bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors required for bone marrow stromal fibroblast colony formation in vitro
Authors: Kuznetsov Sergei A. , Friedenstein Alexander J. , Gehron Robey Pamela
Published: November 2003

Marrow stromal fibroblasts (MSFs) are essential for the formation of the haemopoietic microenvironment and bone; however, regulation of MSF proliferation is poorly understood. MSF colony formation was studied in primary mouse and human marrow cell cultures. After a brief exposure to serum, MSF colony formation occurred in the absence of both serum and non‐adherent marrow cells, if medium conditioned by marrow cells was present (serum‐free conditioned medium, SF‐CM). In mouse and human cultures stimulated to proliferate by SF‐CM, neutralizing antibodies against PDGF, TGF‐β, bFGF and EGF specifically suppressed MSF colony formation. The degree of supression was species‐dependent, with the most profound inhibition achieved in mouse cultures by anti‐PDGF, anti‐bFGF and anti‐EGF, and in human cultures by anti‐PDGF and anti‐TGF‐β. Serum‐free medium not conditioned by marrow cells (SFM) did not support MSF colony formation. In mouse cultures in SFM, human recombinant bFGF and bovine natural bFGF were able to partially substitute for the stimulating effect of SF‐CM. Other growth factors, including TGF‐β1, TGF‐β2, PDGF, EGF, IL‐6, IGF‐I and IGF‐II, showed no activity when tested alone. In human cultures in SFM, none of the growth factors, alone or in combination, stimulated MSF colony formation. Mouse and human MSFs grown in SF‐CM formed bone and a haemopoietic microenvironment when transplantated into immunodeficient mice in vivo, and therefore were functionally equivalent to MSFs generated in the presence of serum. These data indicate that stimulation of the initial proliferation of an MSF precursor cell is complex, and requires participation of at least four growth factors: PDGF, bFGF, TGF‐β and EGF. In addition, mouse and human MSF precursor cells have different requirements for each of the growth factors.
A frequent mutation in the protein S gene results in cryptic splicing
Authors: Mustafa Stefan , Pabinger Ingrid , Mannhalter Christine
Published: November 2003

Protein S is a vitamin K dependent coagulation inhibitor. One of several defects in the protein S gene (PROS1) associated with hereditary deficiency is a G → A transition at position 5 of the splice donor in intron J. Although the mutation has been reported to cause allelic exclusion, we demonstrated low amounts of alternatively spliced ectopic PROS1 transcripts in carriers of this mutation. Sequencing of mutant RNA indicated the use of a cryptic splice site upstream of the common splice donor. The use of the cryptic splice site results in the deletion of 32 nucleotides at the 3′ end of exon 10. The new reading frame contains several premature termination signals.
Which compartments are involved in Philadelphia‐chromosome positive chronic myeloid leukaemia? An answer at the single cell level by combining May‐Grünwald‐Giemsa staining and fluorescence in situ hybridization techniques
Authors: Haferlach Torsten , Winkemann Martin , Nickenig Christina , Meeder Marlies , Ramm‐Petersen Lotte , Schoch Robert , Nickelsen Maike , Weber‐Matthiesen Klaus , Schlegelberger Brigitte , Schoch Claudia , Gassmann Winfried , Löffler Helmut
Published: November 2003

Chronic myeloid leukaemia (CML) is believed to represent a stem cell disorder involving all three cell lineages. The typical chromosomal aberration, the Philadelphia chromosome, is the translocation (9;22)(q34;q11). Several studies with cytogenetics, fluorescence in situ hybridization (FISH), or polymerase chain reaction have investigated the presence of the t(9;22) in different cell compartments. However, questions still remain. In six cases of CML we combined the standard May‐Grünwald‐Giemsa staining with FISH at the single‐cell level and were able to demonstrate that not only all maturation stages of granulopoiesis, erythropoiesis, and megakaryocytes, but also plasma cells, eosinophils, basophils and monocytes carried the Philadelphia chromosome in 53–98% of samples. Using immunological identification of single cells we were able to demonstrate that the t(9;22) is detectable in 34% of CD3‐positive T lymphocytes, in 32% of CD19‐positive B lymphocytes, and in 82% of CD34‐positive precursor cells. The results give new insight into the biology of CML and may have implications for future therapeutic strategies.
Long‐term clinical outcome of B‐cell chronic lymphocytic leukaemia patients in clinical remission phase evaluated at phenotypic level
Authors: Brugiatelli M. , Claisse J. F. , Lenormand B. , Morabito F. , Callea V. , Malloum K. , Chevret S. , Binet J. L. , Dighiero G. , Travade P.
Published: November 2003

