bluuebunny/crossref_metadata_2025 huggingface dataset:
How patients manage gastrointestinal symptoms after pelvic radiotherapy
Authors: Gami B. , Harrington K. , Blake P. , Dearnaley D. , Tait D. , Davies J. , Norman A. R. , Andreyev H. J. N.
Published: November 2003

Background : Approximately 13 000 patients undergo pelvic radiotherapy annually in the UK. It is not clear how frequently patients develop a permanent change in bowel habit after pelvic radiotherapy that affects their quality of life because the measures of gastrointestinal toxicity used in trials in the past have generally been inadequate. It has been suggested that patients who are symptomatic are only rarely referred to a gastroenterologist and it is not known how patients manage their symptoms.

Methods : Patients who had completed radiotherapy for pelvic cancer at least 1 year previously were invited to answer 30 structured questions in a face‐to‐face interview to determine the frequency of gastrointestinal symptoms and what orthodox, dietary and complementary therapies they used to deal with them. They were also asked to score the effectiveness of the measures they had taken.

Results : One hundred and seven patients were recruited [35 males; median age, 65 years (range, 35–80 years); 72 females; median age, 67.5 years (range, 31–87 years)]. Eight had been treated for a gastrointestinal primary tumour, 34 for a urological tumour and 65 for gynaecological tumours. Eighty‐seven patients (81%) described new‐onset gastrointestinal problems starting after radiotherapy. These symptoms affected the quality of life in 56 patients (52%). Significant effects on the quality of life were caused by diarrhoea or constipation (n = 53), faecal leakage (n = 19), abdominal, rectal or perineal pain (n = 14) and rectal bleeding (n = 6). Fifty‐nine patients had seen a doctor for their symptoms (86% found this helpful), 12 had seen a dietician or nurse (50% found this helpful) and 14 had seen alternative practitioners (88% found this helpful). Dietary manipulation generally did not improve symptoms, except in a small group of patients (14/15) who avoided raw vegetables to great benefit.

Conclusions : At least 1 year after pelvic radiotherapy, gastrointestinal symptoms which have an adverse effect on the quality of life may be more common than generally reported. Patients found that advice from doctors and alternative practitioners was equally valuable. Dietary manipulation was generally unhelpful for gastrointestinal symptoms after pelvic radiotherapy, although the role of eliminating raw vegetables may benefit from further evaluation.
Oral ulceration – aetiopathogenesis, clinical diagnosis and management in the gastrointestinal clinic
Authors: Field E. A. , Allan R. B.
Published: November 2003

Oral ulceration is a common complaint of patients attending out‐patient clinics. The aim of this review is to provide the gastroenterologist with a differential diagnosis of oral ulceration, and a practical guide for the management of recurrent aphthous stomatitis, including topical and systemic therapy. The association of recurrent aphthous stomatitis with Behçet's disease and other systemic disorders, including coeliac disease, is discussed. Recent evidence concerning the immunopathogenesis of Behçet's disease is reviewed, including renewed interest in the role of Streptococcus sanguis and possible infectious triggering of an inappropriate immunoinflammatory response, resulting in tissue damage. The efficacy and limitations of conventional treatment for this mutisystem disorder are outlined together with the potential role of novel biological agents, such as anti‐tumour necrosis factor‐α therapy. Oral ulceration, as a manifestation of inflammatory bowel disease and a complication of drug therapy, is described. Guidance is given concerning indications for referral of patients with oral ulceration to an oral physician/surgeon for further investigations, including biopsy if appropriate.
The impact of current tobacco use on the outcome of paracetamol poisoning
Authors: Schmidt L. E. , Dalhoff K.
Published: November 2003

Background : Tobacco smoke contains a number of substances that are capable of inducing cytochrome P450. Consequently, current tobacco use may enhance the hepatotoxicity from a paracetamol overdose by increasing the oxidative metabolism of paracetamol.

Aim : To evaluate, by multivariate analysis, the effect of current tobacco use on the morbidity and mortality from paracetamol‐induced hepatotoxicity.

Methods : A retrospective study was carried out on the basis of the hospital charts of 602 patients admitted with single‐dose paracetamol poisoning for whom information on current tobacco use was available.

Results : In patients admitted with paracetamol poisoning, the rate of current daily tobacco use of 70% (424 of 602 patients) was considerably higher than the rate of 31% in the background population (chi‐squared test: P < 0.0001). Current tobacco use was an independent risk factor for the development of hepatic encephalopathy (odds ratio, 2.68; 95% confidence interval, 1.28–5.62) and mortality (odds ratio, 3.64; 95% confidence interval, 1.23–10.75). Current tobacco use was independently associated with high peak values of alanine transaminase and the international normalized ratio.

Conclusions : Current tobacco use was very frequent in patients admitted with paracetamol poisoning. It was an independent risk factor of severe hepatotoxicity, acute liver failure and death following paracetamol overdose.
Role of early ultrasound in detecting inflammatory intestinal disorders and identifying their anatomical location within the bowel
Authors: Parente F. , Greco S. , Molteni M. , Cucino C. , Maconi G. , Sampietro G. M. , Danelli P. G. , Cristaldi M. , Bianco R. , Gallus S. , Bianchi Porro G.
Published: November 2003

Background : Although bowel ultrasound is a widely accepted diagnostic tool in bowel diseases, its value as the primary imaging procedure in patients with symptoms/signs suggestive of inflammatory bowel disorders is still unclear.

Aim : To investigate the accuracy of bowel ultrasound compared with barium X‐ray studies, computed tomography, endoscopy and bowel surgery in the initial assessment of inflammatory bowel disorders.

Methods : Four hundred and eighty‐seven patients hospitalized consecutively for symptoms or signs suggestive of a bowel disorder between December 1999 and March 2002 were initially enrolled in the study. All patients underwent bowel ultrasound as the first imaging procedure within 36 h of admission; radiographic evaluations, endoscopy and/or surgery were then performed as appropriate and the results of these investigations were used as the gold standard.

Results : Three hundred and thirty‐six patients had pathological findings of the bowel detectable at ultrasound as the final diagnosis. The main organic disorders found were Crohn's disease (56%), ulcerative/indeterminate colitis (30%), bowel tumours (5%), appendicitis/diverticulitis (2%) and other inflammatory conditions (8%). The overall sensitivity and specificity of bowel ultrasound were 85% and 95%, respectively, whereas the positive and negative predictive values were 98% and 75%, respectively. Comparisons of ultrasound with X‐ray or endoscopic results by disease localization showed that the diagnostic performance of ultrasound was higher for inflammatory conditions of the ileum and sigmoid/descending colon (sensitivity of 92% and 87%, respectively), whereas abnormalities localized in the rectum, duodenum and proximal jejunum were often missed by ultrasound.

Conclusions : In expert hands, bowel ultrasound is highly predictive of inflammatory disease localized in the ileum or colon, and may well be used as the primary imaging method when Crohn's disease or ulcerative colitis is suspected on a clinical basis.
Constitutive expression of granulocyte‐colony stimulating factor receptor on a human B‐lymphoblastoid cell line
Authors: Morikawa Keiko , Morikawa Shigeru , Miyawaki Toshio , Nagasaki Makoto , Torii Ikuko , Imai Katsuyuki
Published: November 2003

The present study demonstrated that a human B‐ cell line derived from non‐Hodgkin's lymphoma, HCF‐MLpN, constitutively expressed G‐CSF receptor on the cell surface. G‐CSF binding to the cell surface was shown by immunofluorescence staining using biotinylated G‐CSF preparation and analysed by flow cytometry. Specific binding of G‐CSF to the cells was shown by pretreatment with unlabelled G‐CSF. In the radioreceptor assay and Scatchard plot analysis using radiolabelled ligand, MLpN cells revealed a single species of binding site with an equilibrium dissociation constant of 167 (153–182) pM and a maximal binding site per cell of 1076 (1044–1116). The G‐CSF receptor mRNA transcript was exhibited in the RNA from MLpN cells by reverse transcriptase polymerase chain reaction procedure. [3H]thymidine incorporation and trypan blue exclusion showed that the G‐CSF receptor was capable of transducing the growth signal to HCF‐MLpN cells. A small fraction of fresh B blasts from six patients with B‐cell lymphoma and leukaemia displayed G‐CSF binding by two‐colour immunofluorescence staining. In contrast, a panel of seven B‐cell lines was negative for the binding to biotinylated G‐CSF preparation. These results suggest that the phenotype of G‐CSF binding may be lost during the culture. The expression of G‐CSF receptor in HCF‐MLpN cells appeared to be exceptional.
Role of allogeneic bone marrow transplantation from an HLA‐identical sibling or a matched unrelated donor in the treatment of children with juvenile chronic myeloid leukaemia
Authors: Locatelli Franco , Pession Andrea , Comoli Patrizia , Bonetti Federico , Giorgiani Giovanna , Zecca Marco , Taibi Rosa Maria , Mongini Maria Elisa , Ambroselli Franco , De Stefano Piero , Severi Francesca , Paolucci Guido
Published: November 2003

Seven children (age range 1.8–11 years) with juvenile chronic myelomonocytic leukaemia (JCML) received an allogeneic bone marrow transplantation (BMT), four from an HLA‐identical sibling and three from a matched unrelated donor. In the four children transplanted using an HLA‐identical sibling, conditioning regimen included busulfan (BU), cyclophosphamide (CY) and melphalan (L‐PAM), whereas graft‐versus‐host disease (GVHD) prophylaxis consisted of cyclosporine‐A (Cs‐A). The preparative regimen was well tolerated and all patients engrafted promptly. None of the patients have relapsed and all four children remain in haematological remission after an observation time of 7, 24, 25 and 48 months, respectively. Of the three children given BMT from an unrelated volunteer, one was <2 years of age and she received the BU/CY/L‐PAM regimen. In view of the increased risk of graft rejection described in patients transplanted from unrelated donors, we chose to prepare the other two patients with fractionated total body irradiation (TBI), thiotepa and CY. Cs‐A, short‐term methotrexate and Campath‐1G in vivo were employed to prevent GVHD in this group of patients. Graft failure with autologous reconstitution of haemopoiesis occurred in the child given the chemotherapy‐based regimen. One of the two girls given TBI relapsed after BMT; therefore only one of the three patients who received a marrow transplant from a matched unrelated donor survives in complete haematological remission 10 months after BMT. Our study suggests that the conditioning regimen we employed for allogeneic BMT from a compatible sibling is an effective means of eradicating the leukaemic clone. In our experience, results obtained using unrelated donors are less satisfactory and, at present, the use of such donors seems to be riskier and associated with a lower success rate as compared with BMT from an HLA‐identical sibling.
Cytomegalovirus‐induced thrombocytopenia and haemolysis in an immunocompetent adult
Authors: Johan van Spronsen Dick , Breed Wim P. M.
Published: November 2003

We describe a patient with severe symptomatic thrombocytopenia and haemolytic anaemia caused by cytomegalovirus (CMV) infection. As far as we know, this is the second case in the literature. Treatment with ganciclovir seemed to be more effective for thrombocytopenia than treatment with corticosteroids. The hypothetical mechanisms leading to thrombocytopenia and haemolysis in CMV infection are briefly discussed. In cases of acute thrombocytopenia and clinical manifestations of an infectious disease, with or without haemolysis, testing for CMV may be useful in connection with therapeutic consequences.
Ultrastructural changes during adhesion and migration of pre‐B lymphoid leukaemia cells within bone marrow stroma
Authors: Hewson John , Bianchi Alessandra , Bradstock Ken , Makrynikola Vicki , Gottlieb David
Published: November 2003

The ultrastructural changes in leukaemic cells on initial contact with, and during migration into, layers of bone marrow stroma in vitro were examined in a variety of types of acute leukaemia and leukaemic cell lines. Bone marrow fibroblasts (BMF) were grown on polycarbonate microporous membranes, and acute leukaemia cells added to cultures and allowed to adhere to BMF for variable periods of time before fixation. Acute lymphoblastic leukaemia (ALL) blasts showed rapid development of surface membrane microvilli on contact with BMF layers. ALL blasts, and the pre‐B ALL cell line NALM‐6, showed evidence of movement in to the BMF layer within 15–30min, with intrusion of extended cytoplasic processes into gaps between BMF cytoplasm. ALL cells were frequently seen within the layers of fibroblasts after 30min incubation, and had pronounced morphological changes, with pseudopodia and attenuated and elongated microvilli interdigitating with the surface of fibroblasts or with strands of extracellular matrix material. Changes were also noted in the surface membrane of BMF adjacent to ALL cells, with invagination of the cytoplasmic membrane and formation of micropits. In contrast to the migratory behaviour of pre‐B ALL cells, migration was not observed with acute myeloid leukaemia cells or other leukaemic cell lines. These cells showed membrane activation, with variable degrees of microvillous formation, and in some cases insertion of pseudopodia into BMF layers, but migration was not observed. Ultrastructural immunogold labelling was carried out to determine the localization of leukaemic adhesion molecules and their ligands on BMF. This demonstrated that β1 integrins were largely localized to the contact surfaces of both ALL blasts and fibroblasts, with VCAM‐1 expressed only on the surface of BMF. These observations confirm the specificity of migratory behaviour for pre‐B leukaemic cells, and indicate that a complex pattern of surface and intracellular events mediate this process, including the expression of β1 integrins and VCAM‐1 at the sites of insertion of leukaemic cells between fibroblast margins.
Intermediate‐dose melphalan (IDM) combined with G‐CSF (filgrastim) is an effective and safe induction therapy for autologous stem cell transplantation in multiple myeloma
Authors: Lokhorst Henk , Sonneveld Pieter , Wijermans Pierre , Van Marwijk Kooy Marinus , Th. Meuwissen Otger , Van Oers Rien , Van Der Griend René , Dekker Adriaan
Published: November 2003

Twenty‐one previously untreated multiple myeloma (MM) patients and 10 previously treated patients with refractory or relapsed disease received two or three cycles of intermediate‐dose melphalan (70 mg/m2) (IDM), administered intravenously every 6 weeks. Seven previously untreated patients received three and all other patients received two courses of IDM. The objective of the study was to reduce the toxicity of high‐dose melphalan (140 mg/m2) (HDM) while maintaining its cytotoxic efficacy and secondly to ensure the possibility of collecting sufficient numbers of peripheral blood stem cells (PBSC) for transplantation. 18 (85%) previously untreated patients responded, of whom four achieved CR (18%). In addition five out of 10 previously treated patients with refractory or relapsed disease responded although bone marrow toxicity in this category was a major drawback. Toxicity was moderate, consisting of alopecia and moderate bone marrow suppression: the granulocyte count dropped below 0.5 × 109/l and platelets below 25 × 109/l for a median of 8 and 6 d, respectively. No serious infections occurred and the majority of patients attended the out‐patient clinic. In 12/14 previously untreated patients sufficient peripheral blood CD34+ cells for harvest were present in the repopulation phase after the first IDM. In nine patients peripheral blood stem cells were collected and eight patients have undergone succesful transplantation.

Repeated IDM followed by filgrastim is highly effective in untreated MM and may be safely administered to reduce tumour load prior to PBSC collection. Autologous stem cells harvested after repeated IDM have a full long‐term repopulating capacity.
Hypereosinophilia during 2‐chlorodeoxyadenosine treatment for hairy cell leukaemia
Authors: Rutella Sergio , Sica Simona , Rumi Carlo , Martucci Robert , Etuk Benedict , De Stefano Valerio , Testa Ugo , Leone Giuseppe , Peschle Cesare
Published: November 2003

A 43‐year‐old male with newly diagnosed hairy cell leukaemia underwent a single course of 2‐chlorodeoxyadenosine (2‐CdA). Skin rash, facial swelling and marked eosinophilia developed 20 d after treatment and were resolved by 7 d of steroid therapy. Eosinophil peak in peripheral blood reached 1230 cells/μl. Flow cytometric analysis of the eosinophil population showed a high expression of the IL‐2 receptor α‐chain (CD25), representing up to 94% of gated cells. HLA‐DR and CD4 antigens were constantly negative; eosinophils strongly reacted with the secretory form of the eosinophil cationic protein (ECP), recognized by EG2 monoclonal antibody. IL‐5 serum levels were markedly elevated at the onset of eosinophilia, returned to normal levels after its disappearance and positively correlated with eosinophil count (r = 0.94, P = 0.016). Eosinophilia is an uncommon finding after treatment with 2‐CdA. It is unclear whether these phenomena represented a true allergic reaction to the drug or the effect of massive tumour cell lysis and haemopoietic pancytopenia with immunosuppression, which induced the release of IL‐5 and possibly other cytokines.
Expression and role in growth regulation of tumour necrosis factor receptors p55 and p75 in acute myeloblastic leukaemia cells
Authors: Carter Anna , Haddad Nuhad , Draxler Ilana , Israeli Ella , Raz Batya , Rowe Jacob M.
Published: November 2003

Tumour necrosis factor (TNF)‐α exerts multiple effects on human acute myeloblastic leukaemia (AML) cells in vitro, including (1) synergistic stimulation of proliferation with interleukin‐3 (IL‐3) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF); (2) inhibition of granulocyte‐CSF (G‐CSF) and stem cell factor (SCF)‐induced growth; (3) suppression of multiplication of clonogenic leukaemic cells; (4) induction of autocrine growth. Recently, two distinct TNF receptors (TNF‐Rs), TNF‐Rp55 and TNF‐Rp75, have been identified. In this study we show that both receptors are expressed on freshly isolated AML blasts, with p75 being the predominant TNF‐receptor type. This study investigates the roles of these two receptors in TNF‐α‐driven growth regulation of AML blasts in vitro. Using a receptor‐specific antibody, it is shown that both receptor types participate in TNF‐α‐mediated stimulation of GM‐CSF/IL‐3‐induced proliferation and in TNF‐α‐induced autocrine growth. In contrast, the TNF‐α‐triggered growth inhibition (antiproliferation) and the potent suppression of G‐CSF‐ and SCF‐induced proliferation exclusively result from activation of TNF‐Rp55. Taken together, these results suggest that the proliferative effects of TNF‐α on AML blasts are mediated through both p55 and p75 TNF receptors, whereas the TNF‐α‐signalled growth inhibition is exclusively transduced via TNF‐Rp55.
Glyburide‐induced acute haemolysis in a G6PD‐deficient patient with NIDDM
Authors: Meloni Gianfranco , Meloni Tullio
Published: November 2003

In a 61‐year‐old man with glucose‐6‐phosphate dehydrogenase (G6PD) deficiency and poorly controlled non‐insulin‐dependent diabetes mellitus, an episode of acute haemolysis occurred after the administration of glyburide (glibenclamide). Erythrocyte fragmentation, with haemoglobin condensation zones next to clear zones, was observed on peripheral blood smears. Since autoimmune haemolysis was excluded on the basis of laboratory data, acute haemolysis was ascribed to G6PD deficiency.
Response to immunosuppressive therapy and an HLA‐DRB1 allele in patients with aplastic anaemia: HLA‐DRB1*1501 does not predict response to antithymocyte globulin
Authors: Nakao Shinji , Takami Akiyoshi , Sugimori Naomi , Ueda Mikio , Shiobara Shintaro , Matsuda Tamotsu , Mizoguchi Hideaki
Published: November 2003

HLA‐DRB1*1501, a subtype of HLA‐DR2, has been shown to be closely associated with a good response to cyclosporine (CyA) therapy in patients with aplastic anaemia (AA). To determine whether this DRB1 allele can also predict a response to antithymocyte globulin (ATG) therapy in AA patients, we analysed the results of HLA‐DRB1 typing in 59 Japanese patients who received ATG within 2 years after diagnosis of AA and also in 52 patients treated with CyA. All patients were divided into three groups: those with DRB1*1501, those with DRB1*1502, and those without either of these two alleles (DR2–). The response rate to ATG in DRB1*1501+ patients (56%) was not significantly higher than that in DRB1*1502+ patients (47%) and in the other DR2– patients (54%). In contrast, the response rate to CyA therapy in DRB1*1501+ patients (92%) was significantly higher than that in the DRB1*1502+ (41%) and in DR2– patients (57%). Multivariate analysis revealed that possessing DRB1*1501 was an independent factor significantly predictive of a good response to CyA. These results indicate that although identifying the DRB1*1501 allele in AA patients prior to therapy is predictive of a good response to CyA therapy, it does not have a predictive value for ATG therapy.
Pathogenicity of IgA and/or IgM antibodies to heparin–PF4 complexes in patients with heparin‐induced thrombocytopenia
Authors: Amiral Jean , Wolf Martine , Fischer Anne‐Marie , Boyer‐Neumann Catherine , Vissac Anne‐Marie , Meyer Dominique
Published: November 2003

Antibodies to heparin–PF4 (H‐PF4) complexes have been tested and isotyped in 38 patients who developed severe heparin‐induced thrombocytopenia (type II HIT). All the patients had a platelet count < 120 × 109/l or a reduction of >30% of the initial value, occurring at least 5 d after the onset of heparin. Thrombocytopenia, which rapidly reversed following the withdrawal of heparin, was associated with thrombosis in nine patients. Although IgG isotypes were found in most cases (n = 26), the presence of only IgM and/or IgA was observed in 12 patients, including three cases showing a thrombotic complication. Our results indicate that type II HIT may be induced by IgA and/or IgM anti‐H‐PF4 antibodies even in the absence of IgG isotypes. This finding demonstrates that platelet Fc receptors (FcγRII) are not necessarily involved in the pathogenicity of heparin‐dependent antibodies and emphasizes the major role of platelet PF4 receptors. The increased expression of the latter following a slight activation by thrombin, and the subsequent binding of IgM and IgA antibodies to H‐PF4 on the platelet surface, may directly trigger platelet activation, aggregation and thrombosis. Alternatively, thrombocytopenia could be indirectly induced through the mediation of neutrophils, monocytes and lymphocytes which expose receptors for IgA (FcαR) or IgM (FcμR). IgM–platelet complexes may also bind and activate complement, leading to platelet activation or destruction. Moreover, the reactivity of the antibodies with glycosaminoglycans–PF4 complexes present on the endothelial surface could also induce endothelial lesions and promote procoagulant activity and predisposition to thrombosis.
Resistance to activated protein C (APCR) in children with venous or arterial thromboembolism
Authors: Nowak‐Göttl U. , Koch H. G. , Aschka I. , Kohlhase B. , Vielhaber H. , Kurlemann G. , Oleszcuk‐Raschke K. , Kehl H. G. , Jürgens H. , Schneppenheim R.
Published: November 2003

Resistance to activated protein C (APCR), in the majority of cases due to the point mutation Arg 506 Gln of the factor V gene, has emerged as the most important hereditary cause of venous thromboembolism. Using an activated thromboplastin time (aPTT) based method in the presence of APC together with a DNA technique based on the polymerase chain reaction, we investigated 37 children with venous (V: n = 19) or arterial (A: n = 18) thromboembolism and 196 age‐matched healthy controls for the presence of this mutation. In the control group 10 children were detected to be heterozygous for the factor V Leiden mutation, indicating a prevalence of 5.1%. 10/19 children (52%) with venous thrombosis and 7/18 (38%) patients with arterial thromboembolism showed the common factor V gene mutation. Additional inherited coagulation disorders were found in 1/10 (V: 10%) and 2/7 (A: 28%) APC‐resistant patients. Inherited coagulation disorders without APCR were diagnosed in 3/9 (V: 33%) and 2/11 (A: 18%) children. Furthermore, we diagnosed exogenous risk factors in 6/10 (V: 60%) and 2/7 (A: 28%) children with thrombosis and APCR. These data are evidence that APCR combined with exogenous reasons may play an important role in the early manifestation of thromboembolism during infancy and childhood.
rVIIa therapy to secure haemostasis during central line insertion in children with high‐responding FVIII inhibitors
Authors: Smith O. P. , Hann I. M.
Published: November 2003

The provision of adequate haemostasis in children with haemophilia A (HA) who have developed high‐responding inhibitors continues to be a great challenge to haematologists and institutional resources. The most exciting development in the management of acute bleeding in these patients, irrespective of inhibitor titre, has been the use of recombinant activated factor VII (rVIIa). Three severe HA children with high‐responding inhibitors underwent four central line insertions (two Hickman catheters and two port‐a‐caths, one replaced because of infection) with rFVIIa cover to enable continuous FVIII infusions. No adverse haemorrhagic events occurred and immune tolerance therapy (ITT) using high‐dose FVIII therapy was initiated and inhibitor eradication was achieved in all three patients at 8, 10 and 7 months. A further patient who had a central line in situ prior to the development of a high‐responding inhibitor was also successfully ‘tolerized’ at 6 months using the same protocol. Interestingly, all four patients had the IVS 22 mutation.
Analysis of p53 and mdm‐2 expression in 18 patients with Sézary syndrome
Authors: Marks David I. , Vonderheid Eric C. , Kurz Benedikt W. , Bigler Robert D. , Sinha Keisha , Morgan Doris A. , Sukman Abby , C. Nowell Peter , Haines Dale S.
Published: November 2003

Sézary syndrome is a leukaemic form of cutaneous T‐cell lymphoma which presents with multiple cytogenetic abnormalities and responds poorly to chemotherapy. Because of the importance of the p53 tumour suppressor in maintaining genomic stability and in sensitizing transformed cells to DNA damaging agents, we looked for alterations which may affect p53 functions in 18 patients with Sézary syndrome. Cytogenetic analysis suggested frequent p53 gene inactivation since 6/18 patients had loss of one copy of 17p. However, single‐strand conformational polymorphism (SSCP) revealed that p53 gene mutations are relatively rare, occurring in only two of 18 Sézary patients. Neither of these two patients was missing a copy of 17p. Possible abnormalities of p53 pathway function through mdm‐2 over‐expression were also investigated. Although all 18 patients had normal levels of mdm‐2 RNA, 4/18 over‐expressed mdm‐2 protein. One patient with advanced disease and the highest percentage of malignant cells overexpressed mdm‐2 protein and possessed a nonsense p53 gene mutation. The five patients with abnormalities of p53 or mdm‐2 were found to have significantly higher absolute lymphocyte counts and higher absolute numbers of Sézary cells (P = 0.021 and 0.027 respectively). In summary, molecular alterations of 17p and potential p53 pathway abnormalities are a common event in Sézary syndrome and appear to be associated with more advanced disease.
Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine‐mobilized blood
Authors: Leitner Agnes , Strobl Herbert , Fischmeister Gustav , Kurz Martin , Romanakis Kostas , Haas Oskar A. , Printz Dieter , Buchinger Petra , Bauer Silvia , Gadner Helmut , Fritsch Gerhard
Published: November 2003

Flow cytometric DNA analysis was performed in combination with three‐colour immunological staining of cell surface antigens on density‐separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell‐cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5 μg per kg body weight of either granulocyte colony‐stimulating factor (G‐CSF) or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34− MNC, the proportion of cells in S/G2M phase increased from <0.1% to 0.75 ± 0.4% (GM‐CSF) and to 1.34 ± 0.75% (G‐CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non‐cycling status of PB and CB progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cell sources. However, in vitro stimulation of these progenitor cells using IL3, GM‐CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2M phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized ‘stem cells’ which may require the BM micro‐environment for adequate proliferation in vivo
A monoclonal antibody directed against a granule membrane glycoprotein (GMP‐140/PADGEM, P‐selectin, CD62P) inhibits ristocetin‐induced platelet aggregation
Authors: BOUKERCHE HABIB , RUCHAUD‐SPARAGANO MARIE‐HéLèNE , R OUEN CHRISTINE , BROCH IER JEAN , KAPLAN CéCILE , MCGREGOR JOHN LOUIS
Published: November 2003

P‐selectin (also called CD62, GMP‐140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin‐induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG1) bound a surface‐labelled glycoprotein (GP) which changed its apparent molecular mass (Mr) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIbα). LYP7 and S12, a monoclonal antibody directed against P‐selectin immunoprecipitated the same band. Using ELISA assay, purified P‐selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of 125I‐labelled LYP7, which was greatly increased on thrombin‐stimulated (2 U/ml) washed platelets (10825±2886, mean ±SD) (Kd=1.5±0.5 nm) compared to resting platelets (2801±1278, mean ±SD) (Kd=1.5±0.6 nm), was found to be normal on thrombin‐stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG1, F(ab′)2 or Fab fragments) inhibited ristocetin‐induced platelet aggregation of platelets in a dose‐dependent fashion without affecting the binding of von Willebrand (vWf ) factor. However, agglutination of formaldehyde‐fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin‐activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P‐selectin, by promoting cell–cell contact, may play an active role in platelet–platelet interactions.
The influence of age, gender and ABO blood group on soluble endothelial cell markers and adhesion molecules
Authors: Blann Andrew , Daly Ray , Amiral Jean
Published: November 2003

The influence of age, gender and ABO blood group on soluble adhesion molecules and markers of endothelial function were tested by measurement of levels of von Willebrand factor, intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1 (VCAM‐1), P‐selectin, E‐selectin and thrombomodulin in venous blood from healthy subjects. Only VCAM‐1 showed a significant multivariate correlation with age (r = 0.2, P = 0.019), and thrombomodulin (P = 0.0001) and E‐selectin (P = 0.026) were lower in the women. Significant differences between ABO blood groups were found for von Willebrand factor (P = 0.024), E‐selectin (P < 0.001) and thrombomodulin (P < 0.001). As these three are all specific endothelial products, our findings may have implications for vascular biology.
Richter's syndrome in a case of atypical chronic lymphocytic leukaemia with the t(11;14)(q13;q32): role for a p53 exon 7 gene mutation
Authors: Cuneo Antonio , De Angeli Cristiano , Grazia Roberti Maria , Piva Nadia , Bigoni Renato , Gan dini Domenica , Matteo Rigolin Gian , Moretti Sabrina , Cavaz zini Pierluigi , Del Senno Laura , C ASTOLDI GIANLUIGI
Published: November 2003

Clinicobiological, histological, cytogenetic and molecular genetic studies were performed in a case of atypical B‐cell chronic lymphocytic leukaemia (B‐CLL) with the t(11;14)(q13;q32) evolving into Richter's syndrome (RS) in order (a) to determine the clonal relationship between the cell of origin for B‐CLL and RS, and (b) to analyse genetic events underlying the disease progression in this patient.

After 4 years following diagnosis, a rapid deterioration of the clinical picture occurred, concomitant with the appearance of large lymphoid blasts in peripheral blood (PB), bone marrow (BM) and ascites samples. A diagnosis of RS was made and cytogenetic analysis revealed karyotype evolution with trisomy 7 and del(17p) in addition to t(11;14). Fluorescence in situ hybridization showed 78% lymphoid blast cells obtained from ascites sample to be trisomic using a chromosome‐7‐specific pericentromeric probe. Whereas no rearrangement of the c‐myc proto‐oncogene was detected at disease progression, direct sequencing of p53 gene exon 5–9 revealed an exon 7 missense point mutation. This abnormality was not present in the CLL phase. Immunological staining with the monoclonal antibody PAb‐1801, detecting the p53 protein product, revealed a negative pattern in the CLL phase, whereas 24% positivity was documented in representative samples obtained at RS.

It is concluded that RS was cytogenetically related with B‐CLL in this patient, suggesting the occurrence of a bona fide transformation and that the mutation of p53 exon 7, in association with the development of 17p deletion, possibly played a role in the development of RS.
Haemopoietic growth factor tyrosine kinase receptor expression profiles in normal haemopoiesis
Authors: Visser Monique , Sonneveld Renate D. , Willemze Roel , Landegent James E.
Published: November 2003

Expression profiles were generated for the haemopoietic tyrosine kinase receptors (HGF‐TKRs or class III TKRs) by PCR on cDNA samples (RT‐PCR) using a degenerate primer set. Each profile consisted of primary and secondary, i.e. enriched for less‐expressed sequences, fingerprints. This method was applied on FACS‐purified haemopoietic CD34+ cells, both from bone marrow (BM) and umbilical cord blood (UCB), and on mature cells from peripheral blood. CD34+ BM cells showed expression of c‐fms, flt3, whereas CD34+ UCB cells expressed c‐fms and, to a lesser extent, c‐kit and flt3. In mature blood cells, only c‐fms was observed in monocytes and a weaker flt3 expression in monocytes and T lymphocytes, whereas no known class III TKRs were detected in B lymphocytes and polymorphonuclear cells (PMNs). In all fractions a novel band could be observed, which appeared to be RET. Expression of RET was confirmed by RT‐PCR and showed the highest levels in monocytes, followed by PMNs and CD34+ cells. B lymphocytes revealed low levels of expression. RET is known to be essential in neural development. Our results suggest a possible role for this receptor in haemopoiesis.
MUTATIONS IN A SUBGROUP OF PATIENTS WITH MILD HAEMOPHILIA A AND A FAMILIAL DISCREPANCY BETWEEN THE ONE‐STAGE AND TWO‐STAGE FACTOR VIII:C METHODS
Authors: Rudzki Zbigniew , Duncan Elizabeth M. , Casey Graeme J. , Neumann Maureen , Favaloro Emmanuel J. , Lloyd John V.
Published: November 2003

A subgroup of patients with haemophilia A who have a familial discrepancy between the one‐stage and two‐stage factor VIII:C results has previously been described. These patients show factor VIII:C levels by one‐stage assay that are 2–7‐fold higher than their two‐stage results. We have studied 10 such families and identified six different mutations in the factor VIII gene in this group. The chemical cleavage method and DNA sequencing was used to identify mutations in factor VIII gene fragments generated by reverse transcription and PCR. All available family members were tested to confirm the presence of the mutation in affected individuals. These patients were found to have one of six single point substitutions causing a missense mutation and alteration to one codon in exons 7, 11, 14 or 18. The mutations comprise three that have not previously been described (Ala284Glu, Arg698Leu, Leu1932Phe) and three that have been previously described (Ser289Leu, Arg531His, Arg698Trp). Alterations to the amino acid composition of the A1, A2 and A3 domains of factor VIII are predicted by these molecular studies. In contrast, a control group of 23 mild haemophilia families with equivalent factor VIII:C results by one‐stage and two‐stage assays did not have any of the above mutations. Detailed studies in seven of these latter families identified four mutations affecting the A3, C1 and C2 domains of factor VIII. These findings suggest a genetic basis to the unusual factor VIII phenotype but do not explain the mechanism of the discrepant factor VIII activity.
An aggressive nasal lymphoma accompanied by high levels of soluble Fas ligand
Authors: Sato Ken , Kimura Fumihiko , Nakamura Yukitsugu , Murakami Hideaki , Yoshida Masayoshi , Tanaka Masato , Nagata Shigekazu , Kanatani Yasuhiro , Wakimoto Naoki , Nagata Naokazu , Motoyoshi Kazuo
Published: November 2003

Fas ligand (FasL), either in the membrane bound form or soluble form, has cytotoxic activity against Fas‐expressing cells. We report a case of nasal lymphoma accompanied by liver damage and pancytopenia. The serum level of soluble FasL (sFasL) was very high on admission, but rapidly decreased to normal levels after chemotherapy for lymphoma. Liver damage and pancytopenia also improved with the decrease in serum sFasL. Since Fas is expressed on both hepatocytes and haemopoietic cells, these facts suggest that FasL was expressed on lymphoma cells and directly associated with pathogenesis of liver damage and pancytopenia through its cytotoxic activity.
A possible change in doubling time of haemopoietic progenitor cells with stem cell development
Authors: Mahmud Nadim , Katayama Naoyuki , Itoh Ryugo , Tanaka Ryuhei , Ohishi Kohski , Masuya Masahiro , Mimami Nobuyuki , Shiku Hiroshi
Published: November 2003

We separated haemopoietic progenitors derived from marrow cells of 5‐fluorouracil (5‐FU)‐treated mice into three groups, based on the stages of stem cell development and studied doubling time, using a serum‐free clonal culture system. Stage I progenitors were those present in primary marrow cells from 5‐FU‐treated mice. Stages II and III progenitors were early and late progenies in culture of stage I progenitors, respectively. The morphological analysis of colonies derived from stage I, II and III progenitors demonstrated an association of progression of stages with loss of multipotentiality. The doubling time of haemopoietic progenitors was estimated by sequential analysis of colony formation and studies of growth fraction. The time required for haemopoietic progenitors to double shortened as their stage of development progressed. Alteration in one doubling time of haemopoietic progenitors at progressive stages of stem cell development was seen in cultures supported by various combinations of growth factors, including interleukin‐3 (IL‐3), IL‐11, and steel factor (SF). Cell‐cycle analysis suggested that reduction of the doubling time of haemopoietic progenitors is probably due to a decrease in the time spent in the G1 phase of the cell cycle. Our results suggest that in early haemopoiesis the doubling time of haemopoietic progenitors may change with stem cell development.
Effect of interferon on the health‐related quality of life of multiple myeloma patients: results of a Nordic randomized trial comparing melphalan–prednisone to melphalan–prednisone + α‐interferon
Authors: WislØff Finn , Hjorth Martin , Kaasa Stein , Westin Jan
Published: November 2003

In a Nordic multi‐centre trial, 583 previously untreated multiple myeloma patients were randomized to receive melphalan–prednisone or melphalan–prednisone + interferon α‐2b at a dose of 5 million units subcutaneously, 3 d/week. A quality‐of‐life study was integrated into the trial, using the EORTC QLQ C‐30 questionnaire supplemented with 11 questions concerning interferon toxicity. The questionnaire was completed prior to treatment and after 1, 6, 12, 24, 36 and 48 months. 90% of the patients participated in the quality‐of‐life study, and 83% completed all questionnaires submitted to them. During the first year of treatment the patients on interferon reported significantly more fever, chills, dry skin, fatigue, pain, nausea/vomiting and appetite loss than the control patients. There was a moderate reduction of the global quality‐of‐life score and slight, non‐significant, reductions of physical, emotional, cognitive, social and role functioning scores. After the first year there were no statistically significant differences in any toxicity, symptom or quality‐of‐life score, except for an increased frequency of dizziness in the interferon group. As only 60% of the patients remained on interferon after 24 months, our data probably underestimate the potential toxicity of the drug. Although there was no significant survival benefit for the interferon patients, a 5–6 months prolongation of the response and plateau phase duration was observed. However, by intention‐to‐treat analysis, there was no late quality‐of‐life benefit for the interferon patients to compensate for the early impairment. Thus, the clinical significance of the plateau‐phase prolongation is uncertain.
Recombinant human c‐Mpl ligand is not a direct stimulator of proplatelet formation in mature human megakaryocytes
Authors: ITO TOSHIHARU , ISHIDA YOJI , KASHIWAGI RIEKO , KURIYA SHIN‐ICHIRO
Published: November 2003

To evaluate the effect of the c‐Mpl ligand on platelet production by megakaryocytes, we investigated proplatelet formation in isolated human megakaryocytes cultured in serum‐free medium, with or without the c‐Mpl ligand, interleukin‐6 and erythropoietin. When interleukin‐6 was added to the culture medium, the percentage of megakaryocytes displaying proplatelets was approximately 1.5‐fold the control value; whereas, in the presence of the c‐Mpl ligand, the percentage of megakaryocytes displaying proplatelets decreased in a dose‐dependent manner and did not increase compared to control values at any dose tested. However, the viability of megakaryocytes after a 4 d culture in the presence of the c‐Mpl ligand was significantly higher than that of the cells cultured without it. The c‐Mpl ligand did not stimulate the proplatelet formation in megakaryocytes in vitro
Analysis of the κ light chain variable region in multiple myeloma
Authors: Kosmas Christos , Viniou Nora‐Athina , Stamato poulos Kostas , Courtenay‐Luck Nigel S. , Papadaki Theodora , Kollia Panagoula , Paterakis George , Anagnostou Dimitra , Yataganas Xenophon , Loukopoulos Dimitris
Published: November 2003

The study of immunoglobulin heavy chain gene rearrangements in multiple myeloma has revealed extensive divergence from the germline sequences, but no intraclonal diversity with disease evolution. Our study investigated the state of the rearranged κ light chain variable region (Vκ) gene segments, as well as abortive Vκ family gene usage in cases of multiple myeloma expressing λ light chain. We studied 11 cases of κ and five cases of λ light chain‐expressing multiple myeloma. Total cellular RNA was extracted from the bone marrow of patients with overt disease and subjected to reverse transcription‐polymerase chain reaction (RT‐PCR) analysis to amplify clonally rearranged variable region sequences. Direct nucleotide sequencing by the dideoxy‐chain termination method was performed on the RT‐PCR products. We did not observe preferential usage of certain Vκ gene families. Mutation frequencies of the Vκ segments varied in number. In the majority of cases, extensive somatic mutations occurred within the complementarity determining regions (CDRs) of Vκ, whereas only a limited degree of divergence from the germline was observed in others. In all cases studied, replacement mutations tended to cluster in the CDRs, a finding compatible with an antigen‐driven somatic hypermutation process. In 3/5 cases of λ light‐chain expressing multiple myeloma, abortively rearranged Vκ gene segments were amplified from genomic DNA; in two cases a non‐templated nucleotide insertion rendering the Vκ sequences out‐of‐frame was observed, and in the third a stop codon was identified in the open reading frame of the Vκ sequence. Somatic mutations were observed in all cases of abortive Vκ genes studied; however, their distribution does not suggest selection by antigen.

We conclude that somatic mutations observed in the Vκ regions of myeloma cells are of variable extent and suggest operation of the antigen selection process. Lack of or minimal somatic hypermutation in a few cases may be in some way implicated in the biological heterogeneity of the disease.
Underestimation of inversion (16) in acute myeloid leukaemia using standard cytogenetics as compared with polymerase chain reaction: results of a prospective investigation
Authors: Ritter Markus , Thiede Christian , Schäkel Ulrike , Schmidt Manuel , Alpen Birgit , Pascheberg Ulrich , Mohr Brigitte , Ehninger Gerhard , Neubauer Andreas ,   for the AML‐SHG study group
Published: November 2003

In order to determine whether the polymerase chain reaction (PCR) is more suitable for the detection of inversion (16) as compared with standard cytogenetics, we prospectively investigated a total of 132 cases of de novo acute myeloid leukaemia (AML) (n=121) and secondary AML after myelodysplastic syndromes (MDS) (n=11) using a sensitive and nested PCR procedure to detect the fusion transcripts CBFβ‐MYH11. All patients were recruited within 10 months in an ongoing multicentre AML‐trial. In addition, several cases from a retrospective molecular analysis were included. The data were compared with standard cytogenetics performed in a central laboratory. Of the 132 prospective AML cases, five patients (3.7%) harboured inv(16) upon conventional cytogenetics. In all cases fusion transcripts CBFβ‐MYH11 were detected using PCR. In addition in two patients fusion transcripts were detected, although cytogenetics revealed a normal karyotype. In the group of patients analysed retrospectively, four patients harboured fusion transcripts specific for CBFβ‐MYH11; cytogenetics were normal in one case, and could not be evaluated in two cases. These data show that PCR may be a better means to detect inv(16) in AML. Since inv(16) may have prognostic impact in AML, detection of this aberration seems important in the clinical management of AML patients.