bluuebunny/crossref_metadata_2025 huggingface dataset:
Marked elevation of thrombin generation in patients with elevated FVIII:C and venous thromboembolism
Authors: O'Donnell James , Mumford Andrew D. , Manning Richard A. , Laffan Michael A.
Published: March 2003

Elevated plasma factor VIII coagulant activity (FVIII:C, > 150 IU/dl) is a risk factor for venous thromboembolism (VTE). We hypothesized that increased FVIII:C may exert a prothrombotic effect by increasing basal thrombin generation. To test this hypothesis we have measured prothrombin fragment 1 + 2 (F1 + 2) and thrombin–antithrombin complex (TAT) in three groups: (i) patients with objectively confirmed VTE and elevated FVIII:C; (ii) patients with VTE and no detectable thrombophilia; and (iii) healthy age‐ and sex‐matched control subjets. In the group of patients with elevated FVIII:C, TAT and F1 + 2 levels were increased in 85% and 78% of individuals respectively. This frequency of coagulation activation is dramatically higher than that reported for other recognized constitutional thrombophilias. In the group of patients with VTE but no proven thrombophilia, increased thrombin generation was present in 30% of individuals. Basal thrombin generation was significantly higher in patients with elevated FVIII:C compared with individuals with VTE but no documented thrombophilia (median TAT = 8·65 µg/l versus 2·95 µg/l, median F1 + 2 = 1·5 nmol/l versus 0·87 nmol/l; P < 0·0001, P < 0·001). Overall FVIII:C levels were strongly correlated with levels of thrombin generation (r= 0·5, P < 0001). The clinical significance of such markedly increased F1 + 2 and TAT levels in patients with high FVIII:C levels remains unclear.
Some factor VIII exon 14 frameshift mutations cause moderately severe haemophilia A
Authors: Nakaya Shelley , Liu Miao‐Liang , Thompson Arthur R.
Published: March 2003

Factor VIII's exon 14 codes for its B domain that includes nearly one‐third of its amino acid sequence that is not necessary for function. Frameshift mutations appear to occur more frequently within exon 14 than in other exons. To assess exon 14 frameshift mutations and their clinical correlates, a series of unrelated, severe or moderately severe haemophilia A patients were screened for heteroduplex formation in amplified exon 14 fragments. In 25 families, a frameshift mutation was identified by sequencing. Occurrence of haemophilia was isolated in 18 of these families. Moderate severity was noted in at least six out of 13 families with an A insertion or deletion at one of two sequences where the frameshift resulted in a sequence of 8–10 As. Inhibitors occurred in five of the other 12 families including one with an A insertion within a sequence of six As. Recurrent insertions into an A8 (codons 1439–1441) or an A9 (codons 1191–1194) sequence or of an A deletion from the A9 sequence are common, recurrent causes of haemophilia A that may have a moderately severe phenotype.
Serum thrombopoietin levels in relation to disease status in patients with immune thrombocytopenic purpura
Authors: Kappers‐Klunne M. C. , De Haan M. , Struijk P. C. , Van Vliet H. H. D. M.
Published: March 2003

Pre‐ and post‐treatment serum thrombopoietin (TPO) concentration was measured in 35 patients with immune thrombocytopenic purpura (ITP). Mean post‐treatment levels were significantly lower (P = 0·02) than pretreatment and not different for treatment modality. No significant correlation between pre‐ or post‐treatment TPO and platelet counts was demonstrable (R = −0·325, P = 0·056 and R = −0·227, P = 0·190 respectively). In patients with very low platelet counts ( 20 × 109/l), pretreatment serum TPO was significantly higher than in patients with higher counts (P = 0·033). The logarithm of the platelet turnover rate, measured in 15 patients, correlated with pretreatment TPO levels (R = 0·64). These findings suggest a contributory role for TPO in the mechanism of ITP.
Use of the collagen‐binding assay for von Willebrand factor  in the analysis of type 2M von Willebrand disease:  a comparison with the ristocetin cofactor assay
Authors: Riddell Anne F. , Jenkins P. Vincent , Nitu‐Whalley Ioana C. , McCraw Angus H. , Lee Christine A. , Brown Simon A.
Published: March 2003

Summary. This study compares the utility of two functional assays for von Willebrand factor (VWF), the ristocetin cofactor assay (VWF:RCo) and the collagen‐binding assay (VWF:CBA). We analysed a group of 32 patients with type 2 von Willebrand disease (VWD) (25 patients with type 2M, six with type 2A and one with type 2B) and 22 normal control subjects. VWF:RCo/VWF antigen (VWF:Ag) ratios and VWF:CBA/VWF:Ag ratios were compared between the patient and control groups. In the six patients with type 2A VWD, both VWF:RCo/VWF:Ag ratios and VWF:CBA/VWF:Ag ratios were discordant (≤ 0·7). In the 25 type 2M VWD patients, the VWF:CBA/VWF:Ag ratios were concordant (> 0·7), but the VWF:RCo/VWF:CBA ratios were discordant (≤ 0·7) (P = 0·001) compared with control subjects. Thus, VWF:RCo/VWF:Ag ratios were discordant in both type 2M and 2A VWD patient groups indicating a functional abnormality. However, VWF:CBA/VWF:Ag ratios were discordant in the type 2A VWD group but not in the type 2M VWD group. Our study showed that VWF:CBA is sensitive to functional variants associated with the loss of high‐molecular‐weight multimers, i.e. type 2A and 2B in VWD, but the assay was unable to discriminate defective platelet‐binding VWD variants with normal multimeric patterns such as type 2M VWD. It was concluded that the VWF:CBA assay should be used in association with rather than as a replacement for the VWF:RCo assay.
Immunocytochemistry for the heavy chain of the non‐muscle myosin IIA as a diagnostic tool for MYH9‐related disorders
Authors: Pecci Alessandro , Noris Patrizia , Invernizzi Rosangela , Savoia Anna , Seri Marco , Ghiggeri Gian Marco , Sartore Saverio , Gangarossa Simone , Bizzaro Nicola , Balduini Carlo L.
Published: March 2003