This retrospective study aimed to evaluate the long‐term prognostic impact of phenotypic remission in B‐cell chronic lymphocytic leukaemia (CLL) patients who have achieved clinical, haematological and bone‐marrow complete remission (CR) after conventional chemotherapy. The clinical and phenotypic data of 77 CLL patients in CR with a median follow‐up from CR achievement of 54 months (range 5–127) were analysed. 32 patients (42%) displayed a normalized phenotype as evaluated by k:λ ratio or by CD5+/CD19+ cell numbers. Patients with normalized phenotype demonstrated a significantly higher incidence of female sex, a lower relapse rate, a trend for higher prevalence of stage A and a lower occurrence of CLL‐related deaths. The relapse‐free survival of patients with normalized phenotype was significantly longer (P=0.02), whereas no difference in overall survival was found between the two groups. Interestingly, Binet's stage at diagnosis was highly predictive of the overall survival following CR achievement. From the results of the present study we conclude that a phenotype normalization at CR obtained with conventional chemotherapy indicates a higher probability of a longer CR but it does not translate into prolonged survival. Clinical features at diagnosis, such as stage distribution, are apparently stronger predictors of the final outcome. These results emphasize, however, the need for a routine assessment of the quality of response since this information could be crucial in designing therapeutic strategies for young patients suffering from advanced CLL.
Measurement of endogenous plasma thrombopoietin in patients with acquired aplastic anaemia by a sensitive enzyme‐linked immunosorbent assay
Authors: Kojima Seiji , Matsuyama Takaharu , Kodera Yoshihisa , Tahara Tomoyuki , Kato Takashi
Published: November 2003

We measured the endogenous plasma concentration of thrombopoietin (TPO) in 76 patients with acquired aplastic anaemia (AA) by a sensitive sandwich enzyme‐linked immunosorbent assay (ELISA). The plasma TPO concentrations were significantly higher in AA patients when compared to healthy control subjects (P < 0.0001) and there was a significant negative correlation between plasma TPO concentrations and platelet counts in 54 AA patients who had not received any platelet transfusions prior to sampling. On the other hand, there was no statistically significant correlation between the TPO concentrations and platelet counts in 22 AA patients who had previously received platelet transfusions.

We studied serial changes of plasma TPO concentration in 24 patients who showed an increase in their platelet counts following bone marrow transplantation or immunosuppressive (IS) therapy. Although a decrease in plasma TPO concentration was observed, levels remained above the range of normal healthy controls even in the patients who attained normal platelet counts following therapy. A decrease in TPO concentrations was observed in only half of the responders to IS therapy. Whether exogenous TPO will result in increased platelet counts in AA patients with high TPO levels remains to be determined.
Protection by α2‐macroglobulin of tissue plasminogen activator against inhibition by plasminogen activator inhibitor‐1
Authors: IEKO M. , SAWADA K. , YASUKOUCHI T. , SAKURAMA S. , TOHMA Y. , SHIROSHITA K. , KUROSAWA S. , OHMOTO A. , KOHNO M. , SATOH M. , KOIKE T.
Published: November 2003

Tissue plasminogen activator (tPA) is widely used in the treatment of acute myocardial infarction (MI). However, its thrombolytic efficacy does not correlate with the dose administered. The interactions between tPA, α2‐macroglobulin (α2‐M), and plasminogen activator inhibitor‐1 (PAI‐1) were investigated both in vitro and in patients undergoing tPA therapy for MI in an attempt to identify variables that might affect the clinical efficacy of tPA.

Purified α2‐M (5.4 mg/ml) protected 16.0% or 22.4% of tPA (12.5 IU/ml) activity from inhibition by PAI‐1 at 4 or 8 IU/ml in vitro. Of nine patients treated with 5–20 mega IU of tPA for MI, the plasma activity of tPA remained increased for 15–30 min after the cessation of infusion in eight; the patient who failed to exhibit a persistent increase in tPA activity had a low plasma concentration of α2‐M. Total tPA activity, derived from the area under the activity‐versus‐time curve (AUC), showed a significant inverse correlation with the ratio of the plasma PAI‐1 activity to the plasma α2‐M concentration. Total tPA activity did not correlate with plasma PAI‐1 activity or plasma α2‐M concentration alone. Results suggest that α2‐M, by binding to tPA, protects the latter against inhibition by PAI‐1.
Immunolocalization of the multi‐sarco/endoplasmic reticulum Ca2+ATPase system in human platelets
Authors: Kovàcs Tündé , Berger Gaetan , Corvazier Elisabeth , Pàszty Katalin , Brown Angie , Bobe Régis , Papp Béla , Wuytack Frank , Cramer Elisabeth M. , Enouf Jocelyne
Published: November 2003

We recently identified a multi‐SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti‐Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti‐SERCA 2b, anti‐SERCA 3, anti‐SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α‐granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes.  Thus, the multi‐SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub‐compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.
Interlaboratory variation in the detection of clinically significant alloantibodies against human platelet alloantigens
Authors: Metcalfe P. , Allen D. , Chapman J. , Ouwehand W. H.
Published: November 2003

This report describes the results of eight workshop exercises which were designed to test the proficiency of laboratories in the detection of antibodies to human platelet antigens (HPA). Detection of the most clinically significant alloantibody, anti‐HPA‐1a, is adequate. However, despite improvements in consistency of test results between laboratories over the last 3 years, there is still a high probability that clinically significant antibodies against other HPA alloantigens will not be detected.
Splenic regrowth in sickle cell anaemia following hypertransfusion
Authors: CAMPBELL P. J. , OLATUNJI P. O. , RYAN K. E. , DAVIES S. C.
Published: November 2003

