bluuebunny/crossref_metadata_2025 huggingface dataset:
Positive diepoxybutane test in only one of two brothers found to be compound heterozygotes for Fanconi's anaemia complementation group C mutations
Authors: DOKAL I. , CHASE A. , MORGAN N. V. , COULTHARD S. , HALL G. , MATHEW C. G. , ROBERTS I.
Published: October 2003

Fanconi's anaemia (FA) is an autosomal recessive disorder characterized by diverse congenital abnormalities, the development of progressive bone marrow failure, and an increased predisposition to malignancy, particularly acute leukaemia. The FA phenotype is so variable that diagnosis on the basis of clinical manifestations alone can be difficult. The modern diagnosis of FA no longer rests entirely on the constellation of clinical and haematological abnormalities first described by Fanconi, but depends on finding elevated chromosomal breakage after incubation of peripheral blood lymphocytes with the chemical clastogens diepoxybutane (DEB) or mitomycin‐C (MMC). The cloning of the gene for FA complementation group C [FAC] provides an opportunity to test the validity of the ‘DEB test’ which in recent times has become the main arbiter as to whether a patient is classified as FA or non‐FA.

We report on two brothers with similar clinical and haematological features who have both been identified as compound heterozygotes for the FAC mutations L554P and ΔG322, but only one of the brothers has a positive DEB test. On the basis of the DEB test one would be classified as FA and the other as non‐FA. The time has come to re‐evaluate the diagnostic criteria of ‘Fanconi's anaemia’.
Hyper‐interleukin (IL)‐6‐naemia in haemophagocytic lymphohistiocytosis
Authors: Imashuku Shinsaku , Hibi Shigeyoshi , Fujiwara Fumihiro , Todo Shinjiro
Published: October 2003

Clinical features in patients with haemophagocytic lymphohistiocytosis (HLH) have been demonstrated to be characterized by hypercytokinaemia. Previously, we reported the impact of high serum levels of interferon (IFN)‐gamma and soluble IL‐2 receptor (sIL‐2R) on patient outcome; however, it was not known if serum levels of interleukin (IL)‐6 also could be a prognostic factor. In a study during the active phase of disease in 25 cases of HLH in children and young adults (median age 3 years, range 0.1–23 years), we noted 12 cases which showed serum IL‐6 >100 (normal <4.0) pg/ml. Five of these cases showed hyper‐IL‐6‐naemia alone without hyper‐IFN‐gamma‐naemia (group A) whereas seven cases showed both hyper‐IL‐6‐ and IFN‐gamma‐naemia (group B). Patient outcome did not differ between the patients with IL‐6 >100 pg/ml and those with IL‐6 <100 pg/ml, suggesting that high serum concentrations of IL‐6 alone do not necessarily indicate poor prognosis in patients with HLH. Among the cases with hyper‐IL‐6‐naemia (>100 pg/ml), underlying disorders causing haemophagocytosis were found to be different between groups A and B.
Saporin, a ribosome‐inactivating protein used to prepare immunotoxins, induces cell death via apoptosis
Authors: Bergamaschi Gaetano , Perfetti Vittorio , Tonon Laura , Novella Annunziata , Lucotti Claudia , Danova Marco , Glennie Martin J. , Merlini Giampaolo , Cazzola Mario
Published: October 2003

The plant toxin saporin is a ribosome‐inactivating protein which inhibits protein synthesis and growth of both normal and tumour cells. Its cytotoxic activity can be increased by coupling with antibodies recognizing cell surface antigens. In this work we performed experiments to test the hypothesis that saporin induces cell death via apoptosis. Exposure to saporin induced apoptosis in different cellular models, such as human peripheral blood B lymphocytes and neutrophils, in the Daudi B‐cell line, and in the haemopoietic cell lines HL‐60 and TF‐1. This was indicated by: (a) the appearance of typical morphological features such as chromatin condensation, nuclear fragmentation and blebbing of plasma membranes; (b) DNA degradation into oligonucleosomal fragments; (c) the appearance of apoptotic cells on DNA flow cytometry as a cell population with reduced DNA content (A0 region). The fraction of cells showing features of apoptosis ranged from 19 ± 5% for TF‐1 cells to 35 ± 8% for neutrophils. In experiments with normal peripheral blood B lymphocytes or with Daudi cells, we compared the activity of native saporin with that of an immunotoxin hybrid molecule obtained by binding the toxin to two bispecific antibodies recognizing both saporin and the B lymphocyte‐specific antigen CD22 (Sap/BsAb complexes). Saporin bound to the antibodies was 2–3 logs more effective than native saporin in inducing apoptosis, with maximal inhibitions being observed at concentrations of 10−6 M for native saporin and 10−9–10−8 M for the hybrid molecules. These findings indicate that treatment with saporin results in apoptosis of target cells and suggest that this may be relevant to the therapeutic use of saporin‐containing immunotoxins. In fact, if used in vivo as an immunotoxin, its cytotoxic activity could be devoid of more extensive and non‐specific tissue damaging effects as would be the case if saporin induced necrosis of target cells.
DOWN‐REGULATION OF FIBRINOGEN BIOSYNTHESIS BY IL‐4, IL‐10 AND IL‐13
Authors: VASSE M. , PAYSANT J. , SORIA J. , MIRSHAHI S. S. , VANNIER J. P. , SORIA C.
Published: October 2003

