bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated platelet‐free calcium in uraemia
Authors: Moosa A. , Greaves M. , Brown C. B. , MacNeil S.
Published: July 2010

Bleeding complications in uraemia are not uncommon. The pathogenesis of haemorrhage in uraemia is still a matter of controversy and the pattern of bleeding suggests a defect of primary haemostasis. Platelet aggregation and biochemistry, including calcium levels, have been studied; however, the results are controversial. We have examined platelet aggregation, platelet‐free calcium and calmodulin in platelet‐rich plasma because of the significant role of calcium and calmodulin in regulating platelet and other cells’functions. Platelet aggregation in uraemic subjects was similar to that of controls. Platelet basal free cytosolic calcium and platelet calcium in response to 10 μM Ca + + ionophore A23187 in eight subjects with uraemia were 117 ± 33 nM and 2025 ± 398 nM (mean ± SEM) respectively. By contrast in seven matched healthy controls basal calcium and ionophore‐stimulated calcium values were 47 ± 14 nM and 1354 ± 414 nM, significantly less than in the patients with uraemia (P < 0.05). The sensitivity of uraemic platelets to A23187 was similar to that of controls. Calmodulin activity in platelet‐rich plasma of 12 subjects with uraemia showed no significant difference from that of controls [1.86 ± 0.29 μg/ml (mean ± SEM) and 2.0 ± 0.37 μg/ml (mean ± SEM) respectively] We conclude that despite elevation of platelet calcium in uraemia, which may be due to a plasma factor such as parathyroid hormone, platelet aggregation is normal and bleeding in uraemia is more likely to be due to other factors, including the effect of reduced haematocrit on platelet endothelial interaction. Disturbances in platelet calcium cannot explain the bleeding manifestations in uraemia but warrant further investigation in order to identify the pathogenic mechanisms responsible.
Interleukin‐4 inhibits apoptotic cell death and loss of the bcl‐2 protein in B‐chronic lymphocytic leukaemia cells in vitro
Authors: Panayiotidis Panayiotis , Ganeshaguru Kanagasabai , Jabbar Shireen A. B. , Hoffbrand A. Victor
Published: July 2010

Summary. When monoclonal B cells from B‐chronic lymphocytic leukaemia (B‐CLL) patients are cultured in vitro, they die by apoptosis. Apoptotic cell death occurred in the B cells from 20/24 B‐CLL patients after 26–30 h in in vitro culture, with 14.3–59.0% (mean 33.6%) of their DNA being fragmented in ∼180 base pair multimers. After 8–10 d culture, 90–100% of the B‐CLL cells were dead. Cell death and DNA fragmentation were inhibited in the presence of 0.5–5 ng/ml human recombinant interleukin‐4 (IL‐4) and viable monoclonal B cells could be maintained in culture up to 3 weeks. At 5 ng/ml. IL‐4 reduced DNA fragmentation after a 26–30 h culture to 2.2–33.3% (mean 14.9%). IL‐4 inhibited apoptosis without stimulating cell proliferation. In four patients the cells were resistant to apoptosis in vitro and they could be maintained for up to 4 weeks in culture medium alone. DNA fragmentation in all patients was increased in the presence of the RNA synthesis inhibitor actinomycin‐D. Western blot analysis of cell lysates showed expression of the bcl‐2 protein in all 11 B‐CLL patients studied. However, during culture, bcl‐2 protein levels were preserved only in patients resistant to apoptosis and were reduced in those susceptible to apoptosis. Reduction of bcl‐2 protein levels was inhibited in cells cultured in the presence of IL‐4. These data offer an explanation for the difference between the long life in vivo and rapid death in vitro of B‐CLL cells and indicate that IL‐4 may participate in the extended survival of these non‐dividing cells in vivo.
Circulating immune complexes involving the ABO system after platelet transfusion
Authors: Heal J. M. , Masel D. , Rowe J. M. , Blumberg N.
Published: July 2010