Summary.  May–Hegglin anomaly (MHA), Sebastian syndrome (SBS) and Fechtner syndrome (FTNS) are autosomal‐dominant macrothrombocytopenias with Döhle‐like leucocyte inclusions. These diseases are due to mutations of the MHY9 gene, encoding the heavy chain of non‐muscle myosin IIA (NMMHC‐A). We investigated the NMMHC‐A localization in blood cells from eight MHA, SBS or FTNS patients with known MYH9 mutations. All the patients showed an altered localization of NMMHC‐A in granulocytes and platelets, suggesting that Döhle‐like bodies are due to the aggregation of NMMHC‐A in the cytoplasm. Therefore, immunocytochemistry for NMMHC‐A is a simple and sensitive method to detect pathological phenotypes of granulocytes and platelets in the diagnosis of MYH9‐related disorders.
On the mechanism of action of recombinant activated factor VII administered to patients with severe thrombocytopenia and life‐threatening haemorrhage: focus on prothrombin activation
Authors: Gerotziafas Grigoris T. , Zervas Kostas , Arzoglou Pantelis , Karavaggeli Eli , Parashou Stella , Van Dreden Patrick , Christakis John , Samama Meyer Michel
Published: March 2003

Summary. We report the ex vivo effect of recombinant activated factor VII (rFVIIa) on prothrombin activation after whole blood clotting. Two patients with severe thrombocytopenia and life‐threatening haemorrhage were successfully managed using a single dose of rFVIIa (90 µg/kg). Western blotting using antiprothrombin antibody showed that rFVIIa did not induce thrombin generation in citrated platelet‐poor plasma. Patient sera showed significantly impaired prothrombin activation before and after rFVIIa administration. rFVIIa administration shortened the prothrombin time, activated partial thromboplastin time and Ivy bleeding time, and normalized the clot retraction. These data indicate that rFVIIa accelerated thrombin generation without significant increase of generated thrombin.
Low levels of activated protein C in patients with systemic lupus erythematosus do not relate to lupus anticoagulants but to low levels of factor II
Authors: Simmelink Marleen J. A. , Fernández José A. , Derksen Ronald H. W. M. , Griffin John H. , De Groot Philip G.
Published: March 2003

Summary. The presence of lupus anticoagulants (LAC) in plasma is a major risk factor for thrombosis. An attractive hypothesis to explain a LAC‐mediated thrombotic tendency is that LAC interfere with activation of protein C, a natural antithrombotic in plasma. We investigated the relationship between LAC and protein C activation in vivo. We selected 20 patients with systemic lupus erythematosus (SLE) with LAC (and not using oral anticoagulants), 36 patients with SLE without LAC and 25 healthy volunteers. In these, we measured circulating levels of activated protein C (APC), prothrombin (FII), free protein S, C4BP, protein C, and antibodies to protein C, protein S, FII and β2‐glycoprotein I (β2GPI). In SLE patients (n = 56), mean levels of APC, FII and free protein S were significantly (P < 0·001) lower than those in healthy volunteers (respectively 13%, 17% and 14%). Mean protein C levels and C4BP levels were similar for SLE patients and healthy volunteers. In contrast to the above hypothesis, the decreased levels of APC could not be attributed to the presence of LAC. Levels of APC were correlated with both FII levels and protein C levels. Decreased levels of APC, FII, protein C and free protein S were related to the presence of anti‐FII antibodies. None of the patients had antibodies against protein C or protein S. In conclusion, although the mean levels of APC, FII and free protein S were significantly decreased in SLE patients, no correlation with LAC was found. However, anti‐FII antibodies were related to decreased levels of APC, FII, protein C, free protein S and C4BP. As FII levels, and not protein C levels, were decreased in SLE patients and correlated with APC levels, we conclude that the decreased FII levels are responsible for the low levels of APC.
Expression of the Ikaros gene family in childhood acute lymphoblastic leukaemia
Authors: Takanashi Mami , Yagi Tomohito , Imamura Toshihiko , Tabata Yasuhiro , Morimoto Akira , Hibi Shigeyoshi , Ishii Eiichi , Imashuku Shinsaku
Published: March 2003

Summary. The Ikaros (Ik) gene family, which includes Ik, Aiolos (Ai), and Helios (He), is a primary regulator of lymphocyte differentiation, and is involved in the development of acute lymphoblastic leukaemia (ALL). We analysed the expression of the Ik gene family isoforms in 97 ALL cases, consisting of 64 childhood and 33 infant ALL cases, using reverse transcription‐polymerase chain reaction (RT‐PCR). Expression of Ik was detected in all cases, 87 of which expressed either Ik1 or Ik2, or both, five of which expressed Ik1/Ik2 and Ik6, and another five of which expressed only Ik6. Therefore, the dominant negative isoform of Ik6 was expressed in 10 of the 38 cases of childhood precursor B ALL, but was absent in other types of childhood ALL (26·3%, χ2‐test, P = 0·0001). In terms of Aiolos and Helios expression, 49 (65·3%) out of the 75 and 40 (50%) out of the 80 ALL cases tested showed non‐spliced Ai1 and He1 respectively. Only one case of T lineage ALL expressed a small‐sized isoform of Helios (designated as He6). It was also found that the expression of Ai1 and He1 was low in Ik6‐positive patients (Fisher's exact test; Ai1 P = 0·005, Hel P = 0·035).
Antithrombotic effect of PMC, a potent α‐tocopherol analogue on platelet plug formation in vivo
Authors: Hsiao G. , Yen M. H. , Lee Y. M. , Sheu J.‐R.
Published: March 2003

