bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene‐transfer to murine lympho‐haemopoietic stem cells
Authors: Bernad Antonio , Varas Florencio , Gallego Jesúas M. , Almendral Jose M. , Bueren Juan A. 
Published: March 2008

SummaryAn efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro, expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5‐FU treatment were incubated under IL‐3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU‐GM and CFU‐S12 progenitor pools was observed (130‐ and 15‐fold, respectively), with no apparent impairment in long‐term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU‐GM susceptibility to the infection by supernatants containing a Moloney‐derived ecotropic retroviral vector carrying the neo’gene. The designed protocol thus involved the infection of freshly harvested 5‐FU‐treated bone marrow, followed by expansion under IL‐3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU‐GM and 50 CFU‐S12 per 105 bone marrow cells, free from non‐genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neo’gene and expressing resistance to G418 5 months after the transplantation indicates that long‐term lympho‐haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self‐renewal and differentiation properties. This gene‐transfer methodology may improve the development of gene therapy protocols where the purging of non‐transduced precursors would guarantee a lasting and uniform expression of exogenous genes.
Optimization of the dilute prothrombin time for the detection of the lupus anticoagulant by use of a recombinant tissue thromboplastin
Authors: Arnout J. , Vanrusselt M. , Huybrechts E. , Vermylen J. 
Published: March 2008

SummaryThe dilute prothrombin time (dPT) is a widely accepted sensitive screening test for the lupus anticoagulant (LA). In general, Simplastin (Organon Teknika), a rabbit brain thromboplastin, diluted 1/500, is used in this test. Recently, human tissue thromboplastin obtained by recombinant DNA technology has become available and we have evaluated the usefulness of one such preparation, Innovin (Dade), for the detection of the LA. dPTs, using several dilutions of Innovin were determined on plasmas from 18 normal individuals and 15 patients with a well‐documented LA. The dPT ratios, calculated as the individual result divided by the mean normal, were statistically compared. Innovin in dilutions from 1/100 onwards was found to be significantly more responsive to the LA than Simplastin (P < 0.05). Intraassay coefficients of variation (CVs) ranged from 1.05% to 14.8% with Innovin, versus 2.7%‐24.3% with Simplastin (P < 0.05); inter‐assay CVs ranged from 5.6%‐11.8% with Innovin, versus 4.2%‐33.8% with Simplastin (P < 0.001). Analysis of 316 consecutive plasma samples from nonanticoagulated patients, on which a LA determination was requested, showed a 100% sensitivity and a 96% specificity of the dPT performed with Innovin, as compared to 81% and 93% respectively for the dPT using Simplastin.
Non‐invasive assessment of tissue iron overload in the liver by magnetic resonance imaging
Authors: Jensen P. D. , Jensen F. T. , Christensen T. , Ellegaard J. 
Published: March 2008

Summary. We investigated the clinical usefulness of a standard magnetic resonance imaging (MRI) system for non‐invasive determination of the liver iron concentration in 38 patients with iron overload and 15 normal controls by measurement of the signal intensity ratio between liver and skeletal muscle (SIR). However, SIR was found dependent on the applied repetition time (TR) of the MRI system, which led us to investigate this relationship in autopsy material of liver and muscle tissue specimens with various iron content. Based on these results, adjustment of SIR measurements to a constant value of TR was achieved. By use of this technique we found a close correlation between MRI and chemically determined liver iron concentration (r2= 0.98) as well as the serum ferritin concentration (r2= 0.86). The reproducibility was sufficiently good for the use of MRI in the follow‐up of iron reductive treatment. The use of iron store parameters in serum was found insufficient as indicators of endpoint for venesection therapy, if 20 μmol Fe/g dry weight was applied as the upper reference limit of the liver iron concentration.It is concluded that MRI based on SIR measurements offers a precise and reproducible non‐invasive method for the determination and follow‐up of iron overload within a wide range of liver iron concentrations. Our findings may increase the clinical use of MRI in haematological patients with iron overload.
Further characterization of platelet‐aggregating cysteine proteinase activity in thrombotic thrombocytopenic purpura
Authors: Consonni R. , Falanga A. , Barbui T. 
Published: March 2008

