perturbation of FGF receptor (FGF‐R) expression on CD34+ MNC with antisense (AS) oligodeoxynucleotides (ODN) was also without apparent effect on cell growth. Neither could we demonstrate any effect of FGF‐1 or 2 on survival of early progenitor cells in serum‐free culture. To explain these findings, we examined progenitor cells for expression of the FGF‐R at the mRNA and protein level using RT‐PCR and flow cytometry. Primitive CD34+/KIT+ MNC had no detectable FGF‐R (FGF‐R1, 2, 3 or 4) mRNA or protein expression. In fact, direct immunofluorescence labelling of MNC for CD34 antigen and FGF‐R1 demonstrated that expression of these markers was mutually exclusive in the populations examined. FGF‐R1 expression was detected on subpopulations of MNC and on cells derived from day‐6 CFU‐GM and BFU‐E colonies. Accordingly, FGF‐R1 is either absent, or present at very low levels, on primitive haemopoietic cells. This fact, combined with our in vitro culture data, suggest that receptors are unlikely to play a significant role in the development of these early cells. Nevertheless, the development of mature cells may be influenced by the FGFs since the FGF‐Rs are expressed on more mature cells.
Abnormal kinetics of colony formation by erythroid burst‐forming units (BFU‐E) in chronic myeloid leukaemia
Authors: MARLEY S. B. , LEWIS J. L. , GOLDMAN J. M. , GORDON M. Y.
Published: October 2003

We have investigated the kinetics of colony formation by progenitor cells in chronic myeloid leukaemia (CML) using erythroid burst‐forming units (BFU‐E) as a model system. For this, we scored the numbers of subcolonies produced by individual BFU‐E in cultures of normal marrow and blood cells and in cultures of CML blood cells. The formation of an erythroid burst consisting of a single subcolony was taken as evidence for immediate terminal differentiation; the formation of multiple subcolonies was taken as evidence for commitment to terminal differentiation only after several cell generations. Therefore the probability of differentiation can be obtained by scoring the numbers of subcolonies in individual erythroid bursts. We found that the probability of differentiation is decreased (P = 0.0004) and the number of subcolonies increased (P = 0.01) in CML BFU‐E compared with normal BFU‐E. The cellularity of the BFU‐E was also increased in CML. Using the probabilities of differentiation and renewal obtained from the BFU‐E cultures the results fitted the predictions of a stochastic branching model. These results indicate that (a) commitment to terminal erythroid differentiation occurs over several cell generations in populations of BFU‐E, (b) the probability of commitment to terminal differentiation (PD) within a particular population of BFU‐E, remains a constant independent of the number of cell generations involved, (c) PD is lower during burst formation by CML BFU‐E than by normal BFU‐E, and (d) commitment to terminal differentiation occurs over more cell generations in CML burst formation than in normal burst formation. Therefore a reduced probability of differentiation may be a primary defect and could explain the expansion of the erythroid progenitor cell compartment in CML.
Tucaresol increases oxygen affinity and reduces haemolysis in subjects with sickle cell anaemia
Authors: ARYA ROOPEN , ROLAN PAUL E. , W OOTTON RAYMOND , POSNER JOHN , BELLINGHAM ALASTAIR J.
Published: October 2003

The primary pathophysiological event in sickling is the intracellular polymerization of deoxygenated haemoglobin S. Tucaresol (589C80;4[2‐formyl‐3‐hydroxy‐phenoxymethyl] benzoic acid), a substituted benzaldehyde, was designed to interact with haemoglobin to increase oxygen affinity and has been shown to inhibit sickling in vitro. We administered tucaresol to sickle cell patients in the steady state to examine the anti‐sickling effect in vivo. Oral doses of tucaresol or placebo were given to nine stable sickle cell patients (aged 17–39 years; tucaresol, six; placebo, three) for 10 d. The first two patients on tucaresol were scheduled to receive a loading dose of 800 mg or 1200 mg (depending on bodyweight) for the first 4 d, followed by maintenance doses of 200 or 300 mg for the next 6 d. Due to concerns over the sharp rise in haematocrit in one patient, subsequent cohorts received 300 mg tucaresol daily throughout the dosing period. The oxygen affinity of haemoglobin S was increased in all patients receiving tucaresol, with between 10% and 24% of the haemoglobin modified, dependent on dose. In all patients on tucaresol, haemolysis was reduced with rises in haemoglobin of 0.9–3.7 g/dl (mean 2.2 g/dl), falls in lactate dehydrogenase of 16–52%, and a halving of the irreversibly sickled cell counts. These effects were apparent within a few days and persisted for 1–2 weeks following discontinuation of the drug. Three of the six patients on tucaresol developed fever and cervical lymphadenopathy, with onset between days 7 and 11 from start of drug. Further evaluation of the tolerability and efficacy of tucaresol in sickle cell patients is necessary.
Exon/intron structure of the human ALL‐1 (MLL) gene involved in translocations to chromosomal region 11q23 and acute leukaemias
Authors: NILSON I. , LöCHNER K. , SIEGLER G. , GREIL J. , BECK J. D. , FEY G. H. , MARSCHALEK R.
Published: October 2003