Summary. It has been proposed that when ABO unmatched platelets are transfused circulating immune complexes (CIC) may be formed between the patient's soluble ABH antigens and the transfused antibodies. Platelets might then be destroyed by bystander mechanisms or by the binding of CIC to the Fc receptor or to C3 binding membrane proteins on the platelet. An ELISA Clq assay was used to detect CIC in 40 patients with haematological diseases who had received multiple platelet transfusions. A significantly larger number of refractory patients were positive (41%) in the assay than non‐refractory patients (13%) or normal blood donors (0%). The presence of circulating IgG anti‐A was sought in six group A refractory patients who had been transfused with ABO unmatched platelets. To determine whether the IgG anti‐A was monomeric or in high molecular weight complexes, the serum was fractionated by gel exclusion chromatography and fractions were tested for the presence of IgG anti‐A. In all six patients the peak of IgG anti‐A binding occurred in fractions of high molecular weight (200–900 kD). Five out of six patients also demonstrated anti‐A activity in fractions corresponding to monomeric IgG (about 150–180 kD). Fractions containing high molecular weight anti‐A were purified using a protein G column and the eluates were tested for the presence of group A antigen using dot immunoblotting. Group A antigen was associated with the purified IgG anti‐A in 4/5 patients tested. Appropriate transfused and non‐transfused controls had no anti‐A in any fractions. Although not unexpected, these studies demonstrate for the first time that refractory patients receiving ABO unmatched platelets have CIC composed of ABO antigens and their corresponding antibodies present in their serum that circulate for at least several days. It also confirms the hypothesis that some CIC in haematological patients are induced by transfusion.
Seasonal changes in red blood cell parameters
Authors: Kristal‐Boneh E. , Froom P. , Harari G. , Shapiro Y. , Green M. S.
Published: July 2010

Summary. Seasonal changes in blood count (haematocrit and haemoglobin values) have been described in studies of small samples. Since Israel is characterized by high summer temperatures, we attempted to determine whether there are circannual changes in blood count, and how they are influenced by mean monthly temperature.

In a cross‐sectional study, venous blood was drawn from 4317 male employees in industry aged 20–64 years. Blood count values were analysed by month. To estimate possible changes in serum osmolality. serum electrolytes were also examined.

Values of haemoglobin and haematocrit in August were significantly lower than during the rest of the year. Circan nual rhythms explained between 10% and 57% of the variances of all the variables as assessed by cosinor rhythmo‐metry, although they had wide confidence intervals and most were not statistically significant. The lower mean haematocrit in August reflected reduction in mean corpuscular volume (MCV) rather than a decrease in red blood cell count (KBC).

The change observed in haematocrit and haemoglobin in August is possibly related to heat acclimatization rather than to endogenous sinusoidal circannual rhythms. Both plasma and red cell volume expansion appear to occur. Possible seasonal changes should be taken into account in evaluation of blood counts.
Serial phenotypic, cytogenetic and molecular genetic studies in Richter's syndrome: demonstration of lymphoma development from the chronic lymphocytic leukaemia cells
Authors: Koduru P. R. K. , Lichtman S. M. , Smilari T. F. , Sun T. , Goh J. C. , Karp L. , Hall W. , Hashimoto S. , Chiorazzi N. , Broome J. D.
Published: July 2010

Summary. In this report we describe a unique longitudinal study on the clinical, phenotypic, cytogenetic and molecular genetic features of malignant cells from diagnosis of chronic lymphocytic leukaemia (CLL) to the development of lymphoma and lymphomatous meningitis. CLL cells at diagnosis were CD5+, CD19+, surface IgG+, kappa+, were karyotypically abnormal and showed clonal rearrangements in the immunoglobulin heavy (IgH) and kappa light chain genes. Phenotypically leukaemic cells and lymphoma cells at RS resembled CLL at diagnosis, but showed cytogenetic evolution. Geometrically leukaemic cells and lymphoma cells retained the initial clonal rearrangements in IGH and kappa genes, but showed additional supervening clonal rearrangements in both of these genes as the disease progressed to RS. Furthermore, the c‐lambda DNA showed clonal rearrangements in the leukaemic cells and lymphoma cells at RS. This complete phenotypic and genotypic analysis of tumour cells during the course of the disease demonstrates the origin of lymphoma from CLL cells through progressive cytogenetic and molecular genetic changes in CLL cells.
Parvovirus B19 induced red cell aplasia in myelofibrosis
Authors: Soutar K. L. , Birnie David H. , Bennett B.
Published: July 2010

Summary. A 38–year‐old female presented with moderate anaemia and a leucoerythroblastic blood film. Subsequent investigation showed myelofibrosis in cellular phase. Her haemoglobin quickly and spontaneously recovered with concurrent serological evidence of recent parvovirus B19 infection. This is the first report in the literature of parvovirus causing red cell aplasia in myelofibrosis.
BCR rearrangement in apparent essential thrombocythaemia
Authors: Richards E. M. , Bloxham D. M. , Nacheva E. , Marcus R. E. , Green A. R.
Published: July 2010