We describe five adult patients with sickle cell anaemia (SS) who developed clinical, radiological and histological evidence of splenic regrowth while receiving regular blood transfusions. Five patients, all homozygous SS, range 23–34 years, were commenced on hypertransfusion therapy. Three patients were transfused because of severe recurrent vaso‐occlusive crises, one for chronic sickle lung and one in an attempt to prevent deterioration of renal function. The mean duration of hypertransfusion prior to documentation of splenic regrowth was 52 months (range 12–97 months). Two patients developed significant hypersplenism. One patient had clinically‐apparent splenomegaly and four patients had splenomegaly documented on ultrasound. Splenic regrowth in hypertransfused adults with sickle cell anaemia is not infrequent and may have important clinical implications.
Clinical features at diagnosis in 430 patients with chronic myeloid leukaemia seen at a referral centre over a 16‐year period
Authors: Savage DAVID G. , Szydlo RICHARD M. , Goldman JOHN M.
Published: November 2003

To determine major presenting features of chronic myeloid leukaemia (CML) in current practice, we have reviewed the records of 430 patients with CML referred to the Hammersmith Hospital for allogeneic bone marrow transplantation since 1981. Approximately 20% of cases were diagnosed incidentally. Symptoms such as fatigue and weight loss were associated with greater degrees of leucocytosis and splenomegaly and lower haemoglobin levels. Most bleeding patients had normal or elevated platelet counts, suggesting that platelet dysfunction was the primary cause of haemorrhage. Although thrombocytosis was common, thrombosis was not seen. Male patients and the relatively young presented with higher WBC counts and larger spleens. The reason that these groups were diagnosed with more advanced leukaemia is not clear. Although retrospective and limited to a select group of relatively young patients, this is the largest series to be reported on CML at diagnosis, and the first such report in modern clinical practice.
Clinical characteristics predict response to antithymocyte globulin in paroxysmal nocturnal haemoglobinuria
Authors: Paquette Ronald L. , Yoshimura Ryan , Veiseh Charles , Kunkel Lori , Gajewski James , Rosen Peter J.
Published: November 2003

Seven patients with paroxysmal nocturnal haemoglobinuria (PNH) were treated with antithymocyte globulin (ATG). Each patient received ATG (20 mg/kg/d) for 8 d and prednisone to prevent or control serum sickness. Three patients experienced a sustained improvement in at least one peripheral blood cytopenia, including one patient who had a complete trilineage response. Several pre‐treatment clinical features appeared to be associated with response to ATG. All responding patients had hypoproliferative features including depressed platelet counts (<30×109/l), and a minor degree of chronic haemolysis as indicated by relatively low reticulocyte counts (<100×109/l), lactate dehydrogenase (<1000 U/l) and total bilirubin (<17μmol/l) levels. Responding patients continued to have chronic low‐grade haemolysis after their response to immunosuppression that was similar to that observed prior to treatment. The non‐responding patients had a classic haemolytic form of PNH characterized by elevated reticulocyte counts (>100×109/l), lactate dehydrogenase (>2000 U/l) and total bilirubin (>17μmol/l) levels.

The impaired haemopoiesis that occurs in hypoproliferative PNH may respond to ATG treatment, but the haemolytic component of the disease, and hence the PNH clone, is not altered by immunosuppressive therapy.
Cryoprecipitate prepared from plasma virally inactivated by the solvent detergent method
Authors: Keeling D. M. , Luddington R. , Allain J.‐P. , Lawrie A. S. , Williamson L. M.
Published: November 2003

There remains a small risk of viral transmission from single‐donor blood components such as fresh frozen plasma (FFP) and cryoprecipitate which have not been subjected to a viral inactivation procedure. It is now possible to subject pooled FFP to viral inactivation by the solvent detergent (SD) treatment method, but with some loss of coagulation factors. To establish whether cryoprecipitate prepared from SD plasma would be suitable for the treatment of hypofibrinogenaemia and von Willebrand's disease (VWD), control and SD cryoprecipitate were assayed for factor VIII, von Willebrand factor (VWF) and fibrinogen content. In SD cryoprecipitate, levels of VWF activity and antigen were only 36% and 37% of control values respectively, whereas fibrinogen was 72%. The highest molecular weight multimers of VWF:Ag were absent from both SD plasma and SD cryoprecipitate. SD cryoprecipitate would thus be unsuitable for treating VWD, but would provide an alternative to untreated individual donor units of cryoprecipitate for the treatment of hypofibrinogenaemic states.
Ex vivo neutrophil function in response to three different doses of glycosylated rHuG‐CSF (Lenograstim)
Authors: Turzanski J. , Crouch S. P. M. , Fletcher J. , Hunter A.
Published: November 2003