High levels of fibrinogen are recognized as an important vascular risk factor; however, it is not known if the increase of plasma fibrinogen is directly responsible for this risk, or is only a marker of vascular inflammation. To support this second hypothesis, Oncostatin M (OSM) is a potent stimulator of fibrinogen biosynthesis and induces smooth muscle cell proliferation. In the same way, we analysed whether interleukin‐4 (IL‐4), interleukin‐10 (IL‐10) or interleukin‐13 (IL‐13), which protect vessel walls from monocytes injuries leading to atherosclerosis, could influence fibrinogen biosynthesis. The two levels of regulation of fibrinogen biosynthesis were tested: firstly, the direct effect of these cytokines on fibrinogen production by the hepatoma cell line Hep G2, and secondly their effect on the secretion of hepatocyte stimulating factor (HSF) activity in the supernatant of lipopolysaccharide (LPS)‐activated monocytes. IL‐4 and IL‐13 added to Hep G2 cells down‐regulated both the increase of fibrinogen secretion induced by IL‐6 and fibrinogen mRNA levels, this effect being more pronounced when Hep G2 were preincubated with the two cytokines before IL‐6 addition. The effect of IL‐10 was evidenced only on mRNA expression. IL‐10 and IL‐13 dose‐dependently decrease HSF activity secreted by LPS‐activated monocytes, whereas IL‐4 had no effect. However, the three cytokines decreased HSF activity when monocytes were incubated with the cytokines before LPS activation. The effects of these cytokines on HSF activity are related to variations of IL‐6 and OSM secretion. Our data strengthen the hypothesis that the fibrinogen level is a marker of vascular disease, since cytokines which have a protective vascular effect down‐regulate fibrinogen production.
The frequency of the haemochromatosis‐associated genotype D6S265‐1:D6S105‐8 in blood donors
Authors: WORWOOD M. , DORAK M. T. , NICKLIN S. , STONE C. , POINTON J. , DARKE C.
Published: October 2003

Homozygosity for HLA‐A3 and the microsatellite markers D6S265 allele 1 and D6S105 allele 8 is associated with a high relative risk for genetic haemochromatosis — indeed we and others have suggested that a haplotype including D6S265‐1, HLA‐A3 and D6S105‐8 is specific for haemochromatosis. To determine the frequency of this haplotype and examine its specificity for haemochromatosis we have analysed data from 7820 blood donors from South Wales. The frequency of homozygosity for D6S265‐1, HLA‐A3 and D6S105‐8 was 1 in 280. Calculations based on the prevalence of haemochromatosis suggest that about 50% of chromosomes carrying D6S265‐1:HLA‐A3:D6S105‐8 also carry the haemochromatosis gene. This information is of value for assessing the risk that the partner of a patient with haemochromatosis also carries the haemochromatosis gene.
Investigation of calmodulin and basic fibroblast growth factor (bFGF) in idiopathic myelofibrosis: evidence for a role of extracellular calmodulin in fibroblast proliferation
Authors: Dalley A. , Smith J. M. , Reilly J. T. , Mac Neil S.
Published: October 2003

The urinary concentration of calmodulin and basic fibroblast growth factor (bFGF) was determined in a total of 53 patients with various chronic myeloproliferative disorders (CMPD), including 22 patients with idiopathic myelofibrosis (IMF). Calmodulin excretion was significantly elevated in IMF (0.29 ± 0.04 μg/mmol creatinine) (P<0.001), when compared to polycythaemia vera (PV) (0.14 ± 0.02), essential thrombocythaemia (ET) (0.13 ± 0.04), chronic myeloid leukaemia (CML) (0.16 ± 0.02), unclassified myeloproliferative disorders (UMPD) (0.11 ± 0.02) and age‐matched controls (0.1 ± 0.02) (P<0.001). In contrast, bFGF was slightly elevated in all CMPD conditions when compared to age‐matched controls. A neutralizing antibody to calmodulin was demonstrated to significantly influence the in vitro proliferation of normal human fibroblasts, an effect dependent on both cell density and the presence of fetal calf serum (FCS). Essentially, the antibody reduced FCS‐induced proliferation of low‐density fibroblasts but had little or no inhibitory effect on high‐density fibroblasts in the absence of FCS. In addition, extracellular calmodulin was shown not to interact with known fibroblast mitogens, namely IFG‐1, EGF, bFGF and PDGF. We conclude that extracellular calmodulin should be considered, in addition to PDGF, TFG‐β and EGF, as a potential mitogen involved in the stromal reaction of idiopathic myelofibrosis.
Clearance of human factor XIa–inhibitor complexes in rats
Authors: Wuillemin Walter A. , Bleeker Wim K. , Agterb erg Jacques , Rigter Gemma , Cate Hugoten , Hack C.Erik
Published: October 2003

The serpins C1 esterase inhibitor (C1Inh), antithrombin (AT), α1‐antitrypsin (α1AT) and α2‐antiplasmin (α2AP) are known inhibitors of coagulation factor XIa (FXIa). Although initial studies suggested α1AT to be the main inhibitor of FXIa, we recently demonstrated C1Inh to be a predominant inhibitor of FXIa in vitro in human plasma. The present study was performed to investigate the plasma elimination kinetics of preformed human FXIa–FXIa inhibitor complexes injected in rats. The amounts of complexes remaining in circulation were measured using enzyme‐linked immunosorbent assays. The plasma half‐life time of clearance (t1/2) was 98 min for FXIa–α1AT complexes, whereas it was considerably shorter, i.e. 19, 18 and 15 min for FXIa–C1Inh, FXIa–α2AP and FXIa–AT complexes, respectively. Thus, due to this different plasma t1/2, preferentially FXIa–α1AT complexes may be detected in clinical samples. Furthermore, measuring FXIa–FXIa inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of FXIa in vivo
CONTRIBUTION OF HAEMOLYSIS TO JAUNDICE IN SEPHARDIC JEWISH GLUCOSE‐6‐PHOSPHATE DEHYDROGENASE DEFICIENT NEONATES
Authors: Kaplan Michael , Vreman Hendrik J. , Hammerman Cathy , Leiter Chava , Abramov Ayala , Stevenson David K.
Published: October 2003