Summary. Platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. PMC (2,␣2, 5, 7, 8‐pentamethyl‐6‐hydroxychromane; 20 µg/g, i.v.) significantly prolonged the latent period of inducing platelet plug formation in mesenteric venules. When fluorescein sodium was given at 10 µg/kg, PMC (20 µg/g) delayed occlusion time by about 1·7‐fold. Furthermore, aspirin (250 µg/g) also showed similar activity in delaying the occlusion time. On a molar basis, PMC was about 14‐fold more potent than aspirin at delaying the occlusion time. PMC was also effective in reducing the mortality of ADP‐induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 5 and 10 µg/g. In addition, intravenous injection of PMC (5 µg/g) significantly prolonged bleeding time by about 1·6‐fold compared with normal saline in severed mesenteric arteries of rats. Continuous infusion of PMC (1 µg/g/min) significantly increased the bleeding time by about 1·6‐fold and the bleeding time was also significantly prolonged for up to 90 min after cessation of PMC infusion. These results suggest that PMC has an effective antiplatelet effect in vivo and may be a potential therapeutic agent for arterial thrombosis, but must be assessed further for toxicity.
Differentiation of functional dendritic cells and macrophages from human peripheral blood monocyte precursors is dependent on expression of p21 (WAF1/CIP1) and requires iron
Authors: Kramer Joan L. , Baltathakis Ioannis , Alcantara Orlando S. F. , Boldt David H.
Published: March 2003

Summary.  Iron is required for monocyte/macrophage differentiation of HL‐60 leukaemia cells. Differentiation requires induction of the cyclin‐dependent kinase inhibitor p21 (WAF1/CIP1), and cell cycle arrest at the G1/S checkpoint. With iron depletion, p21 induction and differentiation are blocked. To establish the roles of iron and p21 in normal monocyte/macrophage differentiation, we examined generation of dendritic cells (DCs) and macrophages from peripheral monocytes. Monocytes were cultured with interleukin 4 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF), then treated with lipopolysaccharide to produce DCs or with M‐CSF to produce macrophages. Iron deprivation was induced by desferrioxamine (DF). Monocyte‐derived DCs had characteristic phenotype and morphology, and stimulated proliferation of naïve allogeneic T lymphocytes. In contrast, DCs generated under iron deprivation were phenotypically undifferentiated and did not stimulate T cells. Similarly, macrophages expressed a characteristic phenotype and morphology, and phagocytosed latex beads, but macrophages generated under iron deprivation failed to develop a mature phenotype and had impaired phagocytosis. Iron deprivation blocked induction of p21 (WAF1/CIP1) expression in both DC and macrophage cultures. Furthermore, p21 antisense oligonucleotides, but not sense oligonucleotides, inhibited both DC and macrophage differentiation. These data indicate that a key role of iron in haematopoiesis is to support induction of p21 which, in turn, is required for DC and macrophage differentiation.
The investigation and management of neonatal haemostasis and thrombosis*
Authors: Williams Michael D. , Chalmers Elizabeth A. , Gibson Brenda E. S.
Published: March 2003

These guidelines address developmental aspects of neonatal haemostasis and thrombosis, the laboratory investigation of the neonate, and the diagnosis and clinical management of haemostatic and thrombotic conditions occurring in this period (defined as the first 4 weeks of life following birth). Relevant scientific papers were identified by a systematic literature review from Medline 1975–2000 using index terms which incorporated the various component subjects of these guidelines. Further publications were obtained from the references cited and from reviews known to the members of the working party and to the Haemostasis and Thrombosis Task Force. Evidence and graded recommendations presented in these guidelines are in accordance with the US Agency for Health Care Policy and Research, as described in the Appendix. It will be noted that there is a lack of a strong evidence base for many of the recommendations suggested, as the appropriate clinical and laboratory trials have not been and perhaps never will be undertaken in neonates. Most of the recommendations are therefore of Grade C evidence levels IV: higher levels are mentioned specifically throughout the document when relevant.
Factor XIII A‐subunit concentration predicts outcome in stroke subjects and vascular outcome in healthy, middle‐aged men
Authors: Kohler Hans P. , Ariëns Robert A. S. , Catto Andrew J. , Carter Angela M. , Miller George J. , Cooper Jackie A. , Mansfield Michael W. , Standeven Kristina F. , Grant Peter J.
Published: March 2003

Summary. There is growing evidence for a role of factor XIII (FXIII) in vascular disease. FXIII measures were determined in (i) a nested case–control study from the Second Northwick Park Heart Study of 63 men with myocardial infarction (MI) and 124 age‐matched controls and (ii) in a case–control study of 475 subjects with acute stroke and 461 controls followed up for 54 months for mortality. In both studies, measures of FXIII A‐ and B‐subunit antigen, FXIII activity and prothrombin fragments (F1 + 2) were made. An in vitro model was used to investigate the effects of thrombin activity on FXIII A‐ and B‐subunit antigen levels. In study 1, patients clinically free of coronary artery disease who later developed MI had lower adjusted FXIII A‐subunit levels at recruitment (129·2%vs 113·3%, P = 0·007). In study 2, stroke patients with large vessel disease had lower A‐subunit antigen levels (102·1%vs 127·2%, P < 0·001), but higher F1 + 2 levels (0·941%vs 0·753%, P < 0·05), than subjects with small vessel disease. Levels of FXIII A‐subunit (100%vs 117%, P < 0·0001) were lower and F1 + 2 higher (1·020%vs 0·702%, P < 0·0001) in stroke patients who had died compared with those still alive at the end of the follow‐up period. Low concentrations of FXIII A‐subunit antigen predicted vascular outcome in otherwise healthy subjects and relate to both size of infarct and poor post‐stroke survival in patients with acute ischaemic stroke. Low in vitro concentrations of FXIII A‐subunit antigen wererelated to increased thrombin generation and, thus, increased risk of thrombotic events.
Mutation in the ATP‐binding site of BCR‐ABL in a patient with chronic myeloid leukaemia with increasing resistance to STI571
Authors: Barthe Christophe , Gharbi Marie‐josée , Lagarde Valérie , Chollet Claudine , Cony‐Makhoul Pascale , Reiffers Josy , Goldman John M. , Melo Junia V. , Mahon François Xavier
Published: March 2003