SUMMARY. Circulating platelet‐aggregating activities have been described by several authors. In a previous study we have confirmed the presence in serum and plasma from patients with thrombotic thrombocytopenic purpura (TTP) of a platelet aggregating cysteine proteinase (PACP). This activity differed in that, among the class of thiol enzymes, it showed characteristics of a lysosomal cathepsin rather than of a cytoplasmic calpain.To further investigate the enzymatic properties of this PACP we have designed a study to evaluate the dependence of the activity on Ca++. Calcium‐dependence is a property of calpains but not of cathepsins. A proteolytic assay was set up and conducted with and without Ca++. The release of acid‐soluble peptide from denatured human globin promoted by TTP samples and standards was fluorimetrically measured. The results show that TTP samples were equally active with and without Ca++ similar to standard palpain (the cathepsin B‐like proteinase), whereas standard calpain only acted with Ca++.An inhibition study performed by the proteolytic assay confirmed the same pattern of sensitivity of PACP to a series of specific cystein proteinase inhibitiors previously shown by the proaggregating assay. The enzymatic behaviour of PACP of TTP resembled a lysosomal (cathepsins) rather than a cytoplalsmic (calpains) cysteine proteinase.
Determination of plasma tissue factor antigen and its clinical significance
Authors: Koyama Takatoshi , Nishida Kenji , Ohdama Shinichi , Sawada Megumi , Murakami Naomi , Hirosawa Shinsaku , Kuriyama Renjiro , Matsuzawa Kimihiko , Hasegawa Ryoichi , Aoki Nobuo 
Published: March 2008

SUMMARY. To investigate the clinical significance of determination of plasma tissue factor (TF) antigen, we have developed a highly sensitive enzyme‐linked immunosorbent assay (ELISA) for plasma TF, using two different monoclonal antibodies against TF apoprotein, 6B4 (catching antibody) and 5G9 (detecting antibody), and tetramethyl benzidine/H2O2 as substrates. Titration curves of recombinant human TF in buffer containing Triton X‐100 were linear within the range from 50 to 2000pg/ml. The total assay time was 3h. Ultracentrifugation and immunoblot analysis indicated that human plasma and urine contained 50 000g sedimentable and non‐sedimentable forms of TF, both of which were detected by our ELISA method.Plasma and urine concentrations of TF in healthy subjects and patients with various diseases were measured by the ELISA method. In healthy subjects, plasma and urinary TF levels were found to be 149± 72pg/ml (n = 30) and 175±60pg TF/urine creatinine mg (n = 95). respectively. TF was increased in plasma of patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, vasculitis associated with collagen diseases, diabetic microangiopathy and chronic renal failure receiving haemodialysis, but not in the plasma of endotoxaemic patients without DIC. The plasma TF/serum creatinine ratio did not show a positive correlation. Measurement of TF antigen in plasma may be useful for evaluating the endothelial damage and cell destruction in TF‐containing tissues.
Molecular detection of clonally rearranged cells in peripheral blood progenitor cell harvests from multiple myeloma patients
Authors: Bird J. M. , Bloxham D. , Samson Diana , Marcus R. E. , Russell N. H. , Kelsey S. M. , Newland A. C. , Apperley J. F. 
Published: March 2008

Summary. Peripheral blood progenitor cells (PBPC) are increasingly used for autologous reconstitution following high‐dose chemotherapy in multiple myeloma but it is unclear whether these cells are less likely to be contaminated with malignant cells than bone marrow (BM). We have investigated this using immunoglobulin heavy‐chain (IgH) gene fingerprinting, a polymerase chain reaction based technique with a sensitivity of 0.1–0.01% (10‐3 ‐ 10‐4). We have looked for patient‐specific IgH rearrangements in leukapheresis samples from eight myeloma patients undergoing PBPC harvest. Seven were in first remission (six partial, one complete) and one in second complete remission. Mobilization of PBPC was accomplished using cyclophosphamide (4 or 7 mg/m2) and rhG‐ or GM‐CSF. Between two and five leukaphereses were performed in each patient. Patient‐specific IgH rearrangements were identified in diagnostic BM in all patients and bands of identical size were found in one or more leukaphereses from 6/8 patients. Overall, 14/32 leukaphereses were shown to be contaminated. Two patients who showed contamination of at least one PBPC harvest had BM harvests in which contaminating cells were not detectable, suggesting that PBPC are not necessarily less likely to be contaminated than marrow stem cells.These results indicate that PBPC harvests from the majority of myeloma patients are likely to contain contaminating cells. Further studies are needed to determine whether these cells are clonogenic and whether they contribute to relapse.
Serum erythropotietin and serum transferrin receptor levels in aplastic anaemia
Authors: Schrezenmeier Hubert , Noe Gudrun , Raghavachar Aruna , Rich Ivan N. , Heimpel Hermann , Kubanek Bernhard 
Published: March 2008

Summary. Serum erythropoietin (EPO) and soluble transferrin receptor levels were serially measured in 74 patients with aplastic anaemia (AA). As control groups we investigated healthy controls (n = 24) and patients with iron‐deficiency (n = 23) or haemolytic anaemia (n = 16). There was a significant negative correlation of log EPO on haematocrit both in AA patients and in the anaemic control group. However, for the same degree of anaemia, log EPO levels in AA were significantly higher than in iron‐deficiency or haemolytic anaemia. EPO levels at diagnosis did not correlate with severity of aplastic anaemia, nor did they predict outcome after immunosuppression. During immunosuppressive treatment of AA with anti‐thymocyte globulin and cyclosporine A, EPO levels were significantly lower compared with pre‐treatment values without a corresponding change in haematocrit. This impaired EPO response to anaemia during immunosuppression might affect recovery of erythropoiesis. In AA patients, EPO levels declined with haemopoietic recovery. However, compared with normal controls, EPO levels in remission patients were still higher with respect to their haematocrit.Results of this study argue against the model of a simple feedback regulation of EPO via hypoxic anaemia. Our data support the hypothesis that cytokines and the erythropoietic progenitor pool are involved in the regulation of EPO production. The results illustrate that serial measurements of EPO along with therapeutic interventions are necessary to identify patients who might benefit from treatment with exogenous recombmant human EPO.
Accessory cells mediate hairy‐cell proliferation by mechanism (s) involving both adhesion and TNFα secretion
Authors: Till K. J. , Cawley J. C. 
Published: March 2008

Summary. The mechanisms responsible for hairy‐cell (HC) growth both in vitro and in vivo are still unclear. In a recent study we showed that monocytes/macrophages induce HC proliferation in vitro. The purpose of the present paper is to examine the specificity of this accessory cell effect and to establish the mechanism(s) involved.We demonstrate that the effect is not confined to monocytes/macrophages but is also potentially seen with a range of other cell types. However, at low accessory cell:HC ratios (< 1:20) only human umbilical vein endothelial cells (HUVEC) and macrophages induce HC proliferation. We suggest that these observations are of pathophysiological significance in relation to the close association frequently observed between HCs and endothelial cells/macrophages in the liver and spleen of patients with hairy‐cell leukaemia (HCL).Regarding the mechanisms of the accessory cell effect, we show that both soluble factors and cell contact are important. A blocking anti‐TNFα antibody abrogated the HC proliferation induced by HUVEC supernatant. indicating the involvement of this cytokine. Interaction of HCs with HUVEC via CD11b and 11c leucocyte integrins was shown to be important in the contact effect.Out demonstration of the involvement of both cytokines and cell contact in HC proliferation is in accord with what is already known about the control of B‐cell growth and differentiation. More specifically, our results suggest that TNFα and interaction with endothelial cells/macrophages via leucocyte integrins are involved in the proliferation of late B‐cells of the maturational stage represented by HCs.
Establishing the incidence of myelodysplastic syndrome
Authors: Williamson P. J. , Kruger A. R. , Reynolds P. J. , Hamblin T. J. , Oscier D. G. 
Published: March 2008

Summary. We studied the incidence of myelodysplastic syndrome (MDS) in a population served by one district general hospital over a 10‐year period (1981‐90). Cases were sought through investigation of individuals presenting with suggestive peripheral blood findings. 279 new cases were diagnosed in a population rising from 203000 to 226 000 over the study period. The observed incidence rate did not change over the study period. The crude incidence rate was 12.6/100000/year. Age‐specific incidence rates per 100000 per year were 0.5 for age < 50, 5.3 for age 50‐59, 15 for age 60‐69, 49 for age 70‐79, and 89 for age 80+. A cohort study of one large general practice revealed several previously unknown and asymptomatic cases, indicating that the observed figures for the population overall may still underestimate the real incidence.We conclude that MDS is more common than reported in previous studies and that incidence continues to rise into very old age. Difficulties in case ascertainment hamper reliable estimates of true incidence.
A cross‐over pharmacokinetic and thrombogenicity study of a prothrombin complex concentrate and a purified factor IX concentrate
Authors: Thomas D. P. , Hampton K. K. , Dasani H. , Lee C. A. , Giangrande P. L. F. , Harman C. , Lee M. L. , Preston F. E. 
Published: March 2008

Summary. A prospective cross‐over study was carried out on 19 patients with haemophilia B, comparing the pharmacokinetics of a purified factor IX concentrate prepared by metal chelate affinity chromatography (9MC) with a conventional three‐factor prothrombin complex concentrate (9A). The highly purified factor IX concentrate was shown to have a half‐life comparable to the PCC; the in vivo recovery of the purified concentrate was significantly greater than that of the complex (P < 0.01). The 20% change in the value of the International Standard for Factor IX Concentrate, introduced in 1988, might have been expected to lower the recovery values. However, the in vivo recovery for both concentrates was somewhat higher than reported previously, particularly in the older literature.In nine patients, serial assays for fibrinopeptide A, prothrombin fragment FI+2 and thrombin‐antithrombin complexes (TAT) were performed to assess the potential thrombogenicity of the two concentrates. Evidence was obtained that there was significantly less activation of coagulation following administration of purified factor IX (9MC), as compared to the activation that occurred after the PCC.
Factor IX gene haplotypes in Brazilian Blacks and characterization of unusual Ddel alleles
Authors: Figueiredo M. S. , Bowen D. J , Silva W. A. , Zago M. A. 
Published: March 2008

SummaryAnalysis of factor IX gene polymorphisms is considered the best approach for prenatal diagnosis and carrier detection of haemophilia B when the identification of the gene mutation is not possible. Studies involving factor IX gene polymorphisms in Black populations are scarce and essentially restricted to the North‐American Black population whose composition is substantially different from that of the Brazilian and presumably other Black populations of South America. In this paper we report the analysis of eight factor IX gene polymorphisms in Brazilian Blacks: 5’ BamHI, DdeI, intron 2 BamHI, XmnI, TaqI, MspI, MnII and HhaI. Characterization of the VNTR‐like Ddel polymorphism revealed six different alleles: B, AB, A2B, A2B2, A3B and A5, the last being described here for the first time. The 5’ BamHI, DdeI, MspI and Hhal polymorphisms showed the highest heterozygosities (0.40‐0.50) and are in linkage equilibrium with one another. 19 complete haplotypes could be identified in this population. Based on the results we propose a systematic strategy for carrier detection and prenatal diagnosis of haemophilia B in this population. The combined analysis of four polymorphisms (5’ BamHI, HhaI, MspI and DdeI) provided an informative genetic marker in 85% of the females. The use of all eight polymorphisms allows information in 95% of females. Additionally, differences in gene frequencies and haplotype distribution suggest dissimilarities in factor IX gene polymorphisms between the Brazilian and the North‐American Black populations.
Detection of trisomy 12 in chronic lymphocytic leukaemia: comparison of a polymerase chain reaction based technique with fluorescence in situ hybridization
Authors: Reining G. , Clodi K. , König M. , Geissler K. , Haas O. A. , Mannhalter C. 
Published: March 2008