We determined the contribution of haemolysis to the development of hyperbilirubinaemia in glucose‐6‐phosphate dehydrogenase (G‐6‐PD) deficient neonates and G‐6‐PD normal controls. Blood carboxyhaemoglobin (COHb), sampled on the third day of life, was measured by gas chromatography, corrected for inhaled carbon monoxide (COHbC), and expressed as a percentage of total haemoglobin concentration (Hb). Serum bilirubin was tested as clinically necessary. 37 non‐jaundiced (peak serum total bilirubin (PSTB) ≤255 μmol/l) and 20 jaundiced (PSTB ≥257 μmol/l) G‐6‐PD‐deficient neonates were compared to 31 non‐jaundiced and 24 jaundiced controls with comparable PSTB values, respectively. COHbC values for the entire G‐6‐PD deficient group were higher than in the controls (0.75 ± 0.17% v 0.62 ± 0.19%, P < 0.001). COHbC and PSTB values did not correlate in the G‐6‐PD‐deficient group (r = 0.15, P > 0.05) but did in the controls (r = 0.58, P < 0.001). COHbC values were increased to a similar extent in the G‐6‐PD‐deficient, non‐jaundiced (0.72 ± 0.16%), the G‐6‐PD‐deficient, jaundiced (0.80 ± 0.19%) and the control, jaundiced (0.75 ± 0.18%) subgroups, compared to the control, non‐jaundiced subgroup (0.53 ± 0.13%) (P < 0.05). Although present in G‐6‐PD deficient neonates, increased haemolysis was not directly related to the PSTB.
Ankyrin Napoli: a de novo deletional frameshift mutation in exon 16 of ankyrin gene (ANK1) associated with spherocytosis
Authors: Miragliadel Giudice E. , Hayette S. , Bozon M. , Perrotta S. , Alloisio N. , Vallier A. , Iolascon A. , Delaunay J. , Morlé L.
Published: October 2003

We report a case of apparently recessive hereditary spherocytosis in an Italian child. The proband exhibited a reduction of overall ankyrin in the red cell membrane. The parents were free of any haematological manifestations. The VNDR associated with the ankyrin gene (ANK1) were consistent with the following diplotypes: AC11/AC14 (father), AC14/AC14 (mother) and AC11/AC14 (child). The cDNA of the patient disclosed the expression of the AC11 allele only. As a consequence, we put forward the hypothesis of a de novo inactivation affecting the ankyrin allele of maternal origin (AC14) and accounting for the disease. PCR amplification of exons, SSCP analysis and nucleotide sequencing disclosed a polymorphism: GAC → AAC; Asp → Asn in codon 328 of exon 10, and a one‐nucleotide deletion : CTG → CG in codon 573 of the exon 16. This frameshift mutation placed in phase the TGA triplet that normally overlaps codons 636 and 637. Termination of translation near the middle of ankyrin mRNA coding sequence resulted, presumably, in its premature degradation. The present allele has been designated allele Napoli.
Duration of filgrastim mobilization and apheresis yield of CD34+ progenitor cells and lymphoid subsets in normal donors for allogeneic transplantation
Authors: ANDERLINI PAOLO , PRZEPIORKA DONNA , HUH YANG , LAUPPE JO , MILLER PAULA , SUNDBERG JANICE , SEONG DAVID , CHAMPLIN RICHARD , KÖRBLING MARTIN
Published: October 2003

Seventy‐seven normal donors underwent leukapheresis for peripheral blood progenitor cell collection beginning on day 4 (n = 45) or day 5 (n = 32) of filgrastim mobilization (12 μg/kg/d). The two groups were comparable for age, weight, blood volumes processed during leukapheresis and target CD34+ cell dose to be collected. The day 5 schedule allowed a more consistent achievement of the target cell dose with one apheresis (P = 0.005) and resulted in the initial collection of a significantly larger number of CD34+ cells (P = 0.009). There was no statistically significant difference in the leukapheresis yield of lymphoid subsets and natural killer cells.
A case of mature B‐cell ALL with coexistence of t(1;19) and t(14;18) and expression of the E2A/PBX1 fusion gene
Authors: Rowe David , Devaraj Prema E. , Irving Julie A. E. , Hogarth Linda , Hall Andrew G. , Turner Gillian E.
Published: October 2003

The translocation t(1;19)(q23;p13) is found in 3–5% of all acute lymphoblastic leukaemias (ALL) and results in the expression of an E2A/PBX1 hybrid gene transcript. This translocation is very closely associated with a pre‐B phenotype. t(14;18) is associated with follicular B‐cell lymphoma and is characterized by over‐expression of the bcl‐2 oncogene. We describe a case of ALL in an adult with a mature B‐cell immunophenotype and a single abnormal cell line with a complex karyotype showing both t(1;19) and t(14;18).

Two reports of this phenomenon have been published previously and molecular analysis, where performed, showed the E2A gene was not rearranged, suggesting the t(1;19) was a molecular variant of the established translocation. In contrast, molecular analysis of our case demonstrated expression of the E2A/PBX1 fusion transcript typically associated with t(1;19) in pre‐B ALL but showed it to be present at an extremely low level, despite the abnormal karyotype being found in the majority of metaphase cells. Analysis of bcl‐2 expression showed a significant up‐regulation.