Summary. The presence of the Philadelphia chromosome is a major determinant of the prognosis of patients with myeloproliferative disorders. We describe a case of apparent essential thrombocythaemia in whom cytogenetic analysis was normal. However, the presence of basophilia, the absence of abnormal megakaryocytes in a trephine biopsy and the female sex of the patient prompted Southern analysis of peripheral granulocyte DNA. This revealed a BCR rearrangement and the patient has therefore undergone allogeneic bone marrow transplantation. This case emphasizes the importance of both cytogenetic and molecular analysis of patients with apparent essential thrombocythaemia.
Clonal chromosomal abnormalities in Fanconi's anaemia: what do they really mean?
Authors: Alter Blanche P. , Scalise Angela , McCombs Jerome , Najfeld Vesna
Published: July 2010

Summary. Patients with Fanconi's anaemia (FA) have aplastic anaemia, leukaemia, myelodysplasia and tumours. Since leukaemia has a very poor prognosis, it is desirable to identify high‐risk patients. To determine the significance of clonal marrow chromosomal abnormalities we began a prospective study in 17 patients: five were normal, eight aplastic, and four myelodysplastic. Three of 11 with adequate cytogenetics had transient abnormal clones. None had leukaemia at 3–24 months. Changing cytogenetic patterns may not be related to leukaemic evolution in patients with a DNA repair defect.
Flow cytometry analysis of dual red blood cell populations after bone marrow transplantation
Authors: Blanchard D. , Bruneau V. , Germond‐Arnoult F. , Bernard D. , Gourbil A. , David B. , Muller J. Y.
Published: July 2010

Flow cytometry represents an alternative method to agglutination assays for the accurate quantification of mixed field populations of erythrocytes observed after bone marrow transplantation. Murine monoclonal antibodies directed against the blood group ABH antigens were selected and processed in order to prepare ready‐to‐use fluorescent reagents. Anti‐A (NaM87‐1F6; IgG3), anti‐B (NaM9‐2E11; IgG3) and anti‐H (NaM19‐7E11; IgM) were purified, labelled with fluorescein isothiocyanate, and used in a direct flow cytometry assay. Anti‐A1 (NaM1‐1C9; IgG3) was no longer active after FITC‐labelling and then was used in an indirect assay. The agglutination was prevented by formaldehyde pretreatment of erythrocytes.

Using artificially‐made double populations of erythrocytes, measured values with mixtures of 1‐100% of cells were very closely related to expected values, showing both the sensitivity and the accuracy of the method. From careful investigation of a series of bone‐marrow transplanted patients, we conclude that engraftments could be demonstrated earlier by flow cytometry than by agglutination, because minor populations (1‐10%) of cells could be determined accurately only with labelled reagents. In addition, the disappearance of the donor cells on a long‐term follow‐up of patients enabled an earlier detection of graft failure in one case. The proposed method provides appreciable help to follow engraftment in patients and may have more general applications for the study of other haemopoietic chimaeras.
Needle liver biopsy in thalassaemia: analyses of diagnostic accuracy and safety in 1184 consecutive biopsies
Authors: Angelucci Emanuele , Baronciani Donatella , Lucarelli Guido , Baldassarri Massimo , Galimberti Maria , Giardini Claudio , Martinelli Filiberto , Polchi Paola , Polizzi Vincenzo , Ripalti Marta , Muretto Pietro
Published: July 2010