SummaryTrisomy 12 is the most frequent chromosomal aberration in chronic lymphocytic leukaemia (CLL) and seems to indicate a poor prognosis. To detect this abnormality we tested the applicability of the polymerase chain reaction (PCR) and compared it to the current standard fluorescence in situ hybridization (FISH). Two DNA regions containing variable numbers of tandem repeats (VNTR) located on (a) the long and (b) the short arm of chromosome 12 were chosen for PCR analysis. 8/72 patients (11%) were trisomy 12 positive compared to 16% by FISH. Chromosomal imbalances were only detected by PCR if at least 20% of the cells carried the numerical aberration.
Familial discrepancy between the one‐stage and two‐stage factor VIII methods in a subgroup of patients with haemophilia A
Authors: Duncan Elizabeth M. , Duncan Bruce M. , Tunbridge Lynn J. , Lloyd John V. 
Published: March 2008

SummaryA higher result for plasma factor VIII:C measured by the one‐stage as compared with the twostage method has been described in some patients with haemophilia A or with von Willebrand's disorder. We used both methods to measure FVIII:C in 95 patients with haemophilia A. The results were equivalent in all 21 patients with severe haemophilia (16 families) and in 45 of the patients with mild or moderate haemophilia (18 families). However, the results were discrepant (FVIII: C by one‐stage assay 2‐7‐fold higher than by two‐stage assay) in the other 29 patients with mild or moderate haemophilia (12 other families). For each patient with discrepant FVIII: C results the classification was the same for all other affected members of his family. In some families with haemophilia A the gene defect leads to a discrepancy between the one‐stage and twostage FVIII: C results and may be more widespread than previously recognized.
Hypercalcaemia in B cell chronic lymphocytic leukaemia
Authors: Fain Olivier , M'Selmi Abdellatif El , Dosquet Christine , Meseure Didier , Lejeune Francloise , Garel Jean‐Michel , Thomas Michel 
Published: March 2008

SummaryHypercalcaemia is common in some lymphoproliferative disorders such as myeloma or T‐cell leukaemialymphoma. but is rarely described in B cell chronic lymphocytic leukaemia (BCLL). We report the case of a patient with BCLL, hypercalcaemia and osteolytic bone lesions. Parathyroid hromone‐related protein (PTHrP) mRNA was identified by Northern blot analysis of liver. spleen and lymph node tumour samples. Serum levels of tumour necrosis factor alpha (TNFα) were increased.
Mutations in the PIG‐A gene causing partial deficiency of GPI‐linked surface proteins (PNH II) in patients with paroxysmal nocturnal haemoglobinuria
Authors: Bessler Monica , Mason Philip J. , Hillmen Peter , Luzzatto Lucio 
Published: March 2008

SummaryParoxysmal nocturnal haemoglobinuria (PNH) is due to the absence or marked reduction of glycan phosphatidylinositol (GPI)‐anchored proteins on the surface of blood cells. Affected patients may have a population of red blood cells that are completely deficient (PNH III) or partially deficient (PNH II) in these proteins, or they may have both. PNH III has recently been shown to be due, in all cases examined, to a somatic mutation in the PIG‐A gene, whose product is required for an early step in GPI anchor synthesis. We now show that two patients with PNH II cells also have somatic mutations of the same gene: these produce a partial rather than a total loss of PIG‐A function.
The in vitro and in vivo effect of recombinant interferon α‐2a on circulating haemopoietic progenitors in polycythaemia vera
Authors: Castello G. , Lerza R. , Cerruti A. , Cavallini D. , Bogliolo G. , Pannacciulli I. 
Published: March 2008

SummaryIn four patients with polycythaemia vera (PV) who received interferon alpha (IFN‐α) (3 MU subcutaneously three times a week) for 5 months, peripheral blood levels of granulocyte‐macrophage colony‐forming units and erythroid burst‐forming units were assessed monthly. Circulating progenitors significantly decreased throughout the treatment period. Moreover, we observed an inhibitory activity of IFN‐α on haemopoietic progenitor cells (HPC) from patients with PV in vitro.The data presented confirm previous research which has shown an inhibitory effect of IFN‐α on HPC from patients with myeloproliferative disorders.Significant improvement in the patients’clinical and haematological conditions should encourage larger studies on IFN‐α administration in PV patients.