A down‐regulation of the E2A/PBX1 hybrid gene as a consequence of the enhanced expression of bcl‐2 may be a possible mechanism for this finding.
Clinical usefulness of desmopressin for prevention of surgical bleeding in patients with symptomatic heterozygous factor XI deficiency
Authors: Castaman Giancarlo , Ruggeri Marco , Rodeghiero Francesco
Published: October 2003

Heterozygous factor XI (FXI) deficiency is sometimes associated with a significant bleeding tendency. Fresh frozen plasma or FXI concentrates are the mainstay of treatment in patients with a clear bleeding history, especially prior to surgery. However, these treatments are not completely free of risk. Furthermore, thrombosis has been reported in patients with FXI deficiency infused with FXI concentrate. No data are available on the possible efficacy of desmopressin in these patients.  Two patients with a clear bleeding history associated with FXI deficiency and no additional haemostatic defects agreed to be treated with desmopressin before carpal tunnel surgery and dental extraction. The reduced basal FXI activity and antigen levels slightly increased after infusion, reaching borderline values. No bleeding was observed after surgical procedures.  Desmopressin treatment seems a reasonable and useful choice in symptomatic, heterozygous FXI‐deficient patients, thus reducing the cost of treatment, the risk of transmission of blood‐borne viruses, and of thrombosis.
Second transplantation with CD34+ bone marrow cells selected from a two‐loci HLA‐mismatched sibling for a patient with chronic myeloid leukaemia
Authors: Fujimori Yoshihiro , Kanamaru Akihisa , Hashimoto Naoaki , Okamoto Takahiro , Okada Masaya , Kawaguchi Kohotaro , Mori Ako , Saheki Kaname , Takatsuka Hiroyuki , Wada Hiroshi , Takemoto Yoshinobu , Kohsaki Masatoshi , Imai Nobuyuki , Kakishita Eizo , Nagai Kiyoyasu
Published: October 2003

A 43‐year‐old man with chronic myeloid leukaemia underwent a second transplant with CD34+ bone marrow cells selected from his two‐loci HLA‐mismatched sibling after rejection of the first graft from an HLA‐matched unrelated donor. By immunomagnetic positive selection, CD34+ marrow cells at 0.95×106/kg with 97% purity and CD3+ T lymphocytes at 1.3×104/kg were collected and transplanted. Engraftment was confirmed to be of CD34+ cell‐donor origin. The patient developed only grade I acute graft‐versus‐host disease (GVHD) and no chronic GVHD to date. These observations suggest that allogeneic CD34+ bone marrow cells are capable of reconstituting haemopoiesis and that CD34+ selection could be applicable to T‐cell depletion.
Flutamide‐induced cyanosis refractory to methylene blue therapy
Authors: Kouides Peter A. , Abboud Camille N. , Fairbanks Virgil F.
Published: October 2003

Flutamide, an anti‐androgen used in prostate cancer therapy, is also a derivative of aniline. Mild, usually asymptomatic, methaemoglobinaemia has been reported. We report a patient receiving flutamide therapy who developed cyanosis, dyspnoea and anaemia, initially attributed to marked methaemoglobinaemia by the CO‐Oximeter method. An unsuccessful trial of methylene blue therapy led to the finding of marked sulphaemoglobinaemia. Sulphaemoglobinaemia has not previously been reported with flutamide use. Recognition of this association is important, given the refractoriness of sulphaemoglobinaemia to methylene blue therapy.
Expression of the multidrug resistance‐associated protein (MRP) mRNA and protein in normal peripheral blood and bone marrow haemopoietic cells
Authors: Legrand Ollivier , Perrot Jean‐Yves , Tang RuoPing , Simonin Ghislaine , Gurbuxani Sandeep , Zittoun Robert , Marie Jean‐Pierre
Published: October 2003

We studied the expression of multidrug resistance‐associated protein (MRP) in normal haemopoietic cells from peripheral blood and bone marrow. The MRP mRNA levels were estimated by RT/PCR and in situ hybridization (ISH) assay, and the protein levels by flow cytometry. 21 samples of peripheral blood and 21 samples of bone marrow (11 normal bone marrow donors, 10 patients in complete remission after chemotherapy for large cell lymphoma or acute myeloid leukaemia) were analysed. In peripheral blood the mean MRP mRNA level in CD3+ cells was statistically higher than in the other cells (3‐fold by the methods used). The levels of MRP in CD3+ varied from one individual to another (4.5–34.8 units by RT/PCR and 5–23 grains/cell by ISH); however, this was proportional to the variation in all the cell lineages of same individual (r = 0.84). In bone marrow the mean MRP levels of the various cell lineages (including CD34+) were similar to the basal level in HL60 cells. Individual expression levels were again variable; however, there was no difference between untreated normal bone marrow and post chemotherapy normal bone marrow. MRP protein expression was determined by flow cytometry with the monoclonal antibody MRPm6. The CD4+ lymphocytes exhibited a higher MRP protein expression than the other cell lineages, including CD8+ cells. There was a good correlation between the three methods used (RT/PCR and ISH, P = 0.0001, r = 0.87; RT/PCR and flow cytometry, P = 0.0001, r = 0.85; ISH and flow cytometry, P = 0.002, r = 0.67).
Transient neutropenia in normal donors after G‐CSF mobilization and stem cell apheresis
Authors: Anderlini Paolo , Przepiorka Donna , Seong David , Champlin Richard , Körbling Martin
Published: October 2003

Thirteen normal adult donors underwent daily leukapheresis for peripheral blood progenitor cell collection for allografting beginning on day 4 or 5 of G‐CSF mobilization (12 μg/kg/d). They had complete blood counts performed 7–10 d after the completion of the procedure. A reduction in the total leucocyte count below baseline levels, accounted for by a decrease in the absolute neutrophil (ANC) and lymphocyte counts, was noted. Neutropenia (ANC < 1.5 × 109/l) occurred in two (15%) of the donors. The lowest ANC observed was 0.6 × 109/l. The neutropenia was asymptomatic and resolved on follow‐up evaluation.
Clonal expansion of γδ T cells expressing two distinct T‐cell receptors
Authors: Hinz Thomas , Marx Sibylle , Nerl Christoph , Kabelitz Dieter
Published: October 2003

92% of peripheral blood mononuclear cells from a patient with chronic lymphocytosis (WBC 22×109/l) were recognized by pan‐γδ monoclonal antibody (mAb) TCRδ1. Further analysis with anti‐Vγ/Vδ mAb indicated that the expanded γδ T‐cell population was stained by mAb specific for Vγ2/3/4, Vγ5 and Vδ1 but not mAb specific for Vγ8, Vγ9, Vδ2 or Vδ3. Cloning and sequencing of reverse transcription PCR products revealed the presence of Vγ4, Vγ5 and Vδ1 in‐frame transcripts. All the cDNA clones obtained from each of these variable elements had identical junction regions, thus clearly demonstrating clonality. As far as we know, this is the first description of a pathological clonal expansion of γδ T cells with two distinct surface‐expressed T‐cell receptor molecules.
Evaluation of a new, integral, whole blood filter (RS2000) system for prestorage leucodepletion of SAG‐M red cells
Authors: RIDER J. R. , MOORE G. , PAYRAT J‐M. , CHAN D. , MORSE C. , STANDEN G. R. , PAMPHILON D. H.
Published: October 2003

Residual donor leucocytes are responsible for many adverse transfusion reactions. Prestorage leucodepletion may ameliorate these effects and enhance product quality. We studied a bottom and top (BAT) system incorporating an integral filter for whole blood leucodepletion. Our evaluation assessed leucodepletion efficiency as well as in vitro SAG‐M red cell quality and storage characteristics.  Sixty‐six units of blood were collected; test units into the Optipac®‐pLuS system and controls into the standard triple pack configuration. Test units were held for 4–6 h at room temperature (rt) or 12–18 h at 4°C. The mean leucocyte counts for the SAG‐M red cells in the quality and storage trial were 0.6×106 (rt hold), 0.05×106 (4°C hold) and 2500×106 (controls). We observed no significant differences between the groups for Na+, ATP, 2,3‐DPG, glucose, lactate and pH during the 49 d storage. The control group, however, showed a greater increase in haemolysis and K+ with time. Autologous in vivo 24 h red cell recovery, after 42 d storage, was >75%. Adjustment of processing parameters in subsequent studies gave leucodepleted SAG‐M red cells with minimal cell loss (9–19%) plus acceptable haemoglobin content (46–76 g/U) and haematocrit (54–62%). This system achieved >3.5 log leucodepletion with all but one unit containing <1×106 leucocytes. The product quality is good and the system suitable for routine use in blood centres.
The development of human megakaryocytes. II. CD4 expression occurs during haemopoietic differentiation and is an early step in megakaryocyte maturation
Authors: Basch Ross S. , Dolzhanskiy Alexandr , Zhang Xin‐Min , Karpatkin Simon
Published: October 2003

CD4 expression is not limited to T cells and monocytes. In both mouse and man the antigen has been detected on some early haemopoietic progenitors and we have shown that some mature megakaryocytes (MK) express CD4, the surface molecule that serves as the high‐affinity receptor for human immunodeficiency virus, type‐1 (HIV‐1). Using a serum‐free culture system in which sorted CD34+ haemopoietic progenitors are cultured with thrombopoietin (TPO), IL‐3, IL‐6 and SCF, we now show that CD4 expression is induced in virtually all developing haemopoietic cells. This phenomenon was particularly striking in the MK lineage, where CD4 expression began whilst the cells were still CD34+ but after they expressed CD41 (GPIIb/IIIa). CD4 expression and endomitotic polyploidization occur at the same time in MK development. In culture, maximum CD4 expression occurred 4–6 d after CD41 expression and lasted for a few days. Expression of CD4 declined gradually thereafter and most MK were CD4− by the end of the culture period. The amount of CD4 on the surface of some MK, as measured by intensity of fluorescence staining, exceeded that of normal monocytes and approached the brightness of T cells. Appearance of the surface antigen correlated with the presence of mRNA for CD4, as measured by RT‐PCR.
Kaposi's sarcoma‐associated herpesvirus DNA sequences in AIDS‐related and AIDS‐unrelated lymphomatous effusions
Authors: Carbone Antonino , Gloghini Annunziata , Vaccher Emanuela , Zagonel Vittorina , Pastore Cristina , Palma Paolo Dalla , Branz Fabio , Saglio Giuseppe , Volpe Rachele , Tirelli Umberto , Gaidano Gianluca
Published: October 2003

Primary effusions presenting as the sole lymphoma localization are also known as body‐cavity‐based‐lymphoma (BCBL), and have been shown to carry Kaposi's sarcoma herpesvirus (KSHV) DNA sequences. The aim of this study was a comparative analysis of the clinical, pathologic and molecular features of BCBL and lymphomatous effusions secondary to tissue‐based lymphomas occurring both in the general population and in HIV‐1‐infected individuals. All the lymphomatous effusion samples (seven AIDS‐related and nine AIDS‐unrelated) were subjected to an identical multiparameter investigation, including collection of clinical data, analysis of morphology and immunophenotype, as well as the study of viral sequences and genetic lesions. In six cases defined as BCBL (four AIDS‐related and two AIDS‐unrelated), the patients exhibited exclusive or predominant involvement of the body cavities. BCBL tended to display indeterminate phenotypes (4/6), whereas all AIDS‐related and AIDS‐unrelated lymphomatous effusions secondary to tissue‐based lymphomas consistently expressed B‐cell phenotype. Detection of KSHV DNA sequences was restricted to cases of BCBL (3/4 AIDS‐related and 1/2 AIDS‐unrelated), whereas EBV association (3/4) and expression of EBV‐encoded antigens (LMP‐1, 2/3; EBNA‐2, 1/3) were confined to the AIDS‐related BCBL. Overall, our results confirm that both AIDS‐related and AIDS‐unrelated BCBL preferentially associate with peculiar clinical, immunophenotypic and molecular features among lymphomatous effusions and therefore should be singled out as a specific clinico‐pathologic entity.
Myelodysplasia following aplastic anaemia–paroxysmal nocturnal haemoglobinuria syndrome after treatment with immunosuppression and G‐CSF: evidence for the emergence of a separate clone
Authors: Jin Jong‐Youl , Tooze Jennifer A. , Marsh Judith C. W. , Matthey Francis , Gordon‐Smith Edward C.
Published: October 2003

Myelodysplasia (MDS) and aplastic anaemia–paroxysmal nocturnal haemoglobinuria (AA/PNH) syndrome developed in a severe aplastic anaemia (AA) patient after treatment with immunosuppressive (IS) therapy. Glycosylphosphatidyl inositol (GPI)‐linked proteins were determined, and during the AA/PNH phase, a high proportion of neutrophils were found to be negative, without clinical evidence of haemolysis. However, MDS developed with cytogenetic abnormalities of monosomy 7, 9q− and a rearranged chromosome 6; the GPI‐linked protein negative cells were completely replaced by positively expressing cells. This represents the emergence of a GPI‐linked protein positive myelodysplasia clone arising separately from an AA/PNH clone.
Fibrinogen Matsumoto II: γ308Asn → Lys (AAT → AAG) mutation associated with bleeding tendency
Authors: Okumura Nobuo , Furihata Kenichi , Terasawa Fumiko , Ishikawa Shinsuke , Ueno Ichiro , Katsuyama Tsutomu
Published: October 2003

Fibrinogen Matsumoto II is a hereditary dysfibrinogenaemia identified in a woman with Basedow's disease and a bleeding tendency. Coagulation tests of the patient's plasma revealed a prolonged thrombin time and a decreased fibrinogen level determined by functional method. Release of fibrinopeptide A and B was normal, whereas fibrin monomer polymerization was delayed. Fibrinogen γ‐chain gene of the propositus was heterozygous for a missense mutation that resulted in Asn → Lys substitution at codon 308. Though the same amino acid substitution was also attributed to fibrinogen Kyoto I and Bicetre II, fibrinogen Matsumoto II showed different clinical manifestations from them.
Unrelated donor bone marrow transplantation for children with relapsed acute lymphoblastic leukaemia in second complete remission
Authors: OAKHILL A. , PAMPHILON D. H. , POTTER M. N. , STEWARD C. G. , GOODMAN S. , GREEN A. , GOULDEN P. , GOULDEN N. J. , HALE G. , WALDMANN H. , CORNISH J. M. M.
Published: October 2003

Allogeneic sibling bone marrow transplantation (BMT) is the recommended treatment for relapsed childhood acute lymphoblastic leukaemia (ALL), but appropriate donors are only available in 30% of cases. Unfortunately, BMT from unrelated donors (UD) has been associated with high rates of severe graft‐versus‐host disease (GvHD) and transplant‐related mortality (TRM). In an attempt to improve outcome in UD‐BMT we have assessed the impact of T‐cell depletion using CAMPATH‐1 (anti‐CD52) monoclonal antibodies in 50 consecutively referred patients with relapsed ALL in second remission. All were previously treated according to MRC protocols UKALL X and XI, and then given chemotherapy on MRC R1 from relapse until UD‐BMT. 19 patients had relapsed on and 31 off therapy.

Patients and donors were fully matched at HLA‐A, ‐B, ‐DR and ‐DQ loci in 29 cases and mismatched in 21 (four mismatched for more than one antigen). Pre‐transplant conditioning comprised CAMPATH‐1G, cyclophosphamide and total body irradiation. Bone marrow was T‐cell depleted in vitro using CAMPATH‐1 antibodies. Additional GvHD prophylaxis consisted of cyclosporin A (42 cases), cyclosporin plus methotrexate (four) or none (four). 47 patients engrafted. The incidence of acute GvHD was very low: two patients with grade II disease in the matched group, four with grade II–IV in the mismatched group. Only four patients have chronic GvHD. The actuarial event‐free survival (EFS) at 2 years is 53%, with no significant difference between the matched and mismatched group. Further leukaemic relapse was the most important cause of failure.

These results are similar to the most favourable published reports for HLA‐matched sibling BMT in relapsed ALL.
Occurrence of dysregulated oncogenes in primary plasma cells representing consecutive stages of myeloma pathogenesis: indications for different disease entities
Authors: Rasmussen Thomas , Theilgaard‐Mönch Kim , Hudlebusch Heidi R. , Lodahl Marianne , Johnsen Hans E. , Dahl Inger Marie S.
Published: October 2003

Summary. This study investigated the expression pattern in primary plasma cells (PCs) of putative oncogenes suggested to be involved in multiple myeloma (MM) development. cDNA archives were generated by global reverse transcription polymerase chain reaction from CD38++/CD19–/CD56–/++ aberrant PCs of a prospective cohort of 96 subjects, including healthy individuals, patients with monoclonal gammopathies of undetermined significance (MGUS), MM and MM with extramedullary manifestations (ExMM). The cDNA archives were analysed quantitatively for expression of the cyclin D1, fibroblast growth factor receptor 3 (FGFR3), C‐MYC, C‐MAF and cyclin D3 oncogenes. In addition, all patients were screened for IGH–MMSET hybrid transcripts. None of the analysed oncogenes was randomly distributed. C‐MYC and cyclin D3 expression increased at the extramedullary transformation stage. Furthermore, C‐MYC and cyclin D3 expression in CD56+ MM was similar to MGUS, whereas CD56– MM was similar to ExMM. FGFR3/IGH–MMSET was only observed among CD56+ MM patients, whereas an increased frequency of C‐MAF dysregulation was seen among CD56– MM. High cyclin D1 expression levels were identified at similar frequencies at all stages, whereas the frequency of patients with low cyclin D1 levels increased during MM development. These data support the stepwise transformation model accumulating genetic alterations and proliferative capacity during MM initiation and development resulting in different clinical entities.
Pregnancy‐associated venous thromboembolism (VTE) in combined heterozygous factor V Leiden (FVL) and prothrombin (FII) 20210 A mutation and in heterozygous FII single gene mutation alone
Authors: Samama Meyer‐Michel , Rached Roberto A. , Horellou Marie‐Hélène , Aquilanti Sandro , Mathieux Valérie G. , Plu‐Bureau Geneviève , Elalamy Ismail , Conard Jacqueline
Published: October 2003

Summary.The risk of venous thromboembolism (VTE) in the absence of prophylaxis was evaluated in a retrospective study of 47 women (84 pregnancies) with combined thrombophilia [heterozygous factor V Leiden (FVL) plus prothrombin (FII) 20210A mutation (group I)] and in 82 women (193 pregnancies) with the FII alone (group II). VTE was more frequent in group I than in group II [17·8% versus 6·2%,P = 0·003, relative risk (RR) 2·9, 95% confidence interval (CI) 1·4–5·9], ante partum (7·1% and 2·1%) and post partum (11·5% and 4·2%). The risk was higher in index cases than in family members (RR 2·5, 95% CI 1·2–5·2 and RR 2·1, 95% CI 0·2–22·3 respectively) Even women who had no history of VTE before pregnancy had an increased risk (RR 2·2, 95% CI 1·0–4·8). Our results suggest that, during ante partum, prophylaxis is indicated in women with combined thrombophilia and with a VTE before pregnancy. In those without VTE before pregnancy, prophylaxis might be decided for each individual case, taking into consideration all risk factors. In women with the FII mutation alone, the low risk may not justify prophylaxis in the absence of previous VTE. In post partum, prophylaxis is indicated in all cases.
Heterocellular hereditary persistence of fetal haemoglobin affects the haematological parameters of β‐thalassaemia trait
Authors: Garner Chad , Dew Tracy K. , Sherwood Roy , Rees David , Thein Swee Lay
Published: October 2003

Summary.To assess and define the effects of heterocellular hereditary persistence of fetal haemoglobin (HPFH) on the haematological phenotype of heterozygous β‐thalassaemia, we have studied a large kindred that included a total of 204 subjects with 60 β‐thalassaemia carriers, of whom 35 were also heterozygous, and five homozygous, for heterocellular HPFH. The study was possible because of the homogeneity of the β‐thalassaemia mutation and the ability to genotype the heterocellular HPFH allele. Heterocellular HPFH had a significant effect on the mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV) and haemoglobin (Hb) A2values in the β‐thalassaemia carriers and accounted for 29%, 30% and 24% of their respective variances. β‐thalassaemia subjects with heterocellular HPFH had higher MCV and MCH values, concomitant with lower levels of Hb A2, and a reduced ineffective erythropoiesis. We conclude that co‐inheritance of heterocellular HPFH leads to a primary increase in γ‐chain synthesis in β‐thalassaemia trait and can be another confounding factor in the use of red cell indices and Hb A2levels in population screening for β‐thalassaemia.
Plasma levels of tumour necrosis factor alpha and interleukin‐6 predict progression‐free survival following thalidomide therapy in patients with previously untreated multiple myeloma
Authors: Thompson Michael A. , Witzig Thomas E. , Kumar Shaji , Timm Michael M. , Haug Jessica , Fonseca Rafael , Greipp Philip R. , Lust John A. , Rajkumar S. Vincent
Published: October 2003

Summary. We studied marrow angiogenesis and plasma levels of angiogenic cytokines in 38 patients receiving thalidomide therapy for previously untreated myeloma. The effect of therapy and the relationship of cytokine levels to myeloma cell proliferation, bone marrow microvessel density and progression‐free survival (PFS) were studied. High pretreatment tumour necrosis factor‐α (TNFα) levels (> 11 pg/ml) and increased interleukin (IL)‐6 of > 2 pg/ml predicted for poorer PFS (TNFα, 48% versus 74% at 2 years, P = 0·01; IL‐6, 24% versus 70% at 2 years, P = 0·01). None of the other parameters predicted response or PFS, and no significant changes in cytokine levels occurred with therapy.
Possible regulation of Wilms’ tumour gene 1 (WT1) expression by the paired box genes PAX2 and PAX8 and by the haematopoietic transcription factor GATA‐1 in human acute myeloid leukaemias
Authors: Siehl Jan M. , Thiel Eckhard , Heufelder Karin , Snarski Emilian , Schwartz Stefan , Mailänder Volker , Keilholz Ulrich
Published: October 2003

Summary. Overexpression of the embryonic transcription factor, Wilms’ tumour protein 1 (WT1), is common in acute myeloid leukaemias (AML). Mutations of Wilms’ tumour gene 1 (WT1) in AML are rare and WT1 expression may be increased by other transcription factors. PAX2, PAX8 and GATA‐1 are known physiological regulators of WT1. In the present study, we analysed either bone marrow or blood samples of 43 AML patients for the expression levels of WT1, PAX2, PAX8 and GATA‐1 by real‐time reverse transcription polymerase chain reaction (LightCycler). Bone marrow samples of patients without haematological malignancies and stem cell preparation samples from healthy donors and lymphoma patients served as controls. PAX2 expression was found in 11 of 43 AML samples, with a clear correlation of PAX2 with WT1 expression levels observed. PAX8 expression was found in two additional samples. GATA‐1 expression was detectable in 41 of 43 AML samples and also in all control samples; no significant differences between these groups were observed and no correlation of GATA‐1 expression with WT1 expression levels was apparent. In conclusion, PAX2, and possibly PAX8, appears to be a candidate for the upregulation of WT1 in a proportion of AML, whereas GATA‐1 expression cannot be explained as an inducer of WT1. In two‐thirds of leukaemias from our series, the basis of WT1 upregulation cannot be explained by the simple upregulation of the known WT1 activators.
The continuing relevance of casework ideas to long‐term child protection work
Authors: Searing Hilary
Published: October 2003

This paper is a personal reflection on how casework ideas have informed my own practice. Traditional social work is under threat. Casework, which was once a core method of working in social work, is particularly vulnerable because it has become a private, personal activity that is often misunderstood. It still has its followers but it is hard to find any modern accounts of casework practice. This paper attempts to remedy this by describing the application of casework ideas to work with families following registration of a child on the child protection register. The casework approach is described and illustrated using two case examples. This shows that casework with involuntary clients remains an important method of working but there are inherent constraints within the statutory role which limit what social workers can achieve in terms of therapy or in tackling poverty and inequality. This is followed by a discussion of contemporary social work practice and ideas for ways forward.
Preferentially rejected children and their development in permanent family placements
Authors: Rushton Alan , Dance Cherilyn
Published: October 2003

Several studies of family placements have indicated poorer outcomes for children who are placed singly in new families. In an effort to understand why this may be so, this paper explores differential outcome among a group of children who were placed singly with adoptive or long‐term foster families during middle childhood. A small number of these children were ‘only children’ at the time of placement but most had siblings living elsewhere. Particular attention is paid to preferential rejection by birth parents in the children's backgrounds. Preferential rejection indicates that one child has been rejected while siblings are accepted. This was found to be associated with poorer progress in placement. The methodological and statistical detail of the analyses are to be found in another paper. We focus here on describing the patterns which emerged and the ways in which the findings may assist practitioners in making and supporting permanent placements.
CspB and CspL, thermostable cold‐shock proteins from Thermotoga maritima
Authors: Phadtare Sangita , Hwang Jihwan , Severinov Konstantin , Inouye Masayori
Published: October 2003

Background:  Cold‐shock proteins (Csps) are important for cellular adaptation to low temperature. Csps help cells adapt to low‐temperature growth through their RNA‐binding and nucleic acid melting abilities, which lead to anti‐termination of transcription.

Results:  We studied the two most thermostable Csps known to date, TmCspB and TmCspL from Thermotoga maritima, a hyperthermophilic eubacterium for which no cold‐shock response has been demonstrated so far. For comparison, we used a well‐characterized Escherichia coli CspE protein. TmCspB and TmCspL are able to bind RNA at both low and high temperatures. They are also able to ‘melt’ nucleic acids secondary structures and as a result decrease E. coli RNA polymerase transcription termination in vivo and E. coli and T. maritima RNA polymerases transcription termination in vitro. Over‐expression of TmCsps allowed E. coli cold‐sensitive mutant cells to acclimate to the low temperatures of 15 °C.

Conclusions:  TmCspB and TmCspL (i) are able to perform essential functions of E. coli Csps in vitro and in vivo, 50–65 °C below the temperature optimum of T. maritima and (ii) can anti‐terminate transcription by T. maritima RNA polymerase at 55 °C, the lower limit of temperature range for growth of T. maritima. We propose that the observed properties of TmCsps are physiologically relevant and that TmCsps are important for adaptation of T. maritima to physiologically low temperatures.
pH‐dependent translocation of α‐tocopherol transfer protein (α‐TTP) between hepatic cytosol and late endosomes
Authors: Horiguchi Masakuni , Arita Makoto , Kaempf‐Rotzoll Daisy E. , Tsujimoto Masafumi , Inoue Keizo , Arai Hiroyuki
Published: October 2003

Background:  α‐Tocopherol transfer protein (α‐TTP), a member of the Sec14 protein family, plays an important role in transporting α‐tocopherol, a major lipid‐soluble anti‐oxidant, in the cytosolic compartment of hepatocytes and is known as a product of the causative gene for familial isolated vitamin E deficiency. It has been shown that the secretion of hepatocyte α‐tocopherol taken up with plasma lipoproteins is facilitated by α‐TTP. To explore the mechanism of α‐TTP mediated α‐tocopherol secretion, we investigated drugs which may affect this secretion.

Results:  We found that, in a hepatocyte cell culture system, intracellular α‐tocopherol transport is impaired by chloroquine, an agent known for its function of elevating the pH in acidic compartments. Under chloroquine treatment, the diffuse cytosolic distribution of α‐TTP changes to a punctate pattern. Double‐staining experiments with endocytosis markers revealed that α‐TTP accumulates transiently on the cytoplasmic surface of late endosomal membranes. This phenomenon is specific for hepatoma cell lines or primarily cultured hepatocytes. Other members of the Sec14 family, such as cellular retinaldehyde‐binding protein (CRALBP) and supernatant protein factor (SPF), do not show this accumulation. Furthermore, we elucidate that the obligatory amino acid sequence for this function is located between amino acids 21 and 50, upstream of the N‐terminal end of the lipid‐binding domain.

Conclusion:  We hypothesize that a liver‐specific target molecule for α‐TTP exists on the late endosomal membrane surface. This transient binding may explain the mechanism of how α‐tocopherol is transferred from late endosomes to cytosolic α‐TTP.
Transcription elongation factor S‐II maintains transcriptional fidelity and confers oxidative stress resistance
Authors: Koyama Hiroshi , Ito Takahiro , Nakanishi Toshiyuki , Kawamura Nobuyuki , Sekimizu Kazuhisa
Published: October 2003

Background:  During transcription elongation, RNA polymerase II is arrested on the template when incorrect ribonucleotides are incorporated into the nascent transcripts. Transcription factor S‐II enhances the excision of these mis‐incorporated nucleotides by RNA polymerase II and stimulates transcription elongation in vitro. This mechanism is considered to be transcriptional proof‐reading, but its physiological relevance remains unknown.

Results:  We report that S‐II contributes to the maintenance of transcriptional fidelity in vivo. We employed a genetic reporter assay utilizing a mutated lacZ gene from which active β‐galactosidase protein is expressed when mRNA proof‐reading is compromised. In S‐II‐disrupted mutant yeasts, β‐galactosidase activity was ninefold higher than that in wild‐type. The S‐II mutant exhibited sensitivity to oxidants, which was suppressed by introduction of the S‐II gene. The mutant S‐II proteins, which are unable to stimulate transcription by RNA polymerase II in vitro, did not suppress the sensitivity of the mutants to oxidative stress or maintain transcriptional fidelity.

Conclusion:  These results suggest that S‐II confers oxidative stress resistance by providing an mRNA proof‐reading mechanism during transcription elongation.