Granulocyte colony‐stimulating factor (G‐CSF) is being considered as adjuvant treatment for infections in non‐neutropenic patients. Normal healthy donors were given rHuG‐CSF (Lenograstim) at 2.5, 5.0 and 7.5 μg/kg subcutaneously daily for 5 d. Polymorphonuclear leucocyte (PMN) function tests were carried out on peripheral blood PMN before the first injection and at 24 h and 96 h. Circulating PMN levels were also measured at these time intervals and found to be significantly increased with all doses by 24 and 96 h. Investigation of cell surface antigen expression revealed no changes in β2 integrin (CD11b/CD18) expression. L‐selectin (CD62L) expression was reduced with all doses by 96 h, this being significant with the 7.5 μg/kg dose. FcγRI (CD64) levels were significantly up‐regulated with the 7.5 μg/kg dose by 96 h whereas FcγRIII (CD16) expression was found to be reduced during G‐CSF treatment. Superoxide anion production was significantly increased in response to N‐formylmethionylleucylphenylalanine (FMLP) and opsonized Staphylococcus epidermidis stimulation at 24 h and 96 h with the 5.0 and 7.5 μg/kg doses. The initial rate of phagocytosis (0–2 min) appeared to have increased with the 7.5 and 5.0 μg/kg doses at 96 and 24 h compared with PMN responses pretreatment, although these increases were not statistically significant. These results show that G‐CSF enhances the functional responses of PMN stimulated by physiological agonists and may help in the treatment of infections.
The factor V Leiden mutation in children with cancer and thrombosis
Authors: Sifontes Maria T. , Nuss Rachelle , Hunger Stephen P. , Wilimas Judith , Jacobson Linda J. , Manco‐Johnson Marilyn J.
Published: November 2003

Thromboembolic phenomena, frequently observed in children with cancer who are undergoing chemotherapy, can cause significant morbidity and, less frequently, mortality. Many contributory factors have been identified. Whether the recently identified and most common coagulation defect predisposing to thrombosis, factor V Leiden, is associated with thrombosis in this setting, has not been explored. The current study was undertaken to determine the prevalence of the factor V Leiden mutation in children with cancer who developed thromboembolic phenomena as compared to those with cancer who did not. Genomic DNA was amplified using the polymerase chain reaction (PCR), followed by digestion of the amplification product with the restriction enzyme MnlI. The digested PCR products were then size‐fractionated to classify samples as heterozygous, homozygous or normal for the factor V Leiden mutation. 67 children with cancer were evaluated for the factor V Leiden mutation. One of 32 children with cancer and thrombosis, and none of 35 who had not experienced thrombotic problems, was found heterozygous for this mutation. We conclude that the factor V Leiden mutation does not play a significant role in the overall incidence of thromboses that occur in children with cancer.
Glucose metabolism in bone marrow cells and granulocytes of adult mice after X‐ray (5 Gy) irradiation: relationship to cell functionality
Authors: Gaitán S. , Escribano S. , Sancho P. , Cuenllas E. , Tejero C.
Published: November 2003

Our experiments focused on the metabolic implications of the residual haemopoietic damage in adult mice given 5 Gy X‐rays. Bone marrow cells from irradiated mice exhibited an increase in protein synthesis and a decrease in ATP levels, which could be related to the enhancement of the proliferative activity of haemopoietic precursor cells. However, the kinetic parameters (Vmax and Km) of glucose uptake, the glycolytic flux and the hexose monophospate (HMP) shunt activity were similar to those found in the control group. On the other hand, a reduction of glucose uptake (Vmax) was found in both resting and stimulated granulocytes from irradiated mice. This reduction was accompanied by a decrease in the glycolytic rate and ATP levels. However, HMP shunt activity was similar in resting granulocytes in both the control and the irradiated mice. The stimulation by PMA produced a significantly higher increase in the activity of the pathway in granulocytes from the irradiated mice and was in accordance with the enhancement of superoxide anion production that has been previously described in these cells.
Successful engraftment of haploidentical stem cell transplant for familial haemophagocytic lymphohistiocytosis using both bone marrow and peripheral blood stem cells
Authors: Collins P. , Watts M. , Brocklesby M. , Gerritsen B. , Veys P.
Published: November 2003

Familial haemophagocytic lymphohistiocytosis (HLH) is a disease with a very poor prognosis unless patients receive a bone marrow transplant. It is often difficult to find an HLA‐matched donor and haploidentical familial donors may be considered. The main complication of this type of transplant is graft rejection. We describe a patient with familial HLH who received a haploidentical transplant using both mobilized peripheral blood and bone marrow stem cells in an attempt to overcome graft rejection by increasing the stem cell dose. The peripheral blood stem cell inoculum was CD34 enriched using a Cellpro column and T‐cell depleted by Campath‐1M, the patient received conditioning for a matched sibling donor transplant with the addition of Campath 1G. There was rapid and full engraftment and the patient remains disease free at 5 months. This technique may be applicable for other fatal inborn errors in the absence of an HLA‐matched donor.
Rapid mutation screening in type 2A von Willebrand's disease using universal heteroduplex generators
Authors: Culpan Doris , Standen Graham , Wood Nigel , Mazurier Claudine , Gaucher Christine , Bidwell Jeffrey
Published: November 2003

Patients with type 2A von Willebrand's disease (VWD) commonly have missense mutations in the A2 domain of the von Willebrand factor (VWF) protein. This domain is encoded by the 3′ region of VWF gene exon 28 and the large majority of patients have heterozygous mutations clustered in the sequence between codons 742 and 909. We describe a DNA‐based diagnostic technique which enables at least 10 previously described mutations to be rapidly identified. The method involves polymerase chain reaction (PCR) amplification of two exon 28 gene segments between codons 717–788 and 803–893, respectively. Each fragment is then hybridized with a synthetic complementary DNA molecule of similar size, termed a Universal Heteroduplex Generator (UHG). The UHG contains base deletions contiguous to the sites of known mutations and, following hybridization, allele‐specific heteroduplexes are generated which can be detected by simple polyacrylamide gel electrophoresis and ethidium bromide staining. A small panel of UHG molecules covering the 3′ region of exon 28 should enable the large majority of type 2A VWD patients to be rapidly diagnosed by genotype.
Heparin‐induced thrombocytopenia and thrombosis: a prospective analysis of the incidence in patients with heart and cerebrovascular diseases
Authors: Kappers‐Klunne M. C. , Boon D. M. S. , Hop W. C. J. , Michiels J. J. , Stibbe J. , Van Der Zwaan C. , Koudstaal P. J. , Van Vliet H. H. D. M.
Published: November 2003

Heparin‐induced thrombocytopenia and/or thrombosis (HITT) are serious complications of heparin treatment. The incidence, as previously reported, varies widely and, in consequence, is not precisely known. Moreover, most reports only concern clinically defined heparin‐induced thrombocytopenia. Therefore we carried out a prospective study of the incidence of serologically confirmed HITT.

All patients admitted to the Departments of Cardiology and Neurology of our institution with an indication for treatment with therapeutic‐dose intravenous unfractionated heparin were enrolled in the study. The patients were examined daily for the occurrence of thromboembolic complications. Regular platelet counts and tests for the presence of heparin‐dependent antibodies were carried out using two different tests: a quantitative platelet factor 4/heparin (PF4/hep) Elisa, and a functional test, the heparin‐induced platelet activation assay (HIPAA). HITT was defined as a rapidly occurring (within 5 d) decrease of the platelet count from normal values of >120 ×109 l to <60 ×109 l or to <100 ×109 l if there was a rapid fall of >50% of starting value or >30% with concomitant acute thrombosis.

The observed incidence of HITT was 1/358 patients (0.3%, 95% confidence limits 0.01–1.5%). However, Elisa PF4/hep specific IgG antibodes were demonstrated in nine (2.5%) and IgM antibodies in seven (2.0%) of 358 patients. 30/358 patients (8.4%) had platelet activating antibodies in the HIPAA.

We conclude that the incidence of serologically confirmed HITT in this study is very low (0.3%) in patients with cardiac and neurologic diseases treated with intravenous unfractionated heparin. The frequency of heparin‐dependent antibodies without concomitant occurrence of thrombocytopenia is much higher.
The significance of human monocyte thrombomodulin during membrane vesiculation and after stimulation by lipopolysaccharide
Authors: SATTA NATHALIE , FREYSSINET JEAN‐MARIE , TOTI FLORENCE
Published: November 2003

Thrombomodulin acts as an essential membrane cofactor of thrombin in the triggering of the natural anticoagulant protein C pathway responsible for the inactivation of procoagulant cofactors VIIIa and Va. Because monocytes play a critical role in the coupling between infection/inflammation and thrombosis, the fate of monocyte thrombomodulin was assessed at the cell plasma membrane and on derived microparticles. A significant basal level of thrombomodulin activity was measured on unstimulated monocytes and microparticles. Lipopolysaccharide treatment resulted in increased thrombomodulin activity on monocytes (∼40%) and microparticles (∼80%), whereas tissue factor and prothrombinase activities were strongly expressed on both. Flow cytometry detection of thrombomodulin antigen confirmed its presence on unstimulated monocytes and microparticles. A decrease (∼30%) in thrombomodulin labelling was noticed on stimulated monocytes. Labelling of microparticles shed from stimulated and unstimulated monocytes remained unchanged, only an increased proportion of microparticles (∼20%) was observed. The absence of early down‐regulation of thrombomodulin following monocyte stimulation suggests that it fulfils an important regulatory function of membrane‐associated procoagulant activities. This would be of particular significance at the surface of microparticles having the ability to diffuse and concentrate by adherence at inflammatory sites.
Influence of genetic predisposition to thrombosis on natural history of acute promyelocytic leukaemia
Authors: REES DAVID , GRIMWADE DAVID , LANGABEER STEPHEN , BURNETT ALAN , GOLDSTONE ANTHONY
Published: November 2003

There is currently no means of identifying the subgroup of APL patients who will succumb to haemorrhagic or thrombotic complications. We have investigated factor V Leiden and thermolabile methylene tetrahydrofolate reductase (MTHFR) to determine whether these are of predictive value for thrombosis in the context of APL. Of 48 patients drawn from the MRC ATRA trial, two were heterozygous for factor V Leiden (allele frequency 2.1%). 10 homozygotes and 17 heterozygotes for thermolabile MTHFR were identified (allele frequency 38.5%). Amongst these patients, one thrombosis occurred (thermolabile MTHFR heterozygote). In the group with no identified increased thrombotic risk, three episodes were recorded. This approach failed to predict thrombotic events in APL, although the exact implications of specific genotypes remain to be established by larger studies.
Antiphospholipid antibody‐dependent C5b‐9 formation
Authors: Stewart Michael W. , Etches Wai S. , Gordon Philip A.
Published: November 2003

The relationship between the presence of antiphospholipid antibodies (APA) and the production of the terminal membrane attack complex (MAC) of complement (C5b‐9) was studied. Serum samples from known high positive APA patients induced platelet activation and destruction which was inhibited by heat‐inactivation of the sera. The response was restored if the heat‐inactivated APA‐positive sera were supplemented with normal sera. Adsorption of the APA‐positive sera with phospholipid (PL)‐coated polystyrene beads inhibited platelet destruction. Addition of monoclonal antibody (mAb) to C5b‐9 (aE11) also inhibited platelet destruction, suggesting that the APA‐dependent platelet destruction might be complement‐mediated. Purified APA, in the presence of normal serum, induced C5b‐9 formation and binding to PL‐coated beads in a dose‐dependent manner as detected by flow cytometry. Prospective analysis of 200 serum samples for C5b‐9 production showed that all sera testing negative for the presence of APA also tested negative for C5b‐9 production. All sera with high levels of IgG binding to PL (GPL) showed evidence of C5b‐9 production. Sera with low or moderate GPL values showed varying levels of C5b‐9 production. These data suggest that complement may play a key role in APA‐dependent platelet activation, in vivo.
Erythropoietin receptor expression on human bone marrow erythroid precursor cells by a newly‐devised quantitative flow‐cytometric assay
Authors: Shinjo Kaori , Takeshita Akihiro , Higuchi Masato , Ohnishi Kazunori , Ohno Ryuzo
Published: November 2003

In order to develop a non‐isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow‐cytometric assay using cells stained with biotin‐labelled and a streptavidine–RED670 conjugate. For quantification, we applied the Kolmogorov‐Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin‐labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by 125I‐Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three‐colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34+CD38− cells (approximately 1600 sites/cell), and decreased in the following order: CD34+CD38− cells > CD34+CD38+ cells > CD34−CD38+ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34+ fraction expressed more EpoR than their CD34− fraction. However, the expression levels of EpoR of these fractions were lower than CD34+CD38− cells. These results indicated that EpoR was highly expressed on CD34+ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.
Cytogenetic study of 30 patients with multiple myeloma: comparison of 3 and 6 day bone marrow cultures stimulated or not with cytokines by using a miniaturized karyotypic method
Authors: BRIGAUDEAU Christophe , TRIMOREAU Frank , GACHARD Nathalie , ROUZIER Evelyne , JACCARD Arnaud , BORDESSOULE Dominique , PRALORAN Vincent
Published: November 2003

Cytogenetics in multiple myeloma (MM) cases are generally difficult to perform due to the low proliferation index of malignant plasma cells (PC) in most cases. Although IL‐6 and GM‐CSF stimulate the in vitro proliferation of malignant plasma cells, their usefulness for improving cytogenetic results in multiple myeloma patients remains questionable, because results which compare various culture conditions in a sufficient number of patients are not available. By using a miniaturized karyotypic method, we compared in 30 multiple myeloma patients the number and percentage of clonal abnormal mitoses obtained from 3 and 6 d bone marrow cultures performed without or with two combinations of cytokines: IL‐6 + GM‐CSF or IL‐6 + GM‐CSF + IL‐2 + IL‐4 + TNFα . The percentage of patients with an abnormal karyotype, which varied with the Durie and Salmon stage of the disease, as well as the type of numerical and structural karyotypic abnormalities that we detected, were in agreement with published results. The detection of clonal karyotypic abnormalities was better after 3 d of culture without cytokine than in all other culture conditions. The higher percentage of patients at all stages of MM with an abnormal karyotype in our study (76.6%) than in previous ones (20% to 60%) is largely explained by the large number of mitoses analysed in six different culture conditions due to the use of a miniaturized karyotypic method.
Frequent de novo mutations of the ANK1 gene mimic a recessive mode of transmission in hereditary spherocytosis: three new ANK1 variants: ankyrins Bari, Napoli II and Anzio
Authors: RANDON J. , MIRAGLIA DEL GIUDICE E. , BOZON M. , PERROTTA S. , DE VIVO M. , IOLASCON A. , DELAUNAY J. , MORLE L.
Published: November 2003