We report the reliability and safety of percutaneous liver biopsy in the evaluation of hepatic iron loading and histology in patients with homozygous β‐thalassaemia prior to and in serial biopsies following allogeneic bone marrow transplantation for this disorder. 501 thalassaemic patients aged 11 ± 4.5 years (range 1‐32 years) underwent 1184 consecutive percutaneous liver biopsies without ultrasound guidance. Overall, 81% of biopsies were evaluable for histological examination and grading of iron. The adequacy of liver biopsy specimens increased with patient age: evaluable specimens were obtained in 73% of patients <5 years of age and in 86% of samples in patients aged >15 years. The degree of iron overload and fibrosis in each biopsy was reported separately by at least two pathologists who did not know the clinical status of each patient. In 103 biopsies, iron grade by light microscopy corresponded to an iron concentration varying between a mean of 32.46 ± 14 μmol/g dry weight liver tissue for iron stores graded by light microscopy as absent to 417.6 ± 150μmol/g dry weight liver tissue for stores graded as severe. The fibrosis score of multiple samples of liver obtained at autopsy within 100 d of the percutaneous biopsy in 41 patients who died following BMT correlated perfectly with that of the first sample in >60% biopsies; in most of the discordant cases fibrosis had been underestimated in the percutaneous biopsy. Liver biopsy demonstrated evidence of chronic hepatitis in 30% of patients with normal transaminase and in 57% of patients with transaminase within twice the normal range. Liver biopsy was complicated in six patients (0.5%) by haemoperitoneum, periocholecystic haematoma, kidney haematoma, or bile peritonitis; no complication was fatal. These data demonstrate that percutaneous liver biopsy provides reliable information regarding liver iron and histology in homozygous β‐thalassaemia with an extremely low risk of complications.
Cobalamin forms and analogues in plasma and myeloid cells during chronic myelogenous leukaemia related to clinical condition
Authors: Gimsing Peter
Published: July 2010

Plasma and buffy coat specimens of CML patients with untreated disease, in chronic and accelerated phase, and in overt blastic crises were analysed for the cobalamin patterns using non‐polar extraction, thin‐layer chromato‐graphy and bioautography. Splenic tissue specimens from four splenectomized patients in accelerated phase were analysed similarly. Cellular extracts were separated into two compartments by adsorption to haptocorrin antibodies. Plasma concentrations of all cobalamin forms were increased in CML. The proportion of methylcobalamin was significantly lower than in a reference population, and a low plasma proportion of methylcobalamin was associated with a poor prognosis. Buffy coat cells and splenic tissue had a higher proportion of 5′‐deoxyadenosylcobalamin and a lower proportion of methylcobalamin than plasma; still, there was relatively more methylcobalamin than in normal tissue. The haptocorrin‐bound compartment differed from the non‐haptocorrin compartment in untreated or chronic phase patients by binding less methylcobalamin. An estimate of cobalamin analogues in plasma was achieved by comparing two different isotope dilution assays employing a cobalamin‐specific binder, intrinsic factor, and a nonspecific binder, hog non‐intrinsic factor. Values for total cobalamin and analogues were increased to the same degree in CML plasma.
Bone marrow pathology in relapsing polychondritis: high frequency of myelodysplastic syndromes
Authors: Diebold J. , Rauh G. , Jäger K. , Löhrs U.
Published: July 2010

Haemopathologic changes were studied in 19 patients (13 male, six female, age 33‐85 years, mean 56 years) with relapsing polychondritis (RP). Anaemia was found in eight, thrombocytopenia in two and splenomegaly in three patients. A total of 17 bone marrow biopsies were obtained from seven individuals. Bone marrow evaluation revealed myelodysplastic syndromes (MDS) with marked trilineage hyperplasia and dysplasia in three cases. Since an excess of myeloblasts or an increase of CD34 positive progenitor cells was not seen, the disorders were designated as‘refractory anaemia’or with regard to the dysplastic megakaryopoiesis‘MDS, unclassifiable'. Two of the three patients died after 10 and 55 months of follow‐up due to infectious complications. In a further patient, bone marrow analysis repeatedly showed an unexplained granulopoietic hyperplasia, which, however, was not dysplastic enough to allow a diagnosis of MDS. The remaining patients had clearly reactive changes.

Our findings support the notion that RP is a heterogenous disorder and suggest that RP may at times represent a paraneoplastic phenomenon of an underlying MDS. Since HLA typing revealed a significantly increased frequency of the antigen DR4 (10/17 patients positive = 59%), we hypothesize that immunological imbalances due to the MDS in conjunction with a specific immunogenetic background may play key roles in the pathogenesis of RP in these patients.
Assessment of intestinal blood‐flux by laser Doppler fluxmetry in mice with altered intestinal iron absorption
Authors: Raja K. B. , Simpson R. J. , Peters T. J.
Published: July 2010

Laser Doppler fluxmetry (LDF) has been used to assess mucosal blood‐flux in the intestine of normal CD1 mice and groups of animals with altered iron metabolism (i.e. iron‐deficient, hypoxic and hypotransferrinaemic [hpx/hpx]). All experimental animals showed a 2‐3‐fold increase in duodenal iron absorption, mainly due to changes in the ‘mucosal transfer’phase. However, only the hypoxic mice exhibited any increases in duodenal blood‐flux. In the hpx/hpx group, blood flux was maintained at control levels despite the anaemia and without any significant alterations in red blood cell velocity. Moreover, these two groups demonstrated a further capacity to increase blood‐flux above basal levels during onset of acute hypoxia (inhalation of 10% 02). Changes in duodenal blood‐flux were apparent on both the mucosal and serosal surfaces, suggesting that the LDF signal reflects flux throughout the thickness of the mouse intestine.

Iron absorption is likely to be more related to plasma flux changes than to blood‐flux changes. Deduction of plasma flux changes from blood‐flux changes is complicated by vasodilation effects on capillary haematocrits. It is clear from the data, however, that plasma flux changes do not parallel changes in iron absorption in the experimental models.

The lack of correlation between the duodenal blood‐flux and iron absorption values in the experimental models argues against the possible involvement of blood‐flux in the control of duodenal iron absorption.
Familial thrombocytosis
Authors: Kikuchi M. , Tayama T. , Hayakawa H. , Takahashi I. , Hoshino H. , Ohsaka A.
Published: July 2010

Four cases of thrombocytosis in three successive generations of a family are described. High peripheral platelet count was found incidentally in the proband with cutaneous malignant lymphoma. Bone marrow examination showed megakaryocytic hyperplasia. Neither Philadephia chromosome nor chimaeric bcr/abl junction was detected in marrow cells. In this family, thrombocytosis was thought to be transmitted by an autosomal dominant mode of inheritance.
Correction of anaemia and thrombocytopenia in a case of adult Type I osteopetrosis with recombinant human erythropoietin (rHuEPO)
Authors: Meletis John , Samarkos Michalis , Michali Evi , Vavourakis Stathis , Meletis Christos , Poziopoulos Christos , Stavrogianni Niki , Konstantopoulos Kostas , Vaiopoulos George , Yataganas Xenophon , Loukopoulos Dimitris
Published: July 2010

A case of adult osteopetrosis Type I was diagnosed in a 22‐year‐old female. She presented for investigation of anaemia with ‘myelophthisic’characteristics and extramedullary haemopoiesis which was resistant to haematinics, nandrolone and low‐dose corticosteroids. She became progressively transfusion‐dependent with gradually worsening thrombocytopenia. She was successfully treated with recombinant erythropoietin. Anaemia as well as thrombocytopenia were corrected. There appeared to be a synergistic action of erythropoietin with steroids.
Parvovirus B19‐associated haemophagocytic syndrome in healthy adults
Authors: Shirono Kenji , Tsuda Hiroyuki
Published: July 2010

Between August 1992 and August 1994 we found seven adult patients with haemophagocytic syndrome associated with human parvovirus B19 (HPV) infection. Five of the patients were previously healthy but the other two had underlying immunosuppressive disease; the former five patients recovered spontaneously without any treatment. Eight cases of HPV‐associated haemophagocytic syndrome have been reported; six of them were children and the remainder were adults with immunosuppressive disease or haematological disorder. Our study suggests that HPV is not a rare cause of virus‐associated haemophagocytic syndrome (VAHS). Moreover, HPV‐associated haemophagocytic syndrome could occur more frequently than previously thought, not only in children and adults with underlying disease but also in adults, even in good health.
Granulocyte colony‐stimulating factor production by adult T‐cell leukaemia cells
Authors: Matsushita Kakushi , Arima Naomichi , Yamaguchi Katsuya , Matsumoto Tadashi , Ohtsubo Hideo , Hidaka Shiroh , Fujiwara Hiroshi , Arimura Kosei , Kukita Toshimasa , Tokito Yuichi , Ozaki Atsuo , Tei Chuwa
Published: July 2010

. We previously demonstrated that, in about 30% of primary adult T‐cell leukaemia (ATL) cases, the leukaemic cells proliferated in response to granulocyte colony‐stimulating factor (G‐CSF). In the present report, we describe five patients with the acute leukaemia type of ATL who showed marked neutrophilia and elevated serum G‐CSF concentrations in the absence of infection. We further examined two of these patients for detailed clinical features and cellular characteristics of the tumour cells. The white blood cell counts of both patients were 62 × 109/l, consisting of approximately 90% neutrophils and 10% ATL cells. Serum concentrations of G‐CSF in the two patients were 138 pg/ml and 93 pg/ml. The G‐CSF concentrations in supernatants of short‐term cultures of the patients' peripheral blood T‐cells were 2·5 pg/ml and 13 pg/ml respectively. Immunostaining with anti‐G‐CSