bluuebunny/crossref_metadata_2025 huggingface dataset:
Review article: the management of primary sclerosing cholangitis
Authors: MITCHELL S. A. , CHAPMAN R. W. G.
Published: October 2003

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by a progressive obliterating fibrosis of the intrahepatic and extrahepatic bile ducts. The pathogenesis of PSC is poorly understood but it is thought to be an immune‐mediated disease. The optimal therapy which successfully improves symptoms, delays progression towards liver failure and transplantation and prevents the onset of cholangiocarcinoma remains elusive. Although current treatments are used to manage cholestasis and its consequences and some of the more general complications of the disease, none of the current therapeutic agents have been shown to retard and reverse the rate of disease progression. The role of cupruretics, corticosteroids, methotrexate, anti‐fibrogenic agents and ursodeoxycholic acid in the treatment of PSC is reviewed. Orthotopic liver transplantation remains the only therapeutic option for advanced PSC but the timing of transplantation remains controversial and the possibility of recurrence of the disease in the graft is increasingly recognised. It is likely that greater insight into the pathogenetic mechanisms involved in PSC will allow therapy to be targetted more specifically at the biliary epithelium.
Colonic delivery of dexamethasone: a pharmacoscintigraphic evaluation
Authors: KENYON C. J. , NARDI R. V. , WONG D. , HOOPER G. , WILDING I. R. , FRIEND D. R.
Published: October 2003

Background: Colonic delivery of corticosteroids may reduce the side‐effects commonly associated with their use. Therefore, we tested the ability of the naturally occurring polysaccharide guar gum to deliver a corticosteroid, dexamethasone, to the colon using pharmacoscintigraphy. Guar gum is metabolized in the colon by resident bacterial enzymes to trigger drug release.

Materials: Each subject (eight per group, parallel study design) was administered one of four dexamethasone (9 mg) tablet formulations, radiolabelled with 153Sm using neutron activation, under fasted conditions. One formulation was designed to release drug rapidly following ingestion while the other three formulations were designed to delay release of dexamethasone to varying degrees. Progression of the formulations down the gastrointestinal tract was followed by gamma scintigraphy. Serum concentrations were measured over time to relate disintegration profiles of the tablets with pharmacokinetic observations.

Results: The immediate release formulation disintegrated in the stomach, on average, within 20  min of dosing. One of the three delayed release preparations (CD1) began to disintegrate in the small intestine 1.7±1.0 h after dosing. The second and third delayed release preparations (CD2 and CD3) did not begin to disintegrate until 5.8±2.3 and 3.6±1.6 h after dosing, respectively. All three colonic delivery preparations completely disintegrated in the colon ranging from 7.8±2.7 h (CD1) to 12.4±3.2 h (CD2) following oral administration. Pharmacoscintigraphic data indicated that 72–82% of the dexamethasone was delivered into the colon although not all the dexamethasone delivered into the colon was absorbed.

Conclusions: Simple guar gum formulations are capable of delivering the corticosteroid dexamethasone to the colon of normal subjects. Locally delivered corticosteroids may be useful in the treatment of ulcerative colitis and Crohn's disease. Pharmacoscintigraphic evaluation is a useful method to discriminate between the in vivo behaviour of colonic delivery systems.
Emergency admissions for upper gastrointestinal disease and their relation to NSAID use
Authors: BLOWER A. L. , BROOKS A. , FENN G. C. , HILL A. , PEARCE M. Y. , MORANT S. , BARDHAN K. D.
Published: October 2003

Background: There are considerable variations in estimates of the number of emergency upper gastrointestinal admissions per annum which are attributable to nonsteroidal anti‐inflammatory drug (NSAID) use.

Aim: To obtain a more accurate estimate of the number of these emergency admissions per annum in UK.

Methods: A retrospective survey of the case notes of all emergency admissions for upper gastrointestinal disease (‘Cases’) to two English District General Hospitals with a combined catchment population of 550000. Records of all community deaths attributed to upper gastrointestinal diagnoses (with the same ICD codes) were also surveyed. Matched controls were identified from emergency admissions not caused by upper gastrointestinal diagnoses. The proportions of patients taking NSAIDs on admission to hospital (or at the time of death at home) and the outcome following admission to hospital were analysed.

Results: 620 emergency upper gastrointestinal admissions were identified and matched with 460 controls. Cases were more likely to be NSAID users than Controls (31% vs. 16%, OR 2.4, 95% CI: 1.8, 3.3; P<0.001). Case NSAID use was higher in females and with increasing age. As severity of mode of presentation worsened, the probability of NSAID use increased (e.g. OR relative to controls for peptic pain 1.9, for perforation 5.9). Blood transfusion requirements were significantly higher (P<0.0001) in Cases taking NSAIDs, although NSAID use did not influence mortality. Extrapolation from these data indicate that there are 65000 emergency upper gastrointestinal admissions per annum in UK; 12000 of these admissions (including 2230 deaths) are attributable to NSAID use. A further 330 attributable deaths occur in the community.

Conclusions: There is a strong association between NSAID use and propensity for upper gastrointestinal emergency admission; NSAID use is associated with significant morbidity and mortality each year in UK.
Association of autoantibodies against platelet glycoproteins Ib/IX and IIb/IIIa, and platelet‐reactive anti‐HIV antibodies in thrombocytopenic narcotic addicts
Authors: Gonzalez‐Conejero R. , Rivera J. , Rosillo M. C. , Cano A. , Rodriguez T. , Vicente V.
Published: October 2003

The levels of platelet‐associated Igs (PAIgs) and plasma circulating antiplatelet antibodies were evaluated by a flow cytometric immunofluorescence assay (FCIFA), an enzyme‐linked immunoassay (ELISA), and a platelet radioactive antiglobulin test (PRAT), in a group of 45 human immunodeficiency virus (HIV)‐infected intravenous drug users (IVDUs), with or without thrombocytopenia (TCP). Direct tests demonstrated an increased amount of PAIgs in 40% of the patients, irrespective of their platelet count. These PAIgs were mainly of IgG class and could not be eluted with ether. Plasma IgG with antiplatelet activity was found in 70% of the thrombocytopenic individuals, whereas it was detected in only one patient without TCP. The relative frequencies of antibodies against the platelet glycoproteins (GPs) Ib/IX and IIb/ IIIa were assessed in plasma from all patients by means of the monoclonal antibody‐specific immobilization of platelet antigens assay (MAIPA). Plasmas from all non‐thrombocytopenic patients were negative when tested by indirect MAIPA. In contrast, 10/23 plasmas from thrombocytopenic patients reacted with either GP IIb/IIIa, GP Ib/IX, or both GPs. Finally, aiming to investigate whether HIV antibodies from these patients are reactive with normal platelets, we performed absorption‐elution experiments, followed by evaluation of HIV antibodies in the indirect eluates by ELISA and Western blot. Interestingly, we detected anti‐HIV antibodies that bind to normal platelet antigens in 50% of the ether eluates prepared from control platelets sensitized with plasma from patients with TCP, but in only 5% of eluates obtained from platelets sensitized with plasma from non‐thrombocytopenic patients.  The present study provides direct evidence that specific autoantibodies against platelet membrane GPs Ib/IX and IIb/IIIa are common in HIV positive thrombocytopenic individuals. The finding in these patients of HIV antibodies cross‐reactive with normal platelets, suggests that mimicry of human antigens by HIV could play a key role in the pathophysiology of the HIV‐related TCP.
Evidence for an active fibrinolytic system in normal human bone marrow
Authors: McWilliam Nicola A. , Robbie Linda A. , Barelle Caroline J. , Adey Gillian , Prasad Sai , Bennett Bruce , Booth Nuala A.
Published: October 2003

Normal human bone marrow from patients undergoing heart surgery was analysed quantitatively for components of the fibrinolytic system, using functional and immunological assays. Marrow was found to contain considerable fibrinolytic activity, reflecting high levels of t‐PA (tissue‐type plasminogen activator). The t‐PA was in an active form, despite the presence of the inhibitors PAI‐1 and PAI‐2. Plasminogen and α2‐antiplasmin (α2‐AP) were also present in marrow. The balance of proteases and inhibitors differed dramatically from that observed in plasma, with higher levels of t‐PA, PAI‐1 and PAI‐2, and lower levels of u‐PA (urokinase), plasminogen, α2‐AP and t‐PA‐PAI‐1 complex in bone marrow, and resulted in favourable conditions for fibrinolysis. The presence of plasmin–α2‐AP complex at concentrations of the same order of magnitude as total plasminogen and α2‐AP demonstrated that active generation of plasmin was indeed occurring. A role for the active fibrinolytic system in normal human bone marrow may be the removal of unnecessary fibrin deposits formed in the cavities of the marrow, in order to maintain flow through this tissue.
Cytoskeletal behaviour in spectrin and in band 3 deficient spherocytic red cells: evidence for a differentiated splenic conditioning role
Authors: Ingrosso Diego , D’Angelo Stefania , Perrotta Silverio , D’Urzo Giovanna , Iolascon Achille , Perna Alessandra F. , Galletti Patrizia , Zappia Vincenzo , Miraglia del Giudice Emanuele
Published: October 2003

Based on quantitative analysis of red cell membrane proteins, hereditary spherocytosis (HS) can be divided into two main groups including isolated or ankyrin combined spectrin deficiency and band 3 reduction. Protein methyl esterification catalysed by protein carboxyl methyltransferase (PCMT type II; EC 2.1.1.77) is a post‐biosynthetic modification which is involved in the metabolism of damaged membrane proteins. We utilized the evaluation of erythrocyte membrane protein methyl esterification as a marker of cytoskeletal disarray in seven HS subjects with spectrin reduction and in seven patients with HS due to band 3 deficiency. Our results support the notion that band 3 deficient erythrocytes are not affected by an extensive cytoskeletal derangement. On the contrary, we found a remarkable increase of membrane methylation in the unsplenectomized, spectrin‐deficient, HS patients, suggesting a striking membrane skeleton disarray. This phenomenon was not observed in the spectrin‐deficient red cells of splenectomized patients. Therefore in spectrin deficient erythrocytes the induction of cytoskeletal damage, specifically recognized by PCMT type II, could be one of the splenic steps producing conditioned spherocytes.
Serum erythropoietin in chronic lymphocytic leukaemia
Authors: Beguin Yves , Lampertz Serge , Bron Dominique , Fillet Georges
Published: October 2003

Anaemia is a frequent complication of advanced chronic lymphocytic leukaemia (CLL) and several cytokines known to inhibit erythropoietin (Epo) formation are produced by CLL B cells. Therefore we measured serum Epo levels in 47 CLL patients to determine whether Epo was a significant factor in the development of their anaemia. Epo levels were increased compared to normal individuals and this elevation appeared adequate for the degree of anaemia. The slope of the regression of Epo versus haemoglobin (Hb) was similar to that of a reference group. Serum transferrin receptor (sTfR) levels were also appropriately elevated for the degree of anaemia and correlated with serum Epo. Advanced stage was not associated with reduction of Epo production but diminished erythropoietic activity was observed in several patients. The results indicate that anaemia in CLL is not characterized by inadequate Epo production.
Microsatellite instability in follicle centre cell lymphoma
Authors: RANDERSON J. , CAWKWELL L. , JACK A. , CHILD J. A. , LEWIS F. , HALL N. , JOHNSON P. , EVANS P. , BARRANS S. , MORGAN G. J.
Published: October 2003

Fluorescent polymerase chain reaction (PCR) was used to assay 12 microsatellite markers (APC×2, DCC, P53×2, RB1, NM23, WT1, D6S260, D6S262, D6S281 and TNFa) to look for evidence of microsatellite instability in 40 cases of follicle centre cell lymphoma (FCC). Evidence of novel alleles seen in the tumour tissue but not the normal uninvolved tissue was seen in seven cases (17%). In only two of these cases (5%) was more than one locus involved but in these cases multiple affected loci were seen (4/12 and 7/12 respectively). The detection of microsatellite instability indicates a DNA repair defect such as that which would be predicted to occur in cells with mutated mismatch repair genes, a novel finding in FCC lymphoma.
Implication of CD44 in adhesion‐mediated overproduction of TGF‐β and IL‐1 in monocytes from patients with bone marrow fibrosis
Authors: Rameshwar Pranela , Chang Victor T. , Gascón Pedro
Published: October 2003

Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis and excessive deposits of extracellular matrix (ECM) proteins which include fibronectin (FN), collagen type I and hyaluronic acid (HA). We have previously reported that adhesion to polystyrene overactivates MF monocytes. We now confirm their activation by increased CD25 expression and tyrosine phosphorylation. We hypothesize that ECM protein‐adhesion molecule interactions induce overproduction of fibrogenic cytokines in MF monocytes leading to BM fibrosis. In this study we found that FN, collagen type I and HA induce 2–3‐fold more TGF‐β and 6–9‐fold more interleukin (IL)‐1 in MF monocytes than normal controls (NC). Since CD44 can function as the natural ligand for these proteins, its role was studied. We found that CD44 mediated most of the TGF‐β and IL‐1 produced. Immunoprecipitation of CD44 revealed three proteins at approximately 110 kD in MF monocytes and one in NC. Our results indicate that adhesion is important in overproduction of TGF‐β and IL‐1, and that their production is at least partly mediated by adhesion molecule–ECM protein interactions. These results implicate at least one adhesion molecule, CD44, in the pathophysiology of BM fibrosis.
Plasma soluble interleukin‐2 receptor level in patients with primary myelodysplastic syndromes: a relationship with disease subtype and clinical outcome
Authors: Ogata Kiyoyuki , Yokose Norio , An Emi , Kamikubo Keiko , Tamura Hideto , Dan Kazuo , Sakamaki Hisashi , Onozawa Yasusuke , Hamaguchi Hiroyuki , Nomura Takeo
Published: October 2003

To assess the hypothesis that the plasma soluble interleukin‐2 receptor (sIL‐2R) level may have predictive value for morbidity/mortality in patients with myelodysplastic syndromes (MDS), we determined the plasma sIL‐2R level of 80 MDS patients and examined their subsequent clinical course. Compared with low‐risk MDS (refractory anaemia (RA) and RA with ringed sideroblasts) patients and normal subjects, the plasma sIL‐2R level was significantly elevated in high‐risk MDS (three other MDS subtypes and acute leukaemia following MDS) patients (high‐risk MDS versus low‐risk MDS, P < 0.01; high‐risk MDS versus normal subjects, P < 0.01). 14/40 low‐risk MDS patients developed at least one of the following during the follow‐up period: erythrocyte transfusion dependence, infections requiring hospitalization, disease progression or MDS‐related death. The plasma sIL‐2R level was higher in these eventful subjects than in event‐free low‐risk subjects (P < 0.0001), and all of 10 low‐risk subjects with a plasma sIL‐2R level > 540 U/ml experienced at least one event. By logistic regression analysis of various parameters in these 40 low‐risk subjects, the plasma sIL‐2R level was identified as the strongest independent parameter for predicting eventful subjects (P < 0.0047). The plasma sIL‐2R level did not show a predictive value in high‐risk MDS. This study revealed that the plasma sIL‐2R level is significantly elevated in high‐risk MDS and suggested that the plasma sIL‐2R level is a valuable predictive factor for the clinical outcome in low‐risk MDS.
Fibrinogen Lincoln: a new truncated α chain variant with delayed clotting
Authors: Ridgway Hayley J. , Brennan Stephen O. , Gibbons Stephen , George Peter M.
Published: October 2003

A patient referred for preoperative investigation of prolonged bleeding and easy bruising was found to have increased thrombin and reptilase times; however, the thrombin catalysed release of fibrinopeptides A and B was normal. Analysis of five other family members, spanning three generations, indicated that three had a similar defect and suggested autosomal dominant inheritance. Non‐reducing SDS‐PAGE of purified fibrinogen from affected individuals showed that the 340 kD form of their fibrinogen ran as a doublet. SSCP (single‐stranded conformational polymorphism) analysis of exon 5 of the Aα gene, which encodes the C‐terminal half of the chain, confirmed the presence of a mutation. Cycle sequencing of PCR amplified DNA revealed a 13 base pair deletion (nt 4758–4770), resulting in a frameshift at Ala 475, which translates as four new amino acids before terminating at a new stop codon (‐476His‐Cys‐Leu‐Ala‐Stop). The presence of a circulating truncated Aα chain was confirmed when SDS‐PAGE gels were probed with an α chain specific antisera; which showed that the variant Aα chain comigrated with γ chains. The truncation results in a variant Aα chain with a deletion of 131 amino acids (480–610), and four new amino acids at the C‐terminal.
Donor leucocyte transfusions for relapse in myelodysplastic syndrome after allogeneic bone marrow transplantation
Authors: Okumura H. , Takamatsu H. , Yoshida T.
Published: October 2003

A 31‐year‐old man with refractory anaemia of excess blasts, which had karyotypic abnormalities, received an allogeneic bone marrow transplant (BMT). At time of relapse, 3 months after BMT, he was treated with donor leucocyte transfusions (DLT). Grade III acute GVHD (graft‐versus‐host disease) occurred 35 d after DLT which was fully reversed with cyclosporin and prednisolone. His condition was complicated by a herpes zoster infection. 2 months after DLT, neutrophil and platelet count were increased and karyotypic abnormalities disappeared. This observation demonstrates that DLT is an effective treatment for relapse of myelodysplastic syndrome (MDS) after BMT.
CD20 and CD40 mediated mitogenic responses in B‐lineage acute lymphoblastic leukaemia
Authors: Smiers Frans J. , VanPaassen Marleen , Hählen Karel , Löwenberg Bob , Touw Ivo P.
Published: October 2003

Activation of CD20, a cross‐membrane ion channel, induces cell cycle progression from G0 to G1 in B lymphocytes. Subsequent activation of CD40, a membrane receptor of the nerve growth factor receptor superfamily, transits the B cells to the S phase. CD40 may also act synergistically in combination with IL‐4 (B lymphocytes) or IL‐3/IL‐7 (B‐cell precursors). We investigated the proliferative responses of B‐lineage acute lymphoblastic leukaemia (ALL) cells to CD20/CD40 activation. In 18/56 ALL cases, CD20 activation resulted in significant increases in DNA synthesis. Similar, although more moderate, effects were seen of activation of CD40 in 10/44 cases. Responses to CD20 or CD40 activation were independent of co‐stimulation with IL‐3, IL‐4 or IL‐7, and various cocktails of the different growth stimuli did not act synergistically.
A case of cryofibrinogenaemia responsive to stanozolol
Authors: Rubegni Pietro , Flori Maria Laura , Fimiani Michele , Andreassi Lucio
Published: October 2003

Cryofibrinogen is a plasma protein complex whose presence in the peripheral blood is generally asymptomatic, but may sometimes cause multiple thromboembolism in the skin, lung and myocardium. The pathological manifestations associated with cryofibrinogenaemia have been treated with plasmapheresis and fibrinolytic drugs such as streptokinase, streptodornase and/or urokinase. Good results have recently been reported with stanozolol. This prompted us to treat a patient suffering from cryofibrinogenaemia with this androgenic hormone. The patient was a 66‐year‐old woman with rapidly evolving leg ulcers. Stanozolol was orally administered at 4 mg b.i.d. for 5 months and then gradually reduced. Plasma cryofibrinogen disappeared after 45 d from the start of therapy and cutaneous ulcers healed in 5 months.
Unrelated donor marrow transplantation for myelodysplasia (MDS) and MDS‐related acute myeloid leukaemia
Authors: Anderson Jeanne E. , Anasetti Claudio , Appelbaum Frederick R. , Schoch Gary , Gooley Ted A. , Hansen John A. , Buckner C. Dean , Sanders Jean E. , Sullivan Keith M. , Storb Rainer
Published: October 2003

Allogeneic marrow transplantation using related marrow donors for myelodysplasia (MDS) and acute myeloid leukaemia (AML) arising from MDS results in 35–56% actuarial disease‐free survival. Because the use of unrelated donors has not been well‐characterized, we report on the outcome of 52 patients with MDS or MDS‐related AML consecutively treated between 1987 and 1993 with unrelated donor marrow transplantation. The median age was 33 (range 1–53) years. 33 patients received chemotherapy and total body irradiation and the remainder busulfan and cyclophosphamide. The donors were phenotypically identical at the HLA‐A, B and Dw/DRB1 loci in 34 cases and mismatched for one HLA locus in 17 cases and two loci in one case. Marrow was non‐T‐cell depleted and methotrexate with cyclosporine or FK506 was used for postgrafting immunosuppression. The 2‐year disease‐free survival, relapse, and non‐relapse mortality rates were 38%, 28% and 48%, respectively. One patient who relapsed survives disease‐free after withdrawal of immunosuppressive therapy. 16/19 survivors have a performance status of 90–100%. Patients with MDS in transformation or with AML had a significantly higher risk of relapse than patients with less advanced disease (P = 0.0014). Increased non‐relapse mortality was significantly associated with higher age, longer disease duration before transplant, lower neutrophil count on admission and, unexpectedly, being seronegative for cytomegalovirus. We conclude that the outcome with transplantation using unrelated donors is similar to reported results using related donors and that a meaningful proportion of eligible patients with an otherwise incurable disease may be cured with this treatment. However, mortality from the transplant procedure is high and future studies should focus on reducing toxicity.
Establishment of a myeloid leukaemia cell line (Kasumi‐4) with t(9;22;11)(q34;q11;q13), inv(3)(q21q26) and the EVI1 gene activation from a patient with chronic myelogenous leukaemia in blast crisis
Authors: Asou Hiroya , Eguchi Mariko , Suzukawa Kazumi , Morishita Kazuhiro , Tanaka Kimio , Date Munehiro , Hamamoto Kazuko , Kamada Nanao
Published: October 2003

A novel human leukaemia cell line (Kasumi‐4) was established from the peripheral blood of a 6‐year‐old girl suffering from chronic myelogenous leukaemia (CML) in blast crisis. The Kasumi‐4 cells had the following characteristic features: undifferentiated blasts which were positive for CD34, CD33 and CD13 surface markers, but negative for myeloperoxidase platelet peroxidase, CD36, CD41 and CD42; chromosome abnormalities of t(9;22;11) (q34;q11;q13), inv(3)(q21q26); and elevated expression of EVI1 gene which is located at chromosome band 3q26. Megakaryocytic maturation was not observed in the liquid culture following the addition of TPA, IL‐3, IL‐6 or GM‐CSF. b2‐a2 type of BCR‐ABL chimaeric messenger RNA was detected by RT‐PCR analysis. This is the first leukaemia cell line with a three‐way translocation containing the Ph chromosome and the second cell line with an inv(3)(q21q26). This cell line appears to be useful for studying the mechanisms of leukaemogenesis involving these chromosomal abnormalities and related oncogenes.
Molecular analysis of blood group Rh transcripts from a rGr variant
Authors: Mouro Isabelle , Colin Yves , Gane Pierre , Collec Emmanuel , Zelinski Teresa , Cartron Jean Pierre , Le Van Kim Caroline
Published: October 2003

The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the rGr phenotype. Nucleotide sequence analysis revealed that the r G allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid r G gene (c‐C‐e or c‐D‐e) carries Ile60, Ser68 and Ser103 (as C and D polypeptides); any of these positions appear to be critical in the formation of the G antigen. In addition, Cys16 was found to be important in the phenotypic expression of C.
Differential regulation of GPI‐linked molecules on leukaemic promyelocytes treated in vitro with all‐trans retinoic acid
Authors: DI NOTO R. , SCHIAVONE E. M. , LO PARDO C. , FERRARA F. , MANZO C. , VACCA C. , DEL VECCHIO L.
Published: October 2003

It has been demonstrated that certain cell‐surface proteins are anchored to the cell membrane by a unique structure known as the glycosylphosphatidylinositol (GPI) anchor whose absence has been reported on blood cells from patients with paroxysmal nocturnal haemoglobinuria. We have investigated the expression of CD16/FcτR‐III and CD66b GPI‐linked molecules at the surface of blast cells from five acute promyelocytic leukaemia (APL) patients before and after in vitro stimulation with all‐trans retinoic acid (ATRA). We observed that whereas CD66b antigen exhibited a strong ATRA‐driven up‐regulation in all cases studied, CD16 expression was unaffected by the treatment with the drug.
All‐trans‐RETINOIC ACID INCREASES ADHESION TO ENDOTHELIUM OF THE HUMAN PROMYELOCYTIC LEUKAEMIA CELL LINE NB4
Authors: Marchetti M. , Falanga A. , Giovanelli S. , Oldani E. , Barbui T.
Published: October 2003

Pulmonary distress symptoms and thrombotic complications are side‐effects of all‐trans‐retinoic acid (ATRA) therapy for remission induction in acute promyelocytic leukaemia (APL). The ATRA‐induced increase of leukaemic cell adhesive molecules may be responsible. To explore this we used a functional assay to study the effect of ATRA treatment on the adhesion of blast cells to cultured human endothelial cells (EC), endothelial cell matrix (ECM), and interleukin 1β‐activated EC (IL1 + EC). NB4 cells, a maturation‐inducible human promyelocytic leukaemia cell line, were treated with 1 μm ATRA or the vehicle (control), labelled with 51Cr and tested in the adhesion assay. ATRA increased NB4 adhesion to EC (P < 0.01), ECM (P < 0.001) and IL1 + EC (P = n.s.). An inhibition study with anti‐EC adhesion receptors MoAbs indicated that anti‐E‐selectin, anti‐VCAM‐1 and anti‐ICAM‐1 effectively inhibited cell adhesion to IL1 + EC (18 ± 7%, 45 ± 6.9% and 29 ± 6% inhibition, respectively) and to unstimulated EC. Preincubation of ATRA‐treated NB4 cells with MoAbs anti‐VLA4 and anti‐LFA1, the VCAM‐1 and ICAM‐1 counter‐receptors respectively, resulted in a significant inhibition of adhesion. Cytofluorimetric analysis of the NB4 cell membrane molecules confirmed the increase under ATRA of VLA4, LFA1, MAC1 and ICAM‐1. Therefore ATRA increases NB4 cell adhesion to the endothelium and the subendothelial matrix. These findings parallel the increment of NB4 surface adhesive molecules, among which VLA4 and LFA1 appear to play an important part. These mechanisms may contribute to the complications of ATRA therapy in APL.
Human T leukaemia virus type 1 (HTLV‐1) specific T‐helper cell response: clonal fluctuations and repertoire heterogeneity
Authors: Li Pira Giuseppina , Westby Mike , Oppezzi Laura , Ferraris Anna , Lancia Flavia , Piatti Gabriella , Fenoglio Daniela , Saverino Daniele , Arp Jacqueline , Dekaban Gregory , Dalgleish Angus , Manca Fabrizio
Published: October 2003

The naive T‐helper (Th) repertoire specific for HTLV‐1 envelope (env) has been examined on antigen specific T‐cell lines and clones from non‐immune individuals. Clonal heterogeneity was determined by analysing the T‐cell receptor (TCR) Vβ gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the Vβ gene usage were determined by comparing the TCR Vβ gene profiles of T‐cell lines at different times. We found that a diverse repertoire for HTLV‐1 env could be triggered in vitro. Diverse Vβ genes were used by the same line tested at different times, suggesting that clonal composition of an antigen‐specific T‐cell line is not constant in vitro. Clones in fact may be up‐ and down‐regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T‐cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen‐specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vβ gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV‐1‐infected patients studied in vivo, the Th cell repertoire specific for HTLV‐1 env may prove very heterogenous, with important implications for vaccine development.
Progression from myelodysplastic syndrome to acute lymphoblastic leukaemia with Philadelphia chromosome and p190 BCR‐ABL transcript
Authors: Kohno Tomoko , Amenomori Tatsuhiko , Atogami Sunao , Sasagawa Ippei , Nakamura Hideo , Kuriyama Kazutaka , Tomonaga Masao
Published: October 2003

We describe a patient with Philadelphia chromosome (Ph1)‐positive acute lymphoblastic leukaemia (ALL) who developed it 2.5 years after being diagnosed with myelodysplastic syndrome (MDS). The patient initially had refractory anaemia (RA), but progressed to refractory anaemia with excess blasts (RAEB) 2 years later, that terminated in ALL. An immunophenotypic analysis of the lymphoblasts revealed CD10 and CD19 positive cells. The karyotype was normal 46,XY in RA phase, 46,XY,20q– during the RAEB phase, and 46,XY, t(9;22)(q34;q11), 20q– during the ALL phase. Furthermore, p190 BCR‐ABL mRNA was detected in the ALL blasts. These findings indicate that this ALL arose from the MDS clone through multiple cytogenetic evolutions, the final event of which was the acquisition of p190 BCR‐ABL type Ph1
Molecular characterization of haemophilia A in southern Chinese
Authors: Chan Vivian , Pang Annie , Chan T. P. T. , Chan V. Wan‐Yin , Chan T. K.
Published: October 2003

41 unrelated southern Chinese haemophilia A patients were studied. The 5′ promoter region, all 26 exons, their immediate 5′ and 3′ flanking splice junctions and the 3′ untranslated region of the FVIII gene were amplified (including 16 different segments of exon 14) using GC‐clamped primers. The GC‐clamped PCR products were screened by denaturing gradient gel electrophoresis (DGGE) and fragments showing an abnormal migration pattern were sequenced. 17 mutations were identified, of which four were splicing defects, involving the first 1–6 nucleotide (nt) in the intervening sequences (IVS), six missense mutations, three nonsense mutations and four frameshift mutations. 14 other patients carried the type 1 inversion, affecting the distal copy of the F8A gene at the telomere of the X chromosome and the same gene in intron 22 of the FVIII gene. All the mothers studied (12/14) were carriers of the inversion. Two of these patients with inversion also have a co‐existing missense mutation. In most cases the clinical severity of the disease corresponds to the genotype.
Expression of rhombotin 2 in normal and leukaemic haemopoietic cells
Authors: Dong W‐F. , Billia F. , Atkins H. L. , Iscove N. N. , Minden M. D.
Published: October 2003

The rhombotin 2 gene was identified by its association with a recurrent chromosome translocation (11;14)(p13;q11), occurring in T‐cell acute lymphoblastic leukaemias (ALLs). High levels of RBTN2 have been found in T‐cell leukaemias carrying the translocation and in some T‐cell ALLs that lack, by cytogenetic and molecular techniques, translocations involving 11p13. In normal murine tissues RBTN2 has been found to be widely expressed, with high levels present in brain and early B cells. Studies carried out in mice lacking RBTN2 have demonstrated the importance of this gene in erythropoiesis.

We have investigated the expression of RBTN2 in human leukaemic cells, and in human and murine normal myeloid progenitor cells. RBTN2 is expressed in the leukaemic cells of patients with pre‐B ALL, T‐ALL and acute myeloblastic leukaemia (AML). By cytogenetic and molecular techniques no abnormalities were noted in 11p13. Using clonogenic assays and single cell PCR we found that RBTN2 is expressed strongly in the precursors of mixed erythrocyte/macrophage/mast, erythrocyte, megakaryocyte, neutrophil and macrophage colonies. In contrast, RBTN2 message was low to undetectable in the mature progeny. These findings indicate that RBTN2 is expressed in leukaemias of both the myeloid and lymphoid lineages. Further, these studies show that in normal myeloid and lymphoid cells the expression of RBTN2 is present in progenitor cells and is lost as the cells terminally differentiate. This latter finding further illustrates that the detection of a RNA in a population of cells should not be interpreted to mean that all of the cells in that population express the RNA.
Decreased L‐selectin expression in CD34‐positive cells from patients with chronic myelocytic leukaemia
Authors: KAWAISHI KUNIKO , KIMURA AKIRO , KATOH OSAMU , SASAKI AYAKO , OGUMA NOBUO , IHARA AKIHIRO , SATOW YUKIO
Published: October 2003

Abnormal adhesive interaction between bone marrow stroma and progenitors, one of the causes of unregulated proliferation in chronic myelocytic leukaemia (CML), may be caused by some alterations in adhesion molecules on CML progenitors. We investigated the expression of adhesion molecules (CD44, VLA‐5, VLA‐4, LFA‐1, ICAM‐1, L‐selectin and c‐kit) on bone marrow CD34++ cells from 16 CML patients by three‐colour flow cytometry. The mean percentage of cells expressing L‐selectin in the CD34++CD38+  ∼  ++ fraction from untreated CML patients was significantly lower, and that in the CD34++CD38− fraction tended to be lower than that from normal controls. Among 11 CML patients treated with interferon‐α (IFN‐α), the mean percentage of the cells expressing L‐selectin in the CD34++CD38− fraction from three patients with a low percentage of Ph1(+) cells in bone marrow was significantly higher than that from five patients with a high percentage of Ph1(+) cells. In addition, L‐selectin expression rate was inversely correlated to the percentage of Ph1(+) cells. There was no significant difference between the untreated patients and normal controls with regard to the expression rates of the other adhesion molecules in each CD34++ fraction except LFA‐1. These data suggest that decreased L‐selectin expression in CML CD34++ cells reflects one of the features of malignant CML progenitors.
The incidence and cause of coagulopathies in an intensive care population
Authors: Chakraverty R. , Davidson S. , Peggs K. , Stross P. , Garrard C. , Littlewood T. J.
Published: October 2003

We studied 235 patients admitted to an adult intensive care unit in order to determine the incidence and cause of coagulation disturbances. Clinical coagulopathy, defined as bleeding unexplained by local or surgical factors, was identified in 13.6% of patients. Laboratory evidence of coagulopathy was more common: a prothrombin time (PT) ratio ≥1.5 was found in 66% of patients and a platelet count <100 × 109/l in 38% of patients. Both factors were predictive of excessive bleeding and poor outcome. In a retrospective analysis of plasmas from 45 of the above patients who had PT ratios ≥1.5 the most common cause was vitamin K deficiency (20%).
Incomplete gamma carboxylation of human coagulation factor VII: differential effects on tissue factor binding and enzymatic activity
Authors: Clarke Bryan J. , Sridhara Sampath
Published: October 2003

The integrity of the γ‐carboxylic glutamic acid (GLA) residues of coagulation factor VII are thought to be essential for both the interaction of factor VII with its cell‐surface lipoprotein receptor tissue factor and for the activated protein to manifest its serine protease activity. During the course of transiently expressing recombinant human factor VII in monkey COS cells it was noted that the factor VII synthesized in the absence of added vitamin K had < 20% of expected procoagulant activity yet retained 65% of its binding activity to recombinant human tissue factor. Similar results were obtained when vitamin K was omitted from human 293 cell cultures permanently expressing recombinant factor VII. In contrast, both transient and permanent expression of factor VII in human 293 cell cultures containing physiological concentrations of vitamin K resulted in the synthesis of fully functional factor VII. Furthermore, factor VII in plasma samples from 24 patients undergoing warfarin therapy bound quantitatively to tissue factor whereas factor VII procoagulant activity averaged 65% of normal. Thus, data from both in vitro and in vivo situations indicated that factor VII molecules with suboptimal GLA content retained most of their ability to bind tissue factor but exhibited reduced procoagulant activity.
APLASTIC ANAEMIA PATIENTS WITH CLONAL X‐CHROMOSOME INACTIVATION PATTERN IN HAEMOPOIETIC CELLS EXHIBIT POLYCLONAL TCRγ AND IgH GENE REARRANGEMENTS
Authors: Melenhorst J. Joseph , Fibbe Willem E. , Smits Shirley , Willemze Roel , Landegent James E.
Published: October 2003

Previously, we reported that 13/18 (72%) female patients with aplastic anaemia (AA) exhibited a clonal X‐chromosome inactivation (XCI) pattern in all haemopoietic lineages. To study the consequences of a clonal haemopoiesis for the randomness of immunoreceptor rearrangements in lymphocytes we determined clonality of T‐cell receptor gamma (TCRγ) and immunoglobulin heavy chain (IgH) gene rearrangements in purified cell fractions. Peripheral blood granulocytes, monocytes, and B and T lymphocytes from 18 female patients in remission from AA were studied by PCR for randomness of XCI and rearrangement at the IgH and TCRγ locus. 13 patients were informative at the phosphoglycerate kinase‐1 (PGK1) and monoamine oxidase A (MAOA) loci. Five of them displayed an clonal XCI pattern in all lineages studied and one patient had a clonal XCI in all lineages, except the T cells. In three cases skin biopsies were also available, exhibiting a polyclonal pattern in two of them, and a reversed skewed pattern in the third. Analysis of the rearrangement patterns at the immunoreceptor loci revealed a polyclonal ladder of bands, irrespective of XCI status in the lymphocyte populations. These results demonstrate that in AA a clonal XCI pattern of the lymphoid compartment is compatible with a polyclonal immunoreceptor rearrangement pattern.
In vitro progenitor analysis in a Diamond Blackfan anaemia patient who responded once but not twice to interleukin‐3 therapy, using short‐term and long‐term cultures and c‐kit analysis
Authors: McGuckin Colin P. , Uhr Mario R. , Ball Sarah E. , Gordon‐Smith Edward C.
Published: October 2003

Interleukin‐3 (IL‐3) therapy as a treatment for Diamond Blackfan anaemia (DBA) patients has been largely disappointing despite early hope it would be suitable for stimulating arrested erythropoiesis. Initial hope came from in vitro discoveries that IL‐3 (+EPO) generated well‐haemoglobinized BFU‐E colonies in some patients, but was soon tempered by the realization that in vitro and in vivo IL‐3 response did not, in the majority of cases, correlate. Nevertheless in vitro testing has been the main focus in analysing the abnormality in the stem and progenitor cell compartment in DBA. Here we report in vitro analysis of a DBA patient who responded once to IL‐3 therapy, but not a second time following relapse, using short‐term culture, long‐term culture and c‐kit analysis. Progenitor numbers before and after the first therapy were in the high normal range, but after relapse were much reduced below normal levels. Long‐term cultures suggested some arrested progenitors had been reactivated into normal cycle by the first therapy, but may not have been replaced by more immature progenitors. c‐kit analysis revealed increased expression in all tested cell populations. These results imply that the first IL‐3 therapy reactivated some erythroid progenitors but left the progenitor pool depleted when more immature cells remained arrested.
COMPARISON OF THE DEOXYURIDINE SUPPRESSION TEST WITH SERUM LEVELS OF METHYLMALONIC ACID AND HOMOCYSTEINE IN MILD COBALAMIN DEFICIENCY
Authors: Carmel Ralph , Rasmussen Karsten , Jacobsen Donald W. , Green Ralph
Published: October 2003

Both the deoxyuridine suppression test (dUST) and the cobalamin‐dependent metabolites, methylmalonic acid (MMA) and homocysteine, are valuable tools for identifying clinical cobalamin deficiency. Examination of these metabolic changes in mild or marginal deficiency can provide useful comparisons of diagnostic frequencies and sensitivities and help define the sequence of metabolic changes in early deficiency. These tests were therefore compared directly with each other in 50 patients with low cobalamin levels and few or no obvious signs of deficiency. Serum homocysteine (P = 0.0003) and MMA levels (P = 0.0004) correlated with dUST results. However, the dUST results were abnormal significantly more often (38/50 patients) when matched against levels of homocysteine (25 abnormal results of 50; P = 0.007) or MMA (20/50; P = 0.008). Abnormalities of one or both serum metabolite levels (30/50 patients) occurred almost as often as dUST abnormalities (P = 0.059). Metabolite levels, even when originally ‘normal’, fell with cobalamin therapy in many cases. The results indicate that both the dUST and serum metabolite levels become abnormal before macrocytic anaemia develops in mild cobalamin deficiency. The dUST appears to be the most frequently abnormal of the tests; metabolite levels appear to rise almost concurrently but they do not become diagnostically abnormal as soon.
ORAL CONTRACEPTIVES ENHANCE THE RISK OF CLINICAL MANIFESTATION OF VENOUS THROMBOSIS AT A YOUNG AGE IN FEMALES HOMOZYGOUS FOR FACTOR V LEIDEN
Authors: Rintelen Claudia , Mannhalter Christine , Ireland Helen , Lane David A. , Knöbl Paul , Lechner Klaus , Pabinger Ingrid
Published: October 2003

In 29 patients (17 females) homozygous Arg 506 Gln mutation (FV Leiden) was identified. 25 had been investigated because of venous thromboembolism (VTE); four asymptomatic patients were found during family studies.

The first VTE had occurred significantly earlier in females (median age [m] 26 years, range 17–49) than in males (m=38 years, range 21–82) (P = 0.01). 12 females (80%) had taken oral contraceptives (OC, oestrogen content 0.02–0.1 mg) for 6–150 months prior to thrombosis. Further triggering conditions in females were hormone replacement (n = 1) and pregnancy (n = 2). In 8/10 males the first VTE had occurred spontaneously — in two after surgery. The sites of VTE were deep vein thrombosis, pulmonary embolism, caval vein thrombosis and superficial thrombophlebitis.

From our data we conclude that OC medication is the most important precipitating factor for VTE in females with homozygous FV Leiden.
GM‐CSF RAISES SERUM LEVELS OF β2‐MICROGLOBULIN AND THYMIDINE KINASE IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKAEMIA
Authors: Itälä Maija , Pelliniemi Tarja‐Terttu , Remes Kari
Published: October 2003

The effects of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) on biochemical tumour markers β2‐microglobulin (β2m), thymidine kinase (TK) and lactate dehydrogenase (LDH) were studied in eight patients with chronic lymphocytic leukaemia (CLL). The serum concentration of β2m rose by a median of 30% (range 8–50%) and serum TK by 101% (range 30–1414%). Serum LDH concentration, on the other hand, significantly decreased in all patients. The significant increases of β2m and TK could not be explained by progression of the disease or impaired renal function. Treatment with GM‐CSF reduces the value of serum β2m and TK in assessment of tumour mass and disease activity.
Investigation of chronic hepatitis C infection in individuals with haemophilia: assessment of invasive and non‐invasive methods
Authors: Hanley John , Jarvis Lisa , Andrews Janet , Dennis Rosemary , Lee Robert , Simmonds Peter , Piris Juan , Hayes Peter , Ludlam Christopher A.
Published: October 2003

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease in individuals with haemophilia. A wide spectrum of disease severity is found in this group, ranging from mild hepatitis to cirrhosis. We have studied a cohort of 87 anti‐HCV positive haemophiliacs who have been infected with HCV for 10–25 years and assessed the relative value of invasive and non‐invasive methods of evaluating liver disease. The severity of liver disease was assessed using ultrasound scan (n = 77), upper GI endoscopy (n = 50), laparoscopic liver inspection (n = 33) and liver biopsy (n = 22). Invasive investigations were performed without any significant bleeding complications.  Evidence of severe liver disease was found in approximately 25% of patients. There was agreement between the severity of liver histology and the information derived from the laparoscopic liver inspection, endoscopy and ultrasound in 86%. Co‐infection with HIV was significantly associated with more severe liver disease (P = 0.006).  This study provides further evidence that liver disease is emerging as a major complication in haemophiliacs and severe liver disease is more common in those co‐infected with HIV. We have shown the potential value of laparoscopic liver inspection, in combination with endoscopy and ultrasound, in staging the extent of liver disease, and suggest that most patients may be managed without resorting to liver biopsy.
Development of anti‐interferon antibodies and breakthrough hepatitis during treatment for HCV infection in haemophiliacs
Authors: Hanley John , Jarvis Lisa , Simmonds Peter , Ludlam Christopher A.
Published: October 2003

The development of anti‐interferon antibodies may lead to treatment failure during interferon therapy. We have studied the development of such antibodies in a group of 39 haemophiliacs receiving interferon‐α2a for chronic hepatitis C virus (HCV) infection.  Anti‐interferon antibodies developed in five (13%) patients and were associated with ‘breakthrough hepatitis’ in three cases. There was an association between the development of anti‐interferon antibodies and infection with HCV genotype 3a (P= 0 .01). This study suggests that the development of anti‐interferon antibodies may lead to treatment failure in a proportion of haemophiliacs with HCV infection. The association with genotype 3a has not previously been reported. Monitoring for the development of breakthrough hepatitis due to anti‐interferon antibodies may provide the opportunity to develop strategies to overcome their effects.