Summary. The Abl kinase inhibitor STI571 (imatinib mesylate) induces haematological remissions in many patients with chronic myeloid leukaemia (CML) but advanced stage CML usually becomes resistant to STI571. We describe a patient in whom progressive resistance to STI571 correlated with the appearance of a mutation in the Bcr‐Abl kinase domain. This was a G to A transition that resulted in a glutamic acid to lysine substitution at position 255 (E255K) in the Abl type 1a protein. We suggest that the acquisition of point‐mutations in the tyrosine kinase domain of Bcr‐Abl may cause progressive clinical resistance to STI571.
Successful non‐myeloablative allogeneic transplantation for treatment of idiopathic hypereosinophilic syndrome
Authors: Ueno Naoto T. , Anagnostopoulos Athanasios , Rondón Gabriela , Champlin Richard E. , Mikhailova N. , Pankratova O. S. , Zoubarovskaya L. S. , Semenova E. V. , Afanasyev B. V. , O'Brien Susan , Andreeff Michael , Zaritskey Andrey Y.
Published: March 2003

Summary. In this report, we describe two patients with idiopathic hypereosinophilic syndrome (HES) who received a non‐myeloablative allogeneic transplantation following a reduced‐intensity preparative regimen of melphalan and fludarabine. In both cases, complete donor chimaerism and remission were achieved, and have lasted for more than 10 months. This report provides proof of principle for the feasibility of non‐myeloablative transplantation for patients with idiopathic HES, who can show co‐morbidity due to eosinophilic infiltration of their organs.
Bone marrow cyclooxygenase‐2 levels are elevated in chronic‐phase chronic myeloid leukaemia and are associated with reduced survival
Authors: Giles Francis J. , Kantarjian Hagop M. , Bekele B. Nebiyou , Cortes Jorge E. , Faderl Stephan , Thomas Deborah A. , Manshouri Taghi , Rogers Anna , Keating Michael J. , Talpaz Moshe , O'Brien Susan , Albitar Maher
Published: March 2003

Summary.  Increased angiogenesis is important in the pathophysiology of haematological malignancies. Cyclooxygenase‐2 (Cox‐2) converts arachidonic acid to prostaglandins, which induce expression of angiogenic factors, including vascular endothelial growth factor (VEGF), basic‐fibroblast growth factor, transforming growth factor‐β and interleukin 6. Cox‐2 may also reduce apoptosis and reduce cellular attachment to the extracellular matrix (ECM). Increased bone marrow (BM) vascularity, increased BM cellular and plasma VEGF levels, and decreased progenitor adherence to BM ECM have all been observed in chronic myeloid leukaemia (CML). We investigated the prognostic significance of levels of Cox‐2 in BM cells from patients with CML. Western blot and solid‐phase radioimmunoassay (RIA) were used to measure Cox‐2 BM levels in 149 patients with chronic phase CML (CP CML). Results were compared with those of normal controls. Expression of Cox‐2 was significantly higher in CML than in normal controls (P < 0·0001). Increasing levels of Cox‐2 were significantly associated with shorter survival (P = 0·0002, Cox proportional hazard model). A multivariate model based on Cox‐2 and degree of splenomegaly was developed for survival in patients with early CP CML. Agents that inhibit Cox‐2 activity merit investigation in patients with CP CML.
Limitations of Gallium‐67 SPECT in histological transformation of chronic lymphocytic leukaemia: an analysis of 13 patients with clinically suspected Richter's syndrome
Authors: Cobo Francesc , Rosiñol Laura , Martínez Antonio , Martín Francisco , Esteve Jordi , Gómez Marta , Nadal Elisabet , Bosch Francesc , Javier Setoain Francisco , Campo Elías , Montserrat Emili
Published: March 2003

Summary.  Gallium‐67 single photon emission computerized tomography (Ga‐67 SPECT) was performed in 13 chronic lymphocytic leukaemia (CLL) patients suspected of evolution into diffuse large B‐cell lymphoma (DLCL) or Richter's syndrome (RS). Six positive and nine negative Ga‐67 SPECTs were observed. Ten patients were biopsied (five in each group). DLCL was not detected in any Ga‐67‐positive patient, including those in whom Ga‐67‐positive areas were biopsied. The only case of DLCL was demonstrated in a Ga‐67‐negative patient. The tumoral proliferative index (Ki67 antigen expression) was moderate and similar in both groups of patients. These results illustrate the limitations of Ga‐67 SPECT in identifying RS.
Myeloma clonotypic B cells are hampered in their ability to undergo B‐cell differentiation in vitro
Authors: Guikema Jeroen E. J. , Vellenga Edo , Bakkus Marleen H. C. , Bos Nicolaas A.
Published: March 2003

Summary.  In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present that express the identical V(D)J rearrangements as the malignant plasma cells in the bone marrow. In the present study, the proliferative capacity of clonotypic B cells from MM patients (n = 10) and the ability to differentiate in vitro was determined using the CD40‐culturing system. For six patients, the presence of clonotypic B cells expressing variant immunoglobulin (Ig) isotypes was assessed by Ig isotype‐specific allele‐specific oligonucleotide reverse transcription polymerase chain reaction (ASO‐RT‐PCR) after culturing with CD40L and interleukin 4 (IL‐4). In three out of six patients, clonotypic B cells expressing variant isotypes were detected both before and after culturing. The ability of clonotypic B cells to undergo B‐cell differentiation was studied by abrogating CD40 signalling accompanied by IL‐10 and IL‐2 stimulation, enhancing differentiation towards Ig‐secreting cells. The numbers of clonotypic B cells were determined by quantitative ASO‐PCR. An increase in cell number was observed upon CD40L and IL‐4 stimulation, whereas the relative number of clonotypic B cells was unaltered. In contrast, upon B‐cell differentiation the relative number of clonotypic B cells decreased. In conclusion, clonotypic B cells can be cultured and isolated in vitro using the CD40 system. Clonotypic B cells responded to CD40 triggering in a similar fashion as to non‐clonotypic normal B cells. However, the ability of clonotypic B cells to undergo in vitro activation and differentiation into Ig‐secreting cells is hampered.
Laminin γ2 chain as a stromal cell marker of the human bone marrow microenvironment
Authors: Siler Ulrich , Rousselle Patricia , Müller Claudia A. , Klein Gerd
Published: March 2003

Summary. Laminins are large heterotrimeric molecules consisting of α, β and γ chains. At present, five α chains, three β chains and three γ chains have been characterized. Laminin‐5 (α3β3γ2) is the only isoform known to date which contains a γ2 chain. In human bone marrow, non‐haematopoietic stromal cells expressed the laminin γ2 chain, whereas bone marrow mononuclear cells did not. Co‐localization of the γ2 chain was detected with the laminin α4 and α5 chains, and co‐immunoprecipitation studies revealed a new isoform consisting of α5, β2 and γ2 chains. The laminin γ2 chain was also co‐localized with α‐sm‐actin in bone marrow, but it was not expressed in endothelial cells or megakaryocytes, indicating that the γ2 chain is exclusively expressed in vivo in bone marrow stromal cells. The laminin γ2 chain containing isoform LN‐5 was shown to be an adhesive substrate for a small subpopulation of bone marrow mononuclear cells and also for peripheral blood platelets. Taken together, these results indicate that (I) laminin isoforms containing the γ2 chain can act as adhesive substrates for human haematopoietic cells, and (II) the laminin γ2 chain can be used as a specific marker molecule for human bone‐marrow‐derived stromal cells.
Platelet autoantibodies and lupus‐associated thrombocytopenia
Authors: Michel Marc , Lee Ketty , Piette Jean‐Charles , Fromont Patricia , Schaeffer Annette , Bierling Philippe , Godeau Bertrand
Published: March 2003

Summary. To characterize the antigenic targets of anti‐platelet antibodies (APA) found in systemic lupus erythematosus (SLE)‐associated thrombocytopenia, 48 patients with immune thrombocytopenia and SLE were compared with 20 patients with SLE who had never been thrombocytopenic. Both cases and controls were tested for circulating APA by an indirect platelet suspension immunofluorescence assay (PSIIFT) and by indirect monoclonal antibody specific immobilization of platelet antigens (MAIPA). A direct platelet suspension immunofluorescence assay (PSIFT) was also used for antibodies bound to platelets in vivo in thrombocytopenic patients; 13 of them with high titres of platelets‐bound APA were investigated by direct and indirect MAIPA and platelet eluate analysis. Circulating APA were detected by PSIIFT in 88% of cases and 55% of controls (P = 0·0066) and platelet‐bound antibodies were detected by PSIFT in 90% of cases. Indirect MAIPA detected specific APA (mainly directed against GpIIbIIIa) in 36% of cases and only 5% of the controls (P = 0·0076). Nine out of the 13 fully investigated thrombocytopenic patients (69%) had a positive direct MAIPA and/or APA detected in platelet eluates. In conclusion, the production of specific anti‐platelet autoantibodies, mainly directed against GpIIb/IIIa, and their binding to platelet membrane plays an important role in the pathogenesis of SLE‐associated thrombocytopenia.
AC133+ umbilical cord blood progenitors demonstrate rapid self‐renewal and low apoptosis
Authors: Forraz Nicolas , Pettengell Ruth , Deglesne Pierre‐Antoine , McGuckin Colin P.
Published: March 2003

Summary. Umbilical cord blood (UCB) provides immediate access to haemopoietic stem/progenitor cells (HSPC) but low cell number restricts use in full adult bone marrow reconstitution. This study investigated early ex vivo expansion kinetics of UCB AC133+ cells (2–4 × 104/ml), mononuclear cells (MNC, 1–2 × 106/ml) and AC133negative cells (AC133neg, 2–4 × 104/ml) in stroma‐free 8 d liquid culture (fetal bovine serum‐supplemented Iscove's‐modified Dulbecco's medium (IMDM) with either ‘K36EG’[c‐Kit ligand, interleukin 3 (IL‐3), IL‐6, erythropoietin, granulocyte colony‐stimulating factor] or ‘TPOFL’ (thrombopoietin, Flt‐3 ligand). Cell enumeration, apoptosis assay and AC133/CD34/CD38 antigen immunophenotyping were performed at d 0, 3, 5 and 8. All three cell populations went through a proliferation lag phase between d 3 and d 5. AC133+ cells recovered better from lag phase with significantly higher fold increase (FI) when compared with MNC and AC133neg populations (K36EG FI: 15·04 ± 5·46; TPOFL FI: 8·59 ± 2·92, P < 0·05). After 8 d, populations lacking AC133+ cells were significantly more inclined to undergo apoptosis under proliferative conditions (P < 0·01). Also, when compared with K36EG, 8 d TPOFL‐expanded AC133+ cells encompassed a significantly higher percentage of AC133+ and CD34+ early HSPC (K36EG: 20·50 ± 2·36; TPOFL: 47·00 ± 7·69; P < 0·05). In conclusion, TPOFL synergism demonstrated the potential for AC133+ HSPC ex vivo expansion inducing self‐renewal, early HSPC maintenance and promoting cell survival status.
CXCL13 (BCA‐1) is produced by follicular lymphoma cells: role in the accumulation of malignant B cells
Authors: Husson Hervé , S. Freedman Arnold , A. Cardoso Angelo , Schultze Joachim , Munoz Olivier , Strola Giuliana , Kutok Jeffery , G. Carideo Elizabeth , De Beaumont Rosalie , Caligaris‐Cappio Federico , Ghia Paolo
Published: March 2003

Summary. Follicular lymphomas (FLs) localize in lymphoid tissues and recapitulate the structure of normal secondary follicles. The chemokine/chemokine receptor pair CXCL13/CXCR5 is required for the architectural organization of B cells within lymphoid follicles. In this study, we showed that CXCL13 was secreted by FL cells. FL cells expressed CXCR5 and migrated in response to CXCL13. Furthermore, we observed a synergistic effect between CXCL13 and CXCL12 (SDF‐1), a chemokine produced by stromal cells in lymphoid tissues. The production of CXCL13 by FL cells and CXCL12 by stromal cells probably directs and participates in the accumulation of FL cells within specific anatomic sites.
Effective binding of free iron by a single intravenous dose of human apotransferrin in haematological stem cell transplant patients
Authors: Sahlstedt Leila , Von Bonsdorff Leni , Ebeling Freja , Ruutu Tapani , Parkkinen Jaakko
Published: March 2003

Summary. Myeloablative treatment results in iron accumulation and the appearance of non‐transferrin‐bound iron (NTBI) in the circulation, which may contribute to treatment‐related organ damage and susceptibility to infections. The aim of this study was to investigate the efficacy of human apotransferrin in the binding of NTBI in patients receiving an allogeneic stem cell transplant after myeloablative conditioning. A single intravenous 100 mg/kg dose of apotransferrin was given to six adult patients on d 3 after the transplantation. Initially, all patients had serum transferrin saturation above 80% and NTBI in their serum. After the apotransferrin injection, serum NTBI became undetectable in all patients and transferrin saturation decreased to 30–50%. Serum transferrin increased by an average of 1·95 g/l. The administered apotransferrin was subsequently converted into monoferric and diferric transferrin forms. NTBI reappeared and transferrin saturation again exceeded 80% 12–48 h after the injection in four patients and after 6 d in one patient. NTBI remained non‐detectable for the whole 12 d follow‐up period in one patient. The apotransferrin injection was well tolerated and no adverse events with probable association with the apotransferrin were observed. Repeated apotransferrin infusions might completely eliminate NTBI and iron‐induced toxicity during myeloablative therapy.
The prospective evaluation of a nested polymerase chain reaction assay for the early detection of Aspergillus infection in patients with leukaemia or undergoing allograft treatment
Authors: Ferns R. Bridget , Fletcher Helen , Bradley Susan , Mackinnon Stephen , Hunt Chris , Tedder Richard S.
Published: March 2003

Summary. Patients with acute leukaemia or undergoing allogenic bone marrow transplantation at University College London Hospital Trust were screened for the presence of aspergillosis by polymerase chain reaction (PCR). Aspergillus DNA, from whole blood samples, was amplified by nested PCR to detect a 135 bp fragment in the mitochondrial region of Aspergillus fumigatus or A. flavus (121 bp). One colony‐forming unit (CFU) per 2 ml of blood or 1–10 fg DNA could be detected. Patients at risk of aspergillosis were classified as probable or possible based on the European Organization for Research and Treatment of Cancer definitions. Antifungal drugs given were recorded. In four of 17 patients studied, infection was not suspected and the PCR was negative. Four patients were considered to have possible aspergillosis infection and were PCR positive on at least one occasion. Of the three patients in the probable group, four of the nine samples tested PCR positive from one patient and in another patient only one of nine samples tested positive. The remaining six patients were not suspected of having fungal infection but each had one or two PCR‐positive results. In summary, six of seven patients thought to have clinical evidence of infection were PCR positive on at least one occasion and treatment with antifungals may have reduced infection below detectable levels.
BDCM: a novel B‐cell line with genetic and functional similarity to dendritic cells
Authors: Kharbanda Sandhya , Salter Russell D. , Dong Xin , Tuma‐Warrino Renee J. , Steinman Richard A.
Published: March 2003

Summary. We describe a B‐cell line, BDCM, which arose spontaneously from culture of a pheresis product from a patient with M5a myeloid leukaemia. Cell growth was associated with autocrine activation of the signal transducer and activator of transcription 1 (Stat‐1) transcription factor. Although the cells expressed several B‐cell surface markers and had a rearranged immunoglobin J region, they also exhibited several characteristics associated with dendritic cells. These included extensive surface projections, cross‐priming ability and strong T cell‐stimulating capability. In addition, the cytokine production profile of BDCM cells was nearly identical to that of mature monocyte‐derived dendritic cells.
The role of primary prophylactic factor replacement therapy in children with severe factor X deficiency
Authors: McMahon Corrina , Smith Judith , Goonan Collette , Byrne Mary , Smith Owen P.
Published: March 2003

Summary. Severe factor X (FX) deficiency is one of the severest inherited coagulation disorders. Clinical manifestations include umbilical cord, mucosal, joint and central nervous system bleeding. Four Irish children with severe FX deficiency presented with umbilical cord bleeding. One developed an intraperitoneal haemorrhage and another an intracranial bleed. Prophylaxis, using intermediate purity Factor IX concentrate, was commenced within the first month of life, necessitating the insertion of central venous access devices in two of the children. All children have normal joint function, suggesting that prophylaxis commenced early in life reduces the incidence of arthropathy and improves quality of life.
Treatment of adult systemic mastocytosis with interferon‐α: results of a multicentre phase II trial on 20 patients
Authors: Casassus Philippe , Caillat‐Vigneron Nadine , Martin Antoine , Simon Jeanne , Gallais Valérie , Beaudry Patrice , Eclache Virginie , Laroche Liliane , Lortholary Pierre , Raphaël Martine , Guillevin Loïc , Lortholary Olivier
Published: March 2003

Summary. Systemic mastocytosis (SM) is characterized by proliferation of mast cells in various organs, which may release a wide variety of mediators, thereby explaining the broad clinical spectrum of disease manifestations. The potentially life‐threatening systemic symptoms and tumoral proliferation are poorly controlled despite the use of several cytotoxic chemotherapies and/or symptomatic treatments. Twenty consecutive adult SM patients with histologically confirmed bone marrow (BM) involvement received interferon‐α subcutaneously (1–5 million units/m2/d, with progressive dose intensification over the first month of treatment) and were evaluated after 6 months of therapy. Seven of them had previously received symptomatic treatments, including steroids, which were ineffective. Among the 13 patients treated for at least 6 months, seven partial and six minor responses, mainly concerning vascular congestion and skin lesions, were obtained, while BM infiltration remained unchanged in 12 patients. The significant reduction of mast‐cell mediator levels after 6 months of treatment was not predictive of clinical remission. The rate of depression was unexpectedly high (seven patients; 35%). Two patients died soon after starting therapy (one myocardial infarction, one septic shock). Six months of interferon‐α may relieve vascular congestion in adults with SM, probably by inhibiting mast‐cell degranulation.
Acquired resistance to activated protein C in breast cancer patients
Authors: Nijziel Marten R. , Van Oerle Rene , Christella M. , Thomassen L. G. D. , Van Pampus Elisabeth C. M. , Hamulyák Karly , Tans Guido , Rosing Jan
Published: March 2003

Summary. In 56 women with a lymph‐node‐positive breast carcinoma and 28 matched healthy control subjects, the sensitivity to activated protein C (APC‐sr) was determined with an APC resistance test that quantifies the effect of APC on thrombin generation initiated via the extrinsic coagulation pathway. Carriers of the Factor V Leiden mutation were excluded from the study. Significant resistance to APC was found in the breast cancer patients: median APC‐sr 2·02 vs 1·03 in the healthy control subjects (P < 0·001). No difference in APC‐sr was found between patients with metastases and without metastases. In patients with metastases, protein S levels were significantly elevated compared with patients without metastases and healthy control subjects: 108·0%vs 96·0% and 94·5% (P = 0·008 and P = 0·007). The APC‐sr correlated with protein S in the control subjects and in patients without metastases but not in patients with metastases. The disturbance of the haemostatic balance probed by the tissue‐factor‐based APC resistance test might contribute to the cancer‐related hypercoagulability.
Fcγ receptor IIa and IIIa polymorphisms in childhood immune thrombocytopenic purpura
Authors: Carcao Manuel D. , Blanchette Victor S. , Wakefield Cindy D. , Stephens Derick , Ellis Janet , Matheson Kimberly , Denomme Gregory A.
Published: March 2003

Summary. Fcγ receptor‐mediated destruction of autoantibody‐sensitized platelets is central to the immune pathophysiology of childhood immune thrombocytopenic purpura (ITP). Allelic variants exist among the random population for some Fcγ receptors. The variants represent single nucleotide polymorphisms, leading to functional differences in the ability to bind immunoglobulin (Ig)G or IgG subclasses. The genotypic frequencies for two Fcγ receptor single nucleotide polymorphisms, FcγRIIa‐131 arginine (R) versus histidine (H) and FcγRIIIa‐158 valine (V) versus phenylalanine (F) were examined in 98 children diagnosed with childhood ITP. The genotype frequencies were compared with those of 130 healthy control subjects. Chi‐square analysis was used to determine whether the allelic frequencies of the high‐affinity receptor variants were associated with childhood ITP. Both the FcγRIIa‐131H and the FcγRIIIa‐158V were significantly over‐represented in children with ITP versus the control subjects (P‐values 0·03). The same statistical difference was noted with the combined FcγRIIa‐131H and FcγRIIIa‐158V allelic gene frequencies. There was no statistical difference between children who later developed chronic ITP compared with children with acute ITP, suggesting that additional factors are responsible for the development of the chronic form of the disease. These observations underscore the importance of Fcγ receptor‐mediated cell clearance in childhood ITP.
Is the factor XIII 34Val/Leu polymorphism a protective factor for cerebrovascular disease?
Authors: Endler Georg , Funk Marion , Haering Daniela , Lalouschek Wolfgang , Lang Wilfried , Mirafzal Merdad , Wagner Oswald , Mannhalter Christine
Published: March 2003

Summary. A frequent polymorphism in the factor XIII (FXIII) A‐subunit gene, leading to a Val to Leu amino acid exchange at position 34, suggested to affect clot stability, has been associated with a decreased risk for venous thromboembolism and myocardial infarction. Its role in the development of stroke is still under investigation. Ninety‐four patients with primary arterial intracerebral haemorrhage (mean age ± standard deviation: 69 ± 14 years; 48 men, 46 women), 718 patients with ischaemic stroke (63 ± 14 years; 395 men, 323 women) and 369 healthy control subjects (59 ± 14 years; 299 men, 170 women) were analysed for FXIII Val34Leu. No differences in genotype distribution between all three groups were observed. Also, no significant differences in the genotype distribution were found between subgroups of patients stratified according to age, sex, aetiology, history of hypertension, antiplatelet or anticoagulant medication and other vascular risk factors. In contrast to previously reported findings in smaller collectives, our data suggest that an association of the FXIII Val34Leu polymorphism with a decreased risk of ischaemic stroke or an increased risk of intracerebral haemorrhage is highly unlikely. Thus, screening for the FXIII Val34Leu polymorphism will not contribute significantly to the risk prediction of cerebrovascular disease.
HIV‐1 matrix protein p17 enhances the proliferative activity of natural killer cells and increases their ability to secrete proinflammatory cytokines
Authors: Vitale Marco , Caruso Arnaldo , De Francesco Maria Antonia , Rodella Luigi , Bozzo Luisa , Garrafa Emirena , Grassi Manuela , Gobbi Giuliana , Cacchioli Antonio , Fiorentini Simona
Published: March 2003

Summary. We investigated the effects of human immunodeficiency type‐1 virus (HIV‐1) matrix protein p17 on freshly isolated and purified human natural killer (NK) cells. HIV‐1 p17 increased the cytokines interleukin (IL) 2, IL‐12 and IL‐15, and induced natural killer cell proliferation, but not cytotoxicity. This effect was specific because it was abrogated by anti‐p17 monoclonal antibody. Moreover, HIV‐1 p17 enhanced the cytokine‐induced production of tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ by NK cells. IL‐4 downregulated IFN‐γ and TNF‐α secretion in IL‐2‐ and IL‐15‐treated NK cells. HIV‐1 p17 restored the ability of NK cells to produce both cytokines when added to the cultures simultaneously with IL‐4. The property of p17 to increase the production of TNF‐α and IFN‐γ might be a mechanism used by HIV‐1 to modulate the immune system to support its replication and spreading.
Early detection of meningeal localization in acute promyelocytic leukaemia patients with high presenting leucocyte count
Authors: Breccia Massimo , Carmosino Ida , Diverio Daniela , De Santis Silvia , De Propris Maria Stefania , Romano Atelda , Petti Maria C. , Mandelli Franco , Lo‐Coco Francesco
Published: March 2003

Summary. Extramedullary relapse occurs infrequently in acute promyelocytic leukaemia (APL) but has been increasingly reported after the advent of all‐trans retinoic acid (ATRA) treatment, probably as a consequence of improved patient survival. We describe our single centre experience of six APL patients who had disease localization in the central nervous system (CNS). In three patients, clinical symptoms (headache and/or nausea) that presented during follow‐up led to the performance of a lumbar puncture and detection of overt CNS infiltration. Two of these patients had simultaneous haematological relapse and one was in molecular remission when CNS leukaemia was documented. One patient with no local symptoms showed CNS infiltration at the time of molecular relapse. Following the introduction of routine lumbar puncture, carried out after front‐line induction in all newly diagnosed patients with white blood cell count (WBC) greater than 10 × 109/l, two additional patients in molecular remission with no local symptoms were found to have initial APL localization in the CNS. Presenting features included in 6/6 patients an elevated WBC count (> 10 × 109/l) and a predominance of the PML/RAR bcr3 type (5/6 patients) and of microgranular morphology (5/6 patients). Our findings highlight the importance of carrying out lumbar puncture in APL patients presenting with high‐risk features.
A novel 7·9 kb deletion causing α+‐thalassaemia in two independent families of Indian origin
Authors: Harteveld Cornelis L. , Van Delft Peter , Wijermans Pierre W. , Kappers‐Klunne Mies C. , Weegenaar Jitske , Losekoot Monique , Giordano Piero C.
Published: March 2003

Summary. We describe the characterization of a novel 7·9 kb deletion that eliminated one of the duplicated α‐globin genes, causing an α+‐thalassaemia phenotype in two independent carriers of Suriname–Indian origin. The molecular characterization of the deletion breakpoint fragment revealed neither involvement of Alu repeat sequences nor the presence of homologous regions prone to recombination, suggesting a non‐homologous recombination event. This α+‐thalassaemia deletion was found to give rise to an atypical haemoglobin H (HbH) disease characterized by a non‐transfusion‐dependent moderate microcytic hypochromic anaemia in combination with a poly adenylation signal mutation of the α‐globin gene (α2 AATAAA → AATA‐‐ ‐‐).
Detection of human herpesvirus‐8 in peripheral blood mononuclear cells from adult Japanese patients with multicentric Castleman's disease
Authors: Yamasaki Satoshi , Iino Tadafumi , Nakamura Minoru , Henzan Hideho , Ohshima Koichi , Kikuchi Masahiro , Otsuka Teruhisa , Harada Mine
Published: March 2003

Summary. Human herpesvirus‐8 (HHV‐8) encodes viral homologues of cellular genes, including viral interleukin 6 (vIL‐6), which induces endogenous human IL‐6 (hIL‐6) secretion. Unregulated overproduction of hIL‐6 in lymph nodes (LN) is thought to be responsible for the systemic manifestations of multicentric Castleman's disease (MCD). In the present study, we assessed the presence of HHV‐8 and HHV‐8‐encoded viral homologues in LN and peripheral blood mononuclear cells (PBMC) from adult Japanese patients with MCD. HHV‐8 DNA was amplified by nested polymerase chain reaction (PCR) and was detected in LN from 13 out of 16 MCD patients (81%). HHV‐8 DNA was also detected in PBMC from six out of seven patients (86%) whose LN were positive for HHV‐8 DNA. Because mRNA could not be successfully extracted from LN sections that were either formalin‐fixed or embedded in paraffin, we examined the expression of mRNA for HHV‐8‐encoded viral homologues, such as vIL‐6, vBCL‐2, vCyclin‐D and viral G‐protein‐coupled receptor (vGPCR) by nested reverse transcription (RT)‐PCR in PBMC from 10 MCD patients. However, mRNA of these HHV‐8‐encoded viral homologues was not detected in any patients tested. Although our results do not indicate a role for HHV‐8‐encoded viral homologues in the pathogenesis of MCD, they do suggest that HHV‐8 infection may be associated with MCD in adult Japanese patients.
Molecular pathology of haemophilia B in Turkish patients: identification of a large deletion and 33 independent point mutations
Authors: Onay U. Venüs , Kavakli Kaan , Kilinç Yurdanur , Gürgey Aytemiz , Aktuğlu Gülten , Kemahli Sabri , Özbek Uğur , Çağlayan S. Hande
Published: March 2003

Summary. Heterogeneous mutations in the coagulation factor IX (FIX) gene result in a bleeding tendency known as haemophilia B. The haemophilia B mutation database has a total of 2353 patient entries, including 10 of the estimated 1000 Turkish patients. In this study, a more comprehensive analysis of the molecular pathology of haemophilia B in Turkey revealed one large deletion and 33 point mutations in the FIX gene of 34 unrelated patients. Haplotype analysis using six polymorphic sites showed that the mutations identified in a total of 45 patients occurred on 13 different haplotypes and that each mutation was family specific.