A subset of spherocytosis cases associated with mutations of the ANK1 gene present an apparently recessive inheritance pattern on a clinical and haematological basis. We identified three novel out‐of‐frame deletions in the ANK1 gene: allele Bari (1361delG), Napoli II (2883delC) and Anzio (3032delCA) in three Italian patients, two of whom have been splenectomized. Analysis of the cDNA showed small or trace amounts of ankyrin mRNAs in Bari, Napoli II and Anzio. The parents were normal clinically and haematologically and did not carry the mutations exhibited by their children. We confirmed the de novo character of the HS mutations based on paternity testing. Recessive HS associated with the ANK1 gene is probably rarer than initially thought, and spherocytosis may often be due to de novo mutations.
Degradation of glycophorin A of human erythrocytes in patients with myelo‐ or lymphoproliferative disorders: possible role of neutrophil proteases
Authors: Bykowska K. , Duk M. , Kuśnierz‐Alejska G. , Sikorska A. , ŁE¸towska M. , Mendek‐Czajkowska E. , ŁOpaciuk S. , Kopeć M. , Lisowska E.
Published: November 2003

We have previously reported that glycophorin A (GPA) of human erythrocytes (carrying blood group M and N determinants) was totally digested by incubation of erythrocytes with human neutrophil elastase (HNE) and cathepsin G (CathG). The membrane‐bound GPA fragments fractionated by SDS‐PAGE gave characteristic patterns of bands detected by immunoblotting with the monoclonal antibody PEP80. Erythrocytes were incubated with HNE and CathG at low enzyme concentrations, similar to those found in vivo. Characteristic electrophoretic patterns of bands derived from a partial GPA digestion were observed and these patterns were different for both enzymes and different from those obtained after total GPA digestion. GPA was also partially digested by incubation of erythrocytes with granulocytes in the presence of Ca2+ and calcium ionophore and electrophoretic pattern of digestion products was similar to that obtained with low doses of HNE. No GPA digestion products were detected after treatment of erythrocytes with plasmin and kallikrein. Untreated erythrocytes of 21 patients with various myelo‐ or lymphoproliferative disorders were tested by SDS‐PAGE of RBC membranes and immunoblotting with the anti‐GPA PEP80 antibody. GPA degradation products, resembling those formed by a mild CathG treatment of control RBC, were detected in nine patients. GPA fragmentation was in some cases accompanied by a reduced expression of blood group MN determinants. No distinct relation was observed between the occurrence of GPA degradation in erythrocytes and increases in plasma concentrations of HNE‐α1‐proteinase inhibitor (α1‐PI) complex considered to be an indication of a release of neutrophil proteinases in vivo. However, the results suggested that a partial GPA degradation in haematological proliferative disorders may occur due to limited proteolysis by neutrophil proteinases, most likely by CathG.
Constitutive expression of Fas ligand in large granular lymphocyte leukaemia
Authors: Perzova Raisa , Loughran Thomas P.
Published: November 2003

The T‐cell type of large granular lymphocyte (LGL) leukaemia is a lymphoproliferative disorder characterized by clonal proliferation of CD3+ LGL, which is often associated with autoimmune disorders. Phenotypic and functional data suggest that leukaemic CD3+ LGL represent activated cytotoxic T lymphocytes (CTL). One mechanism whereby CTL mediate target cell killing is through the Fas/Fas ligand apoptotic pathway. Fas ligand is expressed by CTL only after activation. In this study we examined seven patients with LGL leukaemia for expression of Fas ligand gene transcripts using reverse transcriptase‐polymerase chain reaction (RT‐PCR) analyses. We found constitutive expression of Fas ligand gene transcripts in each of the seven patients. Similar up‐regulation of Fas ligand gene expression has been observed in mice with autoimmune lymphoproliferative syndromes caused by Fas mutations. However, sequence analyses of the death domain of the Fas gene in LGL leukaemia patients revealed no evidence for mutations. Our findings provide further support for the hypothesis that leukaemic LGL are CTL activated by chronic antigenic stimulation. Constitutive expression of Fas ligand may contribute to the pathogenesis of the neutropenia observed in LGL leukaemia.
Two subsets of peripheral blood plasma cells defined by differential expression of CD45 antigen
Authors: Schneider Ulrike , Lessen Antje van , Huhn Dieter , Serke Stefan
Published: November 2003

The purpose of this study was to optimize the flow cytometric determination of circulating normal and malignant plasma cells (PC). We investigated peripheral blood (PB) samples of 65 patients with multiple myeloma or monoclonal gammopathy of unknown significance and 47 control subjects using CD38, CD45, B‐B4, CD56, VLA‐4, VLA‐5 and CD19 monoclonal antibodies (MoAbs). Mono‐ or polyclonality was determined by staining of intracellular kappa and lambda light chains. Two subpopulations of PBPC were distinguished by differential expression of CD45. CD45 positive (CD45+) PC showed a more immature morphology and were detected in all groups. They were polyclonal in the control subjects and either poly‐ or monoclonal in the myeloma patients. In contrast, CD45 negative (CD45−) PBPC only occurred in myeloma patients and were consistently monoclonal, their presence being significantly associated with high disease activity (P<0.001). Although detection of CD45− PBPC using CD38 or B‐B4 MoAbs lead to similar results, CD45+ PBPC often were recognized to a lesser extent by B‐B4 than by CD38 MoAbs.