The acute lymphoblastic leukaemia (ALL)‐1 gene on human chromosome 11q23 is the site of many locally clustered chromosomal alterations associated with several types of acute leukaemias, including deletions, partial duplications and translocations. Structurally variant proteins derived from the altered gene presumably cause the malignant transformation of early haemopoietic progenitor cells. According to previously published reports, the gene consisted of at least 21 exons spread over approximately 100 kb. In this report a set of genomic fragments was isolated that represent a total of 35 exons (exons 3–37) encompassing > 95% of the protein‐coding region (except exons 1 and 2) and the 3′‐non‐translated region of the gene. The distances between these exons were determined and a detailed restriction map was produced. The majority of the exon/intron boundaries were sequenced and an intron‐phase analysis was performed. The results form the basis for a greater understanding of the translocations and other structural alterations of the gene that conserve the open reading frame and thus produce presumably oncogenic variants of the ALL‐1 protein.
Expression and prognostic value of the retinoblastoma tumour suppressor gene (RB‐1) in childhood acute lymphoblastic leukaemia
Authors: Sauerbrey Axel , Stammler Gerd , Zintl Felix , Volm Manfred
Published: October 2003

In a retrospective analysis, acute lymphoblastic leukaemia (ALL) blast cells of 102 children were investigated for the expression of the retinoblastoma susceptibility (RB)‐1 gene at mRNA level by dot blot hybridization and semiquantitative RT‐PCR. 56 patients were analysed by dot blot hybridization and 35 representative patients out of this group by semiquantitative RT‐PCR. Two additional groups of patients (23 patients with initial and 23 patients with relapsed ALL) were also investigated by semiquantitative RT‐PCR. RB‐1 gene expression was detectable in all investigated ALL at different levels. According to the relative mRNA expression the patients were discriminated by the median value in groups with low or high RB‐1 expression. The Kaplan‐Meier estimates showed that patients with low RB‐1 expression had a lower probability of remaining in first remission (P=0.03) and a significantly higher risk to succumb to their disease (P=0.03). Furthermore, the comparison of the results between initial and relapsed ALL showed that the relapses had significantly lower RB‐1 mRNA expression (P=0.02). The overall survival of the patients was shorter in both groups when RB‐1 gene expression was low. A multivariate analysis, including age, sex, immunological subtype, initial white blood cell count and RB‐1 expression, identified RB‐1 as an independent prognostic predictor (P=0.017) in addition to the initial white blood cell count (P=0.00001). In conclusion, low RB‐1 expression is an unfavourable prognostic predictor in initial and relapsed childhood ALL. The RB‐1 gene expression in relapsed ALL is significantly lower than in initial ALL.
Fanconi's anaemia presenting as acute myeloid leukaemia in adulthood
Authors: Cavenagh J. D. , Richardson D. S. , Gibson R. A. , Mathew C. G. , Newland A. C.
Published: October 2003

We describe a 28‐year‐old male patient who presented with apparently de novo acute myeloid leukaemia (AML) who was subsequently found to have Fanconi’s anaemia (FA). The gene for complementation group A (FAA) has recently been localized to chromosome 16q24.3 and utilizing genetic markers closely linked to this locus we were able to conclude that this patient was likely to belong to complementation group A. FA presenting as AML is an exceptionally rare event and all previously described cases have occurred in patients less than 21 years of age. We conclude that the diagnosis of FA should always be considered in younger patients presenting with AML. It is important that the correct diagnosis is made in these individuals because the administration of conventional chemotherapy may well have devastating consequences for them. Correlations between the specific mutations causing FA and clinical phenotypes are likely to become apparent as more genetic analyses are performed in this group of patients.
A randomized study comparing the effect of GM‐CSF and G‐CSF on immune reconstitution after autologous bone marrow transplantation
Authors: San Miguel J. F. , Hernández M. D. , Gonzalez M. , López‐Berges M. C. , Caballero M. D. , Vazquez L. , Orfao A. , Nieto M. J. , Corral M. , Del Cañizo M. C.
Published: October 2003

Haemopoietic growth factors (HGFs) have been shown to accelerate recovery from severe neutropenia after autologous bone marrow transplantation (ABMT) but their effect on immune reconstitution is not well defined. The present study compares, through randomized trial, the in vivo effect of GM‐CSF and G‐CSF administration on the immune recovery of patients who underwent ABMT. For that purpose, we have sequentially analysed 14 different T, B and NK lymphoid cell subsets using appropriate dual staining during the first year following transplant (days +6, +17, +31, +66, +90, +120, +180, +360). 24 patients with lymphoproliferative disorders (20 lymphomas and four multiple myelomas) and who had undergone ABMT were included in the study. The median age was 43 years (range 22–62 years). All lymphoma patients were homogenously conditioned with BEAM. Our results show that both GM‐CSF and G‐CSF aid T‐cell (CD3+/αβ) recovery though their contribution varies depending on the T‐cell subset analysed. G‐CSF contributed to a significantly faster recovery of CD8+ cells (P=0.03). The CD8+ cell regeneration was produced mainly by activated cells (CD38+/HLA‐DR+) which lacked the CD11b antigen. In contrast, GM‐CSF favoured the regeneration of CD4+ cells (through both the CD45RO+ and CD45RA+ subset), leading to a higher CD4+:CD8+ ratio (P=0.007). No statistically significant differences were detected in the three groups of patients as regards both the recovery of NK cells and NK activity. Furthermore, the use of HGF did not seem to exert a significant influence on the recovery of B lymphocytes. This recovery was based on the CD5+ subpopulation that showed a rapid rise after the first month. We suggest that G‐CSF and GM‐CSF not only influence myeloid recovery, but also regeneration of the immune system after ABMT.
A population‐based study of 272 children with acute myeloid leukaemia treated on two consecutive protocols with different intensity: best outcome in girls, infants, and children with Down's syndrome
Authors: Lie Sverre O. , Jonmundsson Gudmundur , Mellander Lotta , Siimes Martti A. , Yssing Minna , G ustafsson Göran
Published: October 2003

From July 1984 the five Nordic countries (Denmark, Finland, Iceland, Norway and Sweden) have registered all children with acute myeloid leukaemia (AML) and treated them on two consecutive protocols of different intensity (NOPHO‐84 and NOPHO‐88). We probably have information on every child with this diagnosis in our region. We found an annual incidence of AML of 0.7 new cases per 100 000 children <16 years of age. We observed a distinct peak of incidence in the first 2 years of life. Children with Down’s syndrome accounted for 13% of all cases.  Eighty of 105 cases treated on NOPHO‐84 achieved remission (78%). In NOPHO‐88, 100/118 patients entered remission (85%). The overall event‐free survival (p‐EFS) for the two studies was 0.32 for NOPHO‐84 and 0.42 for NOPHO‐88. The majority of relapses occurred within 2 years of diagnosis. When looking for prognostic factors the strongest significant adverse factor found was male sex. Children with Down’s syndrome (n=35) had a very favourable outcome if they received therapy according to protocol, and infants (n=26) had a superior outcome compared to children 1–2 years or <10 years of age at diagnosis.
Antigen topography is critical for interaction of IgG2 anti‐red‐cell antibodies with Fcγ receptors
Authors: KUMPEL BELINDA M. , Van DeWINKEL JAN G. J. , WESTERDAAL NOMDO A. C. , HADLEY ANDREW G. , DUGOUJON J. MICHEL , BLANCHER ANTOINE
Published: October 2003

IgG antibodies to the Rh D polypeptide on red cells are normally IgG1 or IgG3, whereas antibodies produced in response to carbohydrate antigens such as the A and B blood groups are predominantly IgG2. The consequences of this isotype restriction for the immune destruction of red cells were investigated. Human IgG2 anti‐D and IgG2 anti‐A were isolated by affinity purification from an unusual anti‐D serum (DEL) and anti‐A sera, respectively. These antibodies were compared with IgG1 and IgG3 monoclonal anti‐D in in vitro functional assays of the interaction between IgG‐coated red cells (EA‐IgG) and cells bearing IgG Fc receptors (FcγR). Dimeric IgG2 anti‐D bound efficiently to cell lines transfected with FcγRIIa‐H131, an allotypic form of FcγRIIa which readily interacts with IgG2, IgG1 and IgG3. Unexpectedly, however, ‐D‐phenotype red cells coated with IgG2 anti‐D did not form rosettes with these cells, whereas EA‐IgG2 anti‐A and EA‐IgG1 and EA‐IgG3 anti‐D effectively formed rosettes with these transfectants at the same sensitization level (100 000 molecules IgG/red cell). In antibody‐dependent cell‐mediated cytotoxicity (ADCC) assays, lysis of EA‐IgG2 anti‐A was mediated via FcγRIIa, whereas lysis of EA‐IgG1 and EA‐IgG3 anti‐D was mediated via FcγRI or FcγRIII; EA‐IgG2 anti‐D was inactive in all functional assays. These experiments suggest that both IgG subclass and antigen structure affect functional IgG–FcγR interactions. The topography of the Rh D antigen, an integral membrane protein, ensures that anti‐D is bound near the lipid bilayer surrounded by the glycocalyx. This may sterically hinder access of FcγRIIa‐H131 to the FcγR recognition site on the relatively inflexible IgG2 anti‐D, but not to that of IgG1 or IgG3 anti‐D. In contrast, IgG2 bound to the A antigen on glycoproteins is not so constrained. The topography of the D and A antigens may thus determine whether functional interactions of red‐cell‐bound IgG2 anti‐D and IgG2 anti‐A with cells bearing Fcγ receptors can occur.