bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross‐resistance to cytosine arabinoside in a multidrug‐resistant human promyelocytic cell line selected for resistance to doxorubicin: implications for combination chemotherapy
Authors: Månsson Emma , Paul Astrid , Löfgren Christina , Ullberg Karin , Paul Christer , Eriksson Staffan , Albertioni Freidoun
Published: March 2003

The pyrimidine analogue cytosine arabinoside (AraC) is one of the most effective drugs used in the treatment of acute leukaemia. Overexpression of the multidrug resistance (MDR‐1) gene and its product, P‐glycoprotein (P‐gp), is associated with cellular resistance to drugs, such as anthracyclines and vinca alkaloids. This resistance can be reversed by cyclosporine analogues or verapamil (ver). We investigated the in vitro cross‐resistance to AraC in a doxorubicin‐resistant HL60 cell line, with an elevated expression of the MDR‐1 gene. The resistant clone showed an eightfold increased resistance to AraC and a two‐ to fourfold resistance to the other analogues, as measured by cytotoxicity test. There was no significant increase in the activity of 5′‐nucleotidase or in the amount of deoxyribonucleotide pools between cell lines. We could, however, detect a reduction in deoxycytidine kinase (dCK) activity (30%, P = 0·021, using deoxycytidine as substrate) and the level of AraC triphosphates was significantly reduced in the resistant cells (70%, P = 0·009). When the cells were exposed to cyclosporin A (CsA) or the cyclosporine analogue PSC 833 (PSC) in combination with AraC, there was more extensive apoptosis, as measured by formation of oligonucleosomal DNA fragmentation and caspase‐3‐like activity, than with exposure to AraC alone. We also found an increased retention of AraC in the resistant cells when incubated with AraC in combination with CsA. Ver in combination with AraC, failed to increase apoptosis for the resistant cell line. Our data suggests that the resistance to AraC for the P‐gp‐expressing cells is a result of a reduction of dCK activity and an increase in efflux, the latter possibly depending on P‐gp. A combination of CsA or PSC with AraC may improve the effect of AraC in vivo.
A comparison of three rapid D‐dimer methods for the diagnosis of venous thromboembolism
Authors: Kovacs Michael J. , MacKinnon Karen M. , Anderson David , O'Rourke Keith , Keeney Michael , Kearon Clive , Ginsberg Jeffrey , Wells Philip S.
Published: March 2003

We compared three rapid d‐dimer methods for the diagnosis of venous thromboembolism. Patients presenting to four teaching hospitals with the possible diagnosis of deep vein thrombosis or pulmonary embolism were investigated with a combination of clinical likelihood, d‐dimer (SimpliRED) and initial non‐invasive testing. Patients were assigned as being positive or negative for deep vein thrombosis or pulmonary embolism based on their three‐month outcome and initial test results. The three d‐dimer methods compared were: (a) Accuclot d‐dimer (b) IL‐Test d‐dimer (c) SimpliRED d‐dimer. Of 993 patients, 141 had objectively confirmed deep vein thrombosis or pulmonary embolism. The sensitivity of SimpliRED, Accuclot and IL‐Test were 79, 90 and 87% respectively. All three d‐dimer tests gave similar negative predictive values. The SimpliRED d‐dimer was found to be less sensitive than the Accuclot or IL‐Test. When combined with pre‐test probability all three methods are probably acceptable for use in the diagnosis of venous thromboembolism.
Comparison of five platforms for enumeration of residual leucocytes in leucoreduced blood components
Authors: Van Der Meer P. F. , Gratama J. W. , Van Delden C. J. , Laport R. F. , Levering W. H. B. M. , Schrijver J. G. , Tiekstra M. J. , Keeney M. , De Wildt‐Eggen J.
Published: March 2003

The need for quality control of leucoreduction of blood products has led to the development of various methods to count low levels of residual leucocytes. We compared five platforms side‐by‐side: the Nageotte haemocytometer and four based on fluorescent staining of nuclei: two flowcytometers (Beckman Coulter, BD Biosciences) with methods based on counting beads, a volumetric flow cytometer (Partec) and the microvolumic fluorimeterImagN2000 (BD Biosciences), all according to their manufacturers' recommended methods. Analysis of double‐filtered red cell concentrates (RCCs) and platelet concentrates (PCs), spiked with various numbers of leucocytes, revealed good linearity for all methods over the range of 1·6–32·7 leucocytes/µl, all withr2 > 0·99. At the rejection level of leucocyte‐reduced blood components, i.e. 1 × 106per unit corresponding with approximately 3·3 leucocytes/µl, the Nageotte haemocytometer had low accuracy (0% for RCCs, 56% for PCs), and was relatively imprecise [coefficient of variance (CV) of 34% and 30% respectively]. The Partec flow cytometer gave good results for RCCs (accuracy 67%, CV 22%), but not for PCs (accuracy 0%, CV 25%). TheImagN2000 had an accuracy of 44% for RCCs and 89% for PCs, but the precision was variable (CV 32% for RCCs, 15% for PCs). The best results were obtained with the Beckman Coulter (RCCs: accuracy 86%, CV 13%, PCs: accuracy 67%, CV 16%), and BD Biosciences platforms (RCCs: accuracy 100%, CV 10%; PCs: accuracy 89%, CV 11%). We conclude that, at the rejection level of 1 × 106leucocytes per unit, the widely used Nageotte haemocytometer performs poorly in terms of inaccuracy and imprecision, and that both counting‐bead‐based, flow cytometric methods performed best.
Interleukin 8 and venous thrombosis: evidence  for a role of inflammation in thrombosis
Authors: Van Aken Benien E. , Reitsma Pieter H. , Rosendaal Frits R.
Published: March 2003