In conclusion, normal and malignant circulating PC can reliably be identified using CD38 and CD45 MoAbs. CD45 expression separates PBPC into two subsets of which the CD45− one only occurs in myeloma patients.
Prevention and treatment of thrombotic complications in essential thrombocythaemia: efficacy and safety of aspirin
Authors: Van Genderen Perry J. J. , Mulder Paul G. H. , Waleboer Marco , Van De Moesdijk Desiree , Michiels Jan J.
Published: November 2003

The efficacy and safety of aspirin in the prevention and treatment of thrombosis in essential thrombocythaemia (ET) was retrospectively analysed in a cohort of 68 ET patients. 41 patients presented with thrombosis, five patients with bleeding; two patients had a paradoxical combination of bleeding and thrombosis at presentation. At presentation, patients with bleeding had significantly higher platelet and leucocyte counts than patients with thrombosis. During long‐term follow‐up the incidence of thrombosis was significantly reduced in patients receiving aspirin, either as monotherapy or in combination with cytoreduction. However, treatment with aspirin (500 mg/d) was associated with an increase in (minor) bleeding complications. In patients receiving aspirin, bleeding occurred particularly at platelet counts exceeding 1000×109/l. The overall 5‐ and 10‐years survival probability was 93% and 84% respectively, indicating that life expectancy in ET is close to normal. Although our data need confirmation in prospective clinical trials, they suggest that aspirin, particularly in lower doses (100 mg/d), may be a safe antithrombotic agent in ET with an acceptable risk for bleeding, if applied to patients with a platelet count <1000×109/l and/or absence of a bleeding history.
Assessing social work effectiveness in child care practice: the contribution of randomized controlled trials
Authors: Newman T , Roberts H
Published: November 2003

This article discusses the role of randomized controlled trials (RCTs) in evaluating the impact of social work interventions with children. While recognizing the difficulties of applying RCTs to all aspects of practice, we argue that controlled trials can provide the most convincing evidence of the impact of social work activities on the welfare of children and families. Accumulating evidence of the effectiveness of interventions, we propose, should constitute the core business of social work research. To this end, it is necessary to recognize the primacy of the randomized controlled trial in exploring the relationship between social work activities and client outcomes.
Early intervention for young children with developmental delay: the Portage approach
Authors: Cameron R J
Published: November 2003

It is now 20 years since the Portage Home Teaching model of early intervention was introduced into the UK. In this paper, an overview of the Portage Model and rationale is provided, achievements over the past two decades are noted and related research and evaluation studies are reviewed. A number of areas for future development are also highlighted. These include: issues of quality control; the multi‐cultural dimension of Portage; multi‐agency collaboration; professional training needs of Portage personnel; and influencing central government policy relating to families who have a young child with special needs.
Beyond child development centres: care coordination for children with disabilities
Authors: Appleton P L , Böll V , Everett J M , Kelley A M , Meredith K H , Payne T G
Published: November 2003

A specific model of care coordination for children with disabilities is described, comprising parental empowerment, a defined client population, individual tailoring of service based on assessment of need, inter‐agency collaboration beyond existing team boundaries, continuity of named professional contact across transitions important to families, and a named care coordinator. The first stages of local implementation of the model are described for children with a disability making the transition into nursery school provision. Qualitative findings from interviews with families and care coordinators are presented, and the possibility of care coordinators providing a Named Person function is examined.
Child development teams: are they fulfilling their purpose?
Authors: Hall D
Published: November 2003

Child development teams (CDTs) have been in existence for a quarter of a century, yet little is known of how they function or what they do. This paper originated in a national seminar on how CDTs function and what they seek to achieve. It discusses the wide range of practice in CDTs across Britain and the apparent deficiencies in service provision for certain types of disability. Since each child has a unique set of needs, the composition of the team must be flexible and responsive to the changing circumstances of each child. The need for a philosophical shift to take account of changing parental expectations is stressed. There is a need for further research on a number of topics, including: the patterns of service provision and the differences between districts; the implementation of what is already known about standards of good practice; the possible role of a charter for disabled children and their families; the training needs of future CDT professionals.
The relationship between audit, research and policy: lessons from a community paediatric audiology service
Authors: Fonseca S , Borgstein B , Dobson M , Hall D
Published: November 2003

The pace of medical change is in danger of paralysing the process of decision making, particularly in services where clinical improvements occur more slowly than the introduction of new interventions. Audit within an individual district enables staff to monitor progress towards desired goals and standards but rarely generates sufficient data to inform decision making about major policy changes. The paper describes how the findings from nine audits of a community paediatric audiology service over a 13‐year period were combined with reviews of the literature, resulting in a series of changes to a children’s audiological service. The interest and commitment of all the staff involved were maintained by involving them in the process and using them as a valuable source of qualitative data. Audit must be thorough and should be based on precise case definition and comprehensive casefinding if the results are to be meaningful. It is a more powerful means of achieving improvements in systems if it is combined with research evidence, and a readiness to change the system if the agreed goals are not being attained.