Summary. Elevated plasma levels of interleukin 8 (IL‐8) were previously shown to be associated with recurrent venous thrombosis. To assess the risk of venous thrombosis, IL‐8 plasma concentrations were measured in patients and control subjects of the Leiden Thrombophilia Study (LETS). This population based case–control study included 474 patients with a first deep‐vein thrombosis and 474 age‐ and sex‐matched controls. The risk of venous thrombosis for subjects with elevated IL‐8 levels (above 90th percentile of controls) compared with subjects with IL‐8 levels below the 90th percentile was increased 1·8‐fold (95%CI 1·2–2·8). Adjusted for age and sex, the odds ratio was 1·9 (95%CI 1·3–2·8). IL‐8 concentrations were weakly correlated with age, male sex, and concentrations of C‐reactive protein, factor VIII coagulation activity and homocysteine, but adjustment for these factors did not substantially affect the association between IL‐8 and venous thrombosis. Our results suggest that IL‐8 is a risk factor for venous thrombosis.
High expression of MCL1 gene related to vascular endothelial growth factor is associated with poor outcome  in non‐Hodgkin's lymphoma
Authors: Kuramoto Ken , Sakai Akira , Shigemasa Kazushi , Takimoto Yasuo , Asaoku Hideki , Tsujimoto TakAko , Oda Kenji , Kimura Akiro , Uesaka Toshihiro , Watanabe Hiromitsu , Katoh Osamu
Published: March 2003

Summary. We evaluated the level of MCL1 gene expression using quantitative reverse transcription polymerase chain reaction in lymph nodes of patients with non‐Hodgkin lymphoma (NHL). MCL1 expression in patients in complete remission (CR) was significantly lower than in patients with progressive disease (PD, P = 0·0043). The disease–free survival rate was significantly higher in patients with MCL1 levels below the median level (P = 0·007). We also found that the level of expression of MCL1 mRNA was related to that of vascular endothelial growth factor mRNA in NHL lymph nodes. Our data suggest that the MCL1 expression level could be considered a prognostic factor in NHL.
Mobilization of peripheral blood stem cells with chemotherapy and recombinant human granulocyte colony‐stimulating factor (rhG‐CSF): a randomized evaluation of different doses of rhG‐CSF
Authors: Demirer T. , Ayli M. , Ozcan M. , Gunel N. , Haznedar R. , Dagli M. , Fen T. , Genc Y. , Dincer S. , Arslan O. , Gürman G. , Demirer S. , Ozet G. , Uysal A. , Konuk N. , Ilhan O. , Koc H. , Akan H.
Published: March 2003

Summary. To date, no randomized study has compared different doses of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) following submyeloablative mobilization chemotherapy. Therefore, we evaluated the effect of different doses of rhG‐CSF following mobilization chemotherapy on yields of CD34+ peripheral blood stem cells (PBSC). Fifty patients were randomized to receive 8 (n = 25) versus 16 µg/kg/d (n = 25) of rhG‐CSF following mobilization chemotherapy. The median number of CD34+ cells collected after 8 µg/kg/d of rhG‐CSF was 2·36 × 106/kg (range, 0·21–7·80), compared with 7·99 (2·76–14·89) after 16 µg/kg/d (P < 0·001). Twenty out of 25 (80%) patients in the low‐dose and 23 out of 25 (92%) in the high‐dose rhG‐CSF arm underwent high‐dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT). Median days to white blood cell engraftment in patients mobilized with 8 µg/kg and 16 µg/kg of rhG‐CSF were 12 (10–20) and 9 (8–11) respectively (P < 0·001). There was no difference between the two groups regarding the other parameters of peritransplant morbidity: days to platelet engraftment (P = 0·10), number of red blood cell (P = 0·56) and platelet transfusions (P = 0·22), days of total parenteral nutrition requirement (P = 0·84), fever (P = 0·93) and antibiotics (P = 0·77), and number of different antibiotics used (P = 0·58). These data showed that higher doses of rhG‐CSF following submyeloablative mobilization chemotherapy were associated with a clear dose–response effect based on the collected cell yields. Based on the parameters of peritransplant morbidity, 8 µg/kg/d was as effective as 16 µg/kg/d except for a rapid neutrophil engraftment in the high‐dose arm. Therefore, in routine clinical practice, despite some advantage in the use of higher doses of rhG‐CSF, lower doses may be used for PBSC collections following chemotherapy‐based mobilization regimens in this cost‐conscious era.
Prognostic value of soluble interleukin 2 receptor levels in Langerhans cell histiocytosis
Authors: Rosso Diego A. , Roy Adriana , Zelazko Marta , Braier Jorge L.
Published: March 2003

Summary. We investigated the prognostic significance of soluble interleukin 2 receptor (sIL‐2r) levels in the pre‐ and post‐treatment serum of paediatric patients with Langerhans cell histiocytosis (LCH). Serum levels of sIL‐2r from 32 LCH patients and 14 healthy controls were determined using enzyme‐linked immunosorbent assay. The LCH patients were classified, evaluated and treated according to the Histiocyte Society's protocols. The following clinical stages were considered: single‐system disease (A) divided into single‐site (A1; n=4), multiple‐site (A2; n=9), and multisystem disease (B) without organ dysfunction (B1; n=5) and with organ dysfunction (B2; n=14). Pretreatment concentrations of sIL‐2r were markedly increased at diagnosis in LCH patients compared with controls [in pg/ml, median (range) 9200 (1124–40000) versus 610 (343–800)], P < 0·0001. Levels differed significantly between stages A [3250 (1124–11000)] and B [22750 (3400–40000)], P < 0·05, and between substages A2 and B2, P < 0·05. There was a significant correlation between clinical stages and sIL‐2r serum levels, r=0·7996 (P < 0·0001). Patients with ≥ 17500 pg/ml of sIL‐2r had a 30‐month survival of 0·417 (SEM: 0·142) compared with those with levels < 17500 pg/ml, who presented a 30‐month survival of 0·848 (SEM: 0·100) (log‐rank, P < 0·0001). In multivariate analysis, sIL‐2r levels ≥ 17500 pg/ml were found to have greater predictive strength than other well‐known prognostic factors.
Myeloma of the central nervous system: association with high‐risk chromosomal abnormalities, plasmablastic morphology and extramedullary manifestations
Authors: Fassas Athanasios B.‐T. , Muwalla Firas , Berryman Tanya , Benramdane Riad , Joseph Lija , Anaissie Elias , Sethi Rajesh , Desikan Raman , Siegel David , Badros Ashraf , Toor Amir , Zangari Maurizio , Morris Christopher , Angtuaco Edgardo , Mathew Sajini , Wilson Carla , Hough Aubrey , Harik Sami , Barlogie Bart , Tricot Guido
Published: March 2003

Summary.  Involvement of the central nervous system (CNS) by multiple myeloma, as defined by the detection of malignant plasma cells in the cerebrospinal fluid in the presence of suggestive symptoms, is considered extremely rare. We report on the characteristics of 18 such patients diagnosed and treated at the University of Arkansas over the last 10 years for an overall incidence of approximately 1%. Their evaluation revealed association of CNS involvement with unfavourable cytogenetic abnormalities (especially translocations and deletion of the chromosome 13), high tumour mass, plasmablastic morphology, additional extramedullary myeloma manifestations and circulating plasma cells. The presence of these features should alert clinicians to the possibility of CNS involvement. The outcome of these patients was extremely poor despite the use of aggressive local and systemic treatment including autologous stem cell transplants. Given this universally poor prognosis, the application of allogeneic transplants should be studied in this clinical setting.
Effect of fluconazole prophylaxis on fungal blood cultures: an autopsy‐based study involving 720 patients with haematological malignancy
Authors: Kami Masahiro , Machida Utako , Okuzumi Katsuko , Matsumura Tomoko , Mori Shin‐ichiro , Hori Akiko , Kashima Takeshi , Kanda Yoshinobu , Takaue Yoichi , Sakamaki Hisashi , Hirai Hisamaru , Yoneyama Akiko , Mutou Yoshitomo
Published: March 2003

Summary. To investigate the utility of blood culture of invasive fungal infections in patients with haematological malignancies, an autopsy survey was conducted in 720 patients who were treated between 1980 and 1999. We identified 252 patients with invasive mycosis. These included Candida (n = 94), Aspergillus (n = 91), Zygomycetes (n = 34), Cryptococcus (n = 7), Trichosporon (n = 11), Fusarium (n = 1), and unknown fungi (n = 20). Of the 94 patients with invasive candidiasis, 20 had positive blood cultures. Of the 11 patients with invasive trichosporonosis, seven had positive blood cultures. The sensitivities of blood cultures were 1·1%, 0% and 14% for detecting invasive aspergillosis, zygomycosis and cryptococcosis respectively. Multiple regression analysis showed a significant correlation between results of Candida blood cultures and some variables, including prophylactic use of absorbable antifungals (P = 0·0181) and infection by Candida albicans (P = 0·0086). The sensitivity of blood cultures decreased when patients received antifungal chemoprophylaxis. Unless these agents are inactivated in culture bottles, conventional blood cultures might produce false‐negative results.
RANK (receptor activator of nuclear factor‐κB) and RANKL expression in multiple myeloma
Authors: Roux Sophie , Meignin Véronique , Quillard Jeanine , Meduri Geri , Guiochon‐Mantel Anne , Fermand Jean‐Paul , Milgrom Edwin , Mariette Xavier
Published: March 2003

Summary.  The new members of the tumour necrosis factor (TNF) receptor‐ligand family, receptor activator of nuclear factor‐κB ligand (RANKL) and its receptor RANK, play a crucial role in osteoclast differentiation and activation. An increased expression of RANKL and/or RANK may be involved in the excessive bone resorption observed in multiple myeloma (MM). We used immunohistochemistry to study RANK and RANKL expression in bone marrow (BM) biopsies obtained at diagnosis in 15 MM patients, six patients with monoclonal gammopathy of undetermined significance (MGUS) and 10 normal BM biopsies. Plasma cells were not labelled with anti‐RANKL or anti‐RANK antibodies. In all biopsies, RANKL was expressed in endosteal bone surface, around vessels and in cells characterized by cytoplasmic expansions. These last cells did not express CD45 and were vimentin positive, corresponding to bone marrow stromal cells. Numerous stromal cells expressed RANKL in MM and MGUS specimens, with a greater expression in MM than in MGUS. Very few cells were stained with anti‐RANKL in normal BM specimens. With the anti‐RANK antibody, small mononuclear cells in the bone microenvironment were positive and were identified as erythroblast cells. In conclusion, we showed that RANKL was expressed in reticular stromal cells, with a greater intensity in myeloma specimens. These results suggest that RANKL overexpressed by bone marrow stromal cells may contribute to the high rate of bone resorption observed in MM.
Effect of inositol hexaphosphate (IP6) on human normal and leukaemic haematopoietic cells
Authors: Deliliers Giorgio Lambertenghi , Servida Federica , Fracchiolla Nicola S. , Ricci Clara , Borsotti Chiara , Colombo Gualtiero , Soligo Davide
Published: March 2003

Summary. Inositol hexaphosphate (IP6), a naturally polyphosphorylated carbohydrate, has been reported to have significant in vivo and in vitro anticancer activity against numerous tumours, such as colon, prostate, breast, liver and rhabdomyosarcomas. To confirm this activity in haematological malignancies and to characterize some of the mechanisms of IP6 action, we analysed its effects on human leukaemic cell lines and fresh chronic myelogenous leukaemia (CML) progenitor cells using a combined cellular and molecular approach. IP6 had a dose‐dependent cytotoxic effect on all of the evaluated cell lines, with accumulation in the G2M phase in two out of five cell lines tested. At the molecular level, cDNA microarray analysis after IP6 exposure showed an extensive downmodulation of genes involved in transcription and cell cycle regulation and a coherent upregulation of cell cycle inhibitors. Furthermore, IP6 treatment of fresh leukaemic samples of bone marrow CD34+ CML progenitor cells significantly inhibited granulocyte–macrophage colony‐forming unit (CFU‐GM) formation (P = 0·0062) in comparison to normal bone marrow specimens, which were not affected. No differentiating effect on HL60 cells was observed. Taken together, our results confirm the antiproliferative activity of IP6 and suggest that it may have a specific antitumour effect also in chronic myeloid leukaemias, via active gene modulation.
Human platelet antigen 1 (HPA 1) genotyping with 5′ nuclease assay and sequence‐specific primers reveals a single nucleotide deletion in intron 2 of the HPA 1a allele of platelet glycoprotein IIIa
Authors: Kjaer Killie Mette , Jaegtvik Sissel , Husebekk Anne , Skogen Bjorn
Published: March 2003

Summary.  We have established a 5′ nuclease assay (5′ NA) for human platelet antigen (HPA) 1a/b allelic discrimination. The assay is based on the simultaneous amplification and detection of the two targets in a one‐tube system. The results are read optically, immediately after termination of the polymerase chain reaction (PCR), and no post‐PCR processing is necessary. This genotyping procedure is less time‐consuming and cheaper than our conventional sequence‐specific primer PCR (SSP‐PCR), which is run as a two‐tube test, with verification of the results after electrophoresis in agarose gel. The reduction of analytical steps, simplification of the procedure and potential for automation were important advantages for our choice of system. This test system is more suitable for large‐scale testing and fits better for our screening programme for HPA 1bb determination. DNA from 1093 individuals were tested in parallel with the SSP‐PCR and the 5′ NA. One thousand and ninety‐one samples gave identical results in SSP‐PCR and 5′ NA. Upon repeated testing, two samples consistently came out as HPA 1bb in SSP‐PCR and HPA 1ab in 5′ NA. DNA sequencing revealed a defect located in an intronic area that corresponds to the consensus primer used for the SSP‐PCR HPA 1a typing.
Effects of desmopressin on thrombogenesis in aspirin‐induced platelet dysfunction
Authors: Peter Frank W. , Benkovic C , Muehlberger Thomas , Vogt Peter M. , Homann Heinz H. , Kuhnen Cornelius , Wiebalck Albrecht , Steinau Hans U.
Published: March 2003

Summary. Aspirin causes a coagulation disorder. Desmopressin has haemostatic effects by increasing the plasma levels of coagulation factor VIII and von Willebrand factor. The precise effects of desmopressin on thrombogenesis are not known. In an in vivo model, we investigated the effect of the drug on thrombus formation and platelet function after aspirin use. Male Lewis rats weighing 250–300 g were used. Four groups with 10 animals each were formed: control, aspirin, desmopressin and aspirin + desmopressin. In each animal, the femoral artery was dissected. A thrombogenic vessel injury was created by inverting a full thickness portion of the proximal edge of the incised artery into the lumen. The following parameters were measured: maximum thrombus size, time period until maximum thrombus size was reached and overall platelet function. In addition, the thrombi generated were investigated histologically. Thrombus formation time was significantly shorter with desmopressin compared with the animals treated with aspirin (P < 0·0001) and controls (P = 0·008). Maximum thrombus size was larger in the desmopressin and desmopressin + aspirin groups when compared with the group treated with aspirin only. Overall platelet function was significantly enhanced with desmopressin compared with controls (P = 0·025) and with aspirin (P < 0·0001). The differences were confirmed histologically. In conclusion, desmopressin significantly accelerates thrombus formation in aspirin‐treated animals. It can also re‐establish thrombus size after the use of aspirin. Overall platelet function is significantly increased by desmopressin.
Activation profiles of factor VIII in concentrates reflect one‐stage/chromogenic potency discrepancies
Authors: Hubbard Anthony R. , Weller Lynne J. , Bevan Sally A.
Published: March 2003

Summary.  We have investigated the possibility that differences in the profile of factor VIII (FVIII) activation, by thrombin, may help to explain the one‐stage/chromogenic potency discrepancies in two therapeutic concentrates. A Method M concentrate and a recombinant B‐domain‐deleted (B‐DD) concentrate were found to have one‐stage/chromogenic ratios of approximately 1·15 and 0·70, respectively, relative to the World Health Organization (WHO) 6th International Standard (IS) FVIII concentrate, whether pre‐diluted in FVIII‐deficient plasma or buffer (± von Willebrand factor, VWF). The activation of FVIII, by thrombin, was followed in a buffer medium (± VWF) and all three concentrates showed similar times to reach peak FVIII coagulation (FVIII:C) activity. However, despite the use of equivalent amounts of FVIII:C for all three concentrates, the B‐DD concentrate reached a higher peak level and maintained higher FVIII:C compared with the WHO 6th IS throughout the incubation period, whereas the Method M concentrate reached a lower peak level and maintained lower FVIII:C throughout the incubation period. We propose that the higher levels of FVIII:C found with the B‐DD concentrate and the lower levels with the Method M concentrate, following activation, may be reflected in the potencies obtained by the chromogenic method and may be consistent with one‐stage/chromogenic ratios of < 1·0 and > 1·0 respectively.
Dual transcription of b2a2 and b3a2 BCR–ABL transcripts in chronic myeloid leukaemia is confined to patients with a linked polymorphism within the BCR gene
Authors: Branford Susan , Hughes Timothy P. , Rudzki Zbigniew
Published: March 2003

Summary.  We propose a mechanism for dual expression of b2a2 and b3a2 BCR–ABL in chronic myeloid leukaemia (CML). We have identified a BCR allele, present in approximately 29% of the population, which includes an adenine to guanine polymorphism in the putative branchpoint of BCR intron 13. CML patients who expressed both b2a2 and b3a2 transcripts had the allele, which was also associated with alternative transcription of the normal BCR allele. We conclude that the BCR intronic polymorphism is associated with activation of a cryptic branchpoint resulting in reduced efficiency of RNA splicing and exon 14 (b3) skipping in BCR and BCR–ABL.
Natural history of juvenile haemochromatosis
Authors: De Gobbi Marco , Roetto Antonella , Piperno Alberto , Mariani Raffaella , Alberti Federica , Papanikolaou George , Politou Marianna , Lockitch Gillian , Girelli Domenico , Fargion Silvia , Cox Thimoty M. , Gasparini Paolo , Cazzola Mario , Camaschella Clara
Published: March 2003

Summary.Juvenile haemochromatosis or haemochromatosis type 2 is a rare autosomal recessive disorder which causes iron overload at a young age, affects both sexes equally and is characterized by a prevalence of hypogonadism and cardiopathy. Patients with haemochromatosis type 2 have been reported in different ethnic groups. Linkage to chromosome 1q has been established recently, but the gene remains unknown. We report the analysis of the phenotype of 29 patients from 20 families of different ethnic origin with a juvenile 1q‐associated disease. We also compared the clinical expression of 26 juvenile haemochromatosis patients with that of 93 C282Y homozygous males and of 11 subjects with haemochromatosis type 3. Patients with haemochromatosis type 2 were statistically younger at presentation and had a more severe iron burden than C282Y homozygotes and haemochromatosis type 3 patients. They were more frequently affected by cardiopathy, hypogonadism and reduced glucose tolerance. In contrast cirrhosis was not statistically different among the three groups. These data suggest that the rapid iron accumulation in haemochromatosis type 2 causes preferential tissue damage. Our results clarify the natural history of the disease and are compatible with the hypothesis that the HFE2 gene has greater influence on iron absorption than other haemochromatosis‐associated genes.
Differentiation of PLB‐985 myeloid cells into mature neutrophils, shown by degranulation of terminally differentiated compartments in response to N‐formyl peptide and priming of superoxide anion production by granulocyte–macrophage colony‐stimulating factor
Authors: Pedruzzi Eric , Fay Michèle , Elbim Carole , Muriel Gaudry  , Gougerot‐Pocidalo Marie‐Anne
Published: March 2003

Summary.  Mature blood neutrophils have a short lifespan in vitro and are not easily transfectable. We obtained terminally mature neutrophils after differentiation of immature transfectable PLB‐985 myeloid cells by treatment with dimethylformamide (0·5%), Nutridoma SP (1%) and fetal calf serum (0·5%). Maturation was shown by functional degranulation, in response to bacterial N‐formyl peptide (fMLP), of specific granules and secretory vesicle contents; the latter emerge during the last step of normal neutrophil differentiation into bone marrow. These differentiated cells also produced quantities of superoxide anion similar to those produced by blood neutrophils, in response to physiological stimuli (fMLP); in addition, the fMLP‐induced respiratory burst was primed by the proinflammatory cytokine granulocyte–macrophage colony‐stimulating factor. Thus, in our experimental conditions, PLB‐985 cells transformed into fully differentiated neutrophils capable of fine regulation by inflammatory agents. This cell model will help in the understanding of the molecular mechanisms underlying neutrophil functions.
Liver transplantation in haemophilia
Authors: Wilde Jonathan , Teixeira Paulo , Bramhall Simon R. , Gunson Bridget , Mutimer D. , Mirza Darius F.
Published: March 2003

Summary.  We report our UK single‐centre experience of liver transplantation in haemophilia patients with chronic hepatitis C (HCV) infection. Between March 1990 and March 2001, 16 patients were referred for transplant assessment and 11 (mean age 46 years: nine haemophilia A, two haemophilia B) have been transplanted. Factor concentrate replacement was administered using a continuous infusion regimen following initial bolus dosing. Concentrate infusion was discontinued at a median of 36 h (range 24–72 h) post transplant. Nine patients remain alive at a median of 5 years post transplant (6 months to 11 years). One patient died 6 years post transplant from myocardial infarction. The other patient died of liver failure as a consequence of HCV infection 3 months following a second transplant, having developed HCV cirrhosis within 1 year of receiving his initial graft. Five of the seven patients who have had annual liver biopsy surveillance have developed histological changes of HCV hepatitis at a median of 3 years post transplant (1 year to 9 years). One of these patients progressed to cirrhosis at 3 years 5 months post transplant. Two patients have shown no evidence of HCV hepatitis at 2 years 8 months and 9 years post transplant respectively. The outcome of liver transplantation in haemophilic patients is good and is associated with relatively little morbidity.
Congenital erythropoietic porphyria: identification and expression of eight novel mutations in the uroporphyrinogen III synthase gene
Authors: Shady Amr A. , Colby Brandon R. , Cunha Luis F. , Astrin Kenneth H. , Bishop David F. , Desnick Robert J.
Published: March 2003

Summary.  Mutations in the uroporphyrinogen III synthase (URO‐synthase) gene cause congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of haem biosynthesis. Molecular analysis of the URO‐synthase gene in seven unrelated CEP patients revealed eight novel mutations. These included four missense mutations (A69T, E81D, G188W and I219S), a deletion (21delG), two insertions (398insG and 672ins28) and one complex mutation (627del6ins39), as well as three previously reported mutations, C73R, T228M, and −86C→A. When the four novel missense mutations were expressed in Escherichia coli, only E81D expressed significant enzymatic activity (30% of expressed wild‐type activity), which was thermolabile. In addition, reverse transcription polymerase chain reaction studies demonstrated that E81D, which altered the penultimate nucleotide in exon 4, impaired splicing and caused about 85% exon 4 skipping. The identification and expression of these mutations provided genotype–phenotype correlations and further evidence of the molecular heterogeneity underlying this erythropoietic porphyria.
Induction and characterization of cutaneous lymphocyte antigen on natural killer cells
Authors: Tsuchiyama Junjiro , Yoshino Tadashi , Toba Ken , Harada Naoki , Nishiuchi Ritsuo , Akagi Tadaatsu , Furukawa Tatsuo , Takahashi Masuiro , Fuse Ichiro , Aizawa Yoshifusa , Harada Mine
Published: March 2003

Summary. Cutaneous lymphocyte antigen (CLA) has been reported to be expressed mainly by memory/effector T lymphocytes infiltrating inflammatory skin lesions and cutaneous T‐cell lymphoma. It has been suggested that CLA is a specific homing receptor, facilitating the T‐cell migration into skin lesions, and also an indicator of the skin‐homing T‐cell subset. In the present study, we investigated the expression of CLA in natural killer (NK) cells defined phenotypically as surface CD3– and CD56+ cells in peripheral blood. CLA was definitely expressed on CD3–CD56+ cells at a level comparable to CD3+ cells in peripheral blood of normal Japanese volunteers. After in vitro stimulation of peripheral blood mononuclear cells with interleukin 2 (IL‐2) and IL‐12, there was a significant increase in the number and percentage of CLA+ NK cells but not CLA+ T cells (P < 0·01). To analyse the characteristics of CLA expressed by NK cells, we investigated a CLA+ NK‐leukaemia cell line, NK‐YS, established from a patient with NK leukaemia/lymphoma with skin infiltration. In the in vitro study, the CLA‐expressing NK‐leukaemic cell line bound to E‐selectin‐transfected cells and was inhibited by HECA 452 antibody or neuraminidase treatment of leukaemic cells. These findings suggest that CLA expressed by NK cells is a homing receptor for the E‐selectin molecule and may explain skin infiltration by NK cells and NK lymphoma cells analogous to T cells. An NK‐cell subset expressing CLA must play an important role in host defence against microorganisms and neoplasms in skin lesions.
Simultaneous induction of matrix metalloproteinase‐9 and interleukin 8 by all‐trans retinoic acid in human PL‐21 and NB4 myeloid leukaemia cells
Authors: Shibakura Misako , Niiya Kenji , Kiguchi Toru , Shinagawa Katsuji , Ishimaru Fumihiko , Ikeda Kazuma , Namba Masayoshi , Nakata Yasunari , Harada Mine , Tanimoto Mitsune
Published: March 2003

Summary. All‐trans retinoic acid (ATRA) has been shown to induce differentiation of human acute promyelocytic leukaemia (APL) cells and eventual elimination of the malignant clone. Matrix metalloproteinase‐9 (MMP‐9) is produced by neutrophils and its expression appears to be linked with myeloid cell differentiation. We investigated effects of ATRA on MMP expression in two human myeloid leukaemia cell lines, PL‐21 and NB4. Both cells could differentiate into neutrophils after exposure to ATRA. Both the activity and antigen levels of MMP‐9 were much higher in NB4 cells than in PL‐21 cells. Stimulation with ATRA significantly increased MMP‐9 levels approximately three‐ to fivefold in both PL‐21 and NB4‐conditioned media. MMP‐9 mRNA levels increased in ATRA‐treated cells and was almost in parallel with the increase in MMP‐9 activity, suggesting that ATRA induced MMP‐9 by activating its gene expression. ATRA can induce interleukin 8 (IL‐8) in APL cells. IL‐8, chemokine for neutrophils and a potent inducer of MMP‐9, was also induced by ATRA in PL‐21 cells. However, recombinant IL‐8 did not induce MMP‐9 expression. In addition, a neutralizing antibody against IL‐8 did not inhibit ATRA‐induced MMP‐9 expression in either cell type. These observations suggest that ATRA can induce both MMP‐9 and IL‐8, but IL‐8 is not involved in ATRA‐induced MMP‐9 expression. As MMP‐9 can truncate and activate IL‐8, simultaneous induction of MMP‐9 and IL‐8 by ATRA could activate leucocytes excessively, causing the hyper‐inflammatory events in retinoic acid syndrome.
CD106 and activated‐CD29 are expressed on myelomatous bone marrow plasma cells and their downregulation is associated with tumour progression
Authors: Luque Rosario , García‐Trujillo José Antonio , Cámara Carmen , Moreno Ana , Eiras Pablo , Roy Garbiñe , Villar Luisa María , Lombardía Manuel , Brieva José Antonio , Bootello Alfredo , Roldán Ernesto
Published: March 2003

Summary.  Malignant plasma cells (PC) from multiple myeloma (MM) patients characteristically home to the bone marrow (BM). High numbers of tumour cells are found in the peripheral blood (PB) only at end‐stage disease (secondary plasma cell leukaemia, PCL) in a minority of patients. Using flow cytometric and fluorescence in situ hybridization (FISH) analysis, a high percentage of tumoral BM PC from untreated patients was found to express CD106. In addition, these cells also expressed an activated form of CD29, as determined using the CD29 activation reporter monoclonal antibody HUTS‐21. Adhesion‐binding experiments showed that CD106+‐activated CD29+ BM PC from these patients adhered to fibronectin (FN) in a CD29/CD49d‐dependent manner. In contrast, marrow PC from progressive patients and BM or circulating malignant cells from secondary PCL patients expressed lower levels or were negative for CD106 and activated CD29, respectively, with a decreased or zero ability to adhere to FN. The expression of constitutive CD29 and CD49d, however, was similar during disease progression. We conclude that BM myelomatous cells co‐express CD106 and a functionally active form of CD29. Moreover, our results suggest that the loss of expression and/or function of these antigens are associated with the progression of MM and may explain the exit of tumoral cells from the BM.
Major histocompatibility complex abnormalities in non‐Hodgkin lymphomas
Authors: Drénou Bernard , Le Friec Gaelle , Bernard Marc , Pangault Céline , Grosset Jean‐Marc , Lamy Thierry , Fauchet Renée , Amiot Laurence
Published: March 2003

Summary. An optimal antitumoral immune response requires the participation of both CD8 and CD4 T lymphocytes, which are activated by peptide antigen presentation via human leucocyte antigen (HLA) class I and class II molecules respectively. Loss of HLA molecules has been observed in different malignancies, and provides a mechanism for escape from immune surveillance. Furthermore, HLA‐G, a class Ib molecule, is considered to be an immune tolerance‐inducing molecule. HLA‐G expression on tumour cells could provide a further mechanism for immune escape. To determine the frequency and the pattern of HLA defects in non‐Hodgkin lymphomas (NHL), HLA expression was prospectively studied in 614 NHL cases, using flow cytometry. Furthermore, HLA‐G expression was tested in 50 cases, including 20 cases selected on the basis of their defective HLA class I expression. In 64 cases (10·4%), lymphomatous cells exhibited lower HLA class I mean fluorescence intensity compared with reactive cells. Their characteristics were (1) the diversity of histological entities; (2) the significant frequency of relapse or transformation; (3) the increased incidence of high‐grade NHL compared with low‐grade; and (4) the severity of the class I defect in 50% of the cases, mainly in high‐grade NHL. A defect in HLA‐DR expression was always associated with a severe class I defect (12 cases; 2%). The HLA‐G protein was detected in three class I defective cases. These HLA alterations frequently appeared as a secondary event at relapse or at transformation, suggesting a direct role in lymphomagenesis.
Influence of cell cycling and cell division on transendothelial migration of CD34+ cells
Authors: Yong Kwee L. , Fahey Anne , Pizzey Arnold , Linch David C.
Published: March 2003

Summary. The migration of haemopoietic stem and progenitor cells across endothelium lining bone marrow sinuses is a critical first step in the homing and successful engraftment of these cells. We have previously shown that freshly isolated mobilized peripheral blood CD34+ cells adhere to the endothelial surface but do not transmigrate unless activated by growth factors. The aim of this work was to examine the relationship between cell cycle progression, cell division and migration across endothelium. We now show that the enhanced migration of cytokine‐activated cells is selective for cells which are in G0G1 phase of the cell cycle. Thus, the transmigrated population of CD34+ cells was enriched for cells in G0G1 phase, and sorted cells in G0G1 migrated more efficiently than those in S+G2M. Conversely, cells in S+G2M were more adherent to endothelium, a finding that may explain their reduced migration. Using the cytoplasmic dye, carboxyfluorescein diacetate succinimidyl ester, to track the divisional kinetics of CD34+ cells, we found that migration occurred preferentially in non‐divided cells. Thus, although CD34+ cells require cytokine activation in order to migrate, cell division is not required for transmigration, which occurs optimally before cells enter S phase. The superior migratory ability of CD34+ cells in G0G1 phase of the cell cycle may have important implications for the homing and engraftment of ex vivo expanded cells.
The uptake of recombinant Factor VIII in the Netherlands
Authors: Zwart‐van Rijkom J. E. F. , Plug I. , Rosendaal F. R. , Leufkens H. G. M. , Broekmans A. W. ,   the Study Group Haemophilia in The Netherlands
Published: March 2003

Summary. In comparison with other biotechnology substitutions, the adoption of recombinant Factor VIII (rFVIII) has been relatively slow. We sent a postal questionnaire to all Dutch haemophilia patients and haemophilia‐treating physicians, to determine which factors predict whether a patient uses plasma‐derived FVIII (pdFVIII) or rFVIII and to investigate patients’ and doctors' opinions on both products. Fifty‐six per cent of patients received rFVIII. This percentage varied widely between centres. Only one doctor would choose to use pdFVIII if he suffered from haemophilia A himself, and 74% would choose to use rFVIII. Younger patients, those not infected with the human immunodeficiency virus or hepatitis C, and those who did not have family members who used pdFVIII switched more often from pdFVIII to rFVIII. Patients who rated themselves as innovative, who had family members who used rFVIII, and those who were treated in a large haemophilia treatment centre were also more likely to have switched. For physicians and patients alike, the respondents generally did not see large differences between rFVIII and pdFVIII, except for the risk of infections and the knowledge of long‐term effects (both larger for pdFVIII). Although haemophilia patients represent one of the most empowered patient groups, physicians appear to have been influential in choosing between pdFVIII and rFVIII.
Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome
Authors: Ladhani Shamez , Lowe Brett , Cole Andrew O. , Kowuondo Ken , Newton Charles R. J. C.
Published: March 2003

Summary. Little is known about the changes in white blood cells and platelets in children with falciparum malaria in endemic areas. We measured the white cell count (WCC) and platelets of 230 healthy children from the community, 1369 children admitted to hospital with symptomatic malaria, and 1461 children with other medical conditions. Children with malaria had a higher WCC compared with community controls, and leucocytosis was strongly associated with younger age, deep breathing, severe anaemia, thrombocytopenia and death. The WCC was not associated with a positive blood culture. In children with malaria, high lymphocyte and low monocyte counts were independently associated with mortality. A platelet count of less than 150 × 109/l was found in 56·7% of children with malaria, and was associated with age, prostration and parasite density, but not with bleeding problems or mortality. The mean platelet volume was also higher in children with malaria compared with other medical conditions. This may reflect early release from the bone marrow in response to peripheral platelet destruction. Thus, leucocytosis was associated with both severity and mortality in children with falciparum malaria, irrespective of bacteraemia, whereas thrombocytopenia, although very common, was not associated with adverse outcome.
Macrophage inflammatory protein‐1 alpha is produced by human multiple myeloma (MM) cells and its expression correlates with bone lesions in patients with MM
Authors: Uneda Shima , Hata Hiroyuki , Matsuno Fumihiko , Harada Naoko , Mitsuya Yumi , Kawano Fumio , Mitsuya Hiroaki
Published: March 2003

Summary. Macrophage inflammatory protein‐1 alpha (MIP‐1α) is a chemokine primarily associated with bone absorption. We examined whether MIP‐1α was produced by purified fresh tumour cells isolated from bone marrow samples from 31 multiple myeloma (MM) patients. High levels of MIP‐1α were found in supernatants of myeloma cell cultures. Immunohistochemical staining showed MIP‐1α in the cytoplasm of myeloma cells. MIP‐1α mRNA expression was detected in 18 of 31 patients. Bone lesions were seen in 16 of the 18 MIP‐1α‐positive patients but only in six of the 13 MIP‐1α‐negative patients (P = 0·0097,χ2‐test). The data indicate that MIP‐1α is produced by myeloma cells and possibly plays a role in the pathogenesis of bone lesions in MM patients.
Allogeneic haematopoietic stem cell transplantation for the treatment of adult T‐cell leukaemia/lymphoma
Authors: Kami Masahiro , Hamaki Tamae , Miyakoshi Shigesaburo , Murashige Naoko , Kanda Yoshinobu , Tanosaki Ryuji , Takaue Yoichi , Taniguchi Shuichi , Hirai Hisamaru , Ozawa Keiya , Kasai Masaharu
Published: March 2003

Summary. The feasibility of allogeneic haematopoietic stem‐cell transplantation (allo‐HSCT) in 11 patients with adult T‐cell leukaemia/lymphoma (ATL) (6 acute, 4 lymphoma, 1 chronic type) was evaluated. The preparative regimens (9 conventional, 2 reduced‐intensity) were tolerable. Five patients developed acute graft‐versus‐host disease (GVHD), and three, extensive chronic GVHD. All 10 patients who survived > 30 d achieved complete remission. Estimated 1‐year overall and disease‐free survival rates were 53 ± 30% and 45 ± 29% respectively. Four patients remain alive and disease‐free at a median follow‐up of 25 months. The others died of transplantation‐related complications. This pilot study suggests that allo‐HSCT in ATL should be evaluated further.
Bone marrow transplantation for β‐thalassaemia major: the UK experience in two paediatric centres
Authors: Lawson Sarah E. , Roberts Irene A. G. , Amrolia Persis , Dokal Inderjeet , Szydlo Richard , Darbyshire Philip J.
Published: March 2003

Summary. Stem cell transplantation (SCT) remains the only cure for thalassaemia major. Recent advances in medical treatment make it even more important that accurate information is available regarding outcome of SCT in relevant patient populations in order to guide informed decisions regarding the most appropriate treatment for individual thalassaemia patients. We report the results of 55 consecutive first related allogeneic bone marrow transplants (BMT) for children with β‐thalassaemia major performed in two UK paediatric centres over 10 years. Between February 1991 and February 2001, 55 children underwent 57 allogeneic BMT. The median age at BMT was 6·4 years and the majority of patients (73%) originated from the Indian subcontinent. Using the Pesaro risk classification, 17 patients were class 1, 27 were class 2 and 11 were class 3. Actuarial overall survival and thalassaemia‐free survival at 8 years were 94·5% (95% CI 85·1–98·1) and 81·8% (95% CI 69·7–89·8) respectively. Despite the majority of patients being in class 2 or 3, transplant‐related mortality was low (5·4%). The principal complication was graft rejection accompanied by autologous reconstitution that occurred in 13·2% of transplants. Following modification of the conditioning regimen in 1993, the rejection rate fell to 4·6% and remained low. Acute graft‐versus‐host disease (GVHD) of grade II–IV occurred in 31% and chronic GVHD in 14·5%. These data compare favourably with survival with medical treatment for thalassaemia major and suggest that allogeneic BMT remains an important treatment option for children with β‐thalassaemia major, particularly when compliance with iron chelation is poor.
Idiotype‐pulsed dendritic cells are able to induce antitumoral cytotoxic CD8 cells in chronic lymphocytic leukaemia
Authors: Vuillier Françoise , Maloum Karim , Thomas Elaine K. , Magnac Christian , Dumas Gérard , Payelle‐Brogard Béatrice , Oppezzo Pablo , Dighiero Guillaume , Scott‐Algara Daniel
Published: March 2003

Summary. Idiotypic structures of immunoglobulins from malignant B cells constitute tumour‐specific antigens, though the function of immunoglobulin‐specific CD8+ T cells in disease control and rejection remains unclear. We have studied five cases of B chronic lymphocytic leukaemia patients affected with indolent (three patients) or aggressive (two patients) disease. We showed that CD8+ T cells with major histocompatibility complex class I‐restricted cytotoxicity against autologous tumour B cells could be generated following repeated stimulations with idiotype‐pulsed dendritic cells in vitro. CD8+ T‐cell lines were able to upregulate CD69 expression and to release interferon (IFN)‐γ upon contact with the autologous B cells, though cytolytic activity was only substantiated for patients with indolent disease. The failure of cytolytic activity in patients with aggressive disease may be explained by a skewed maturation of memory CD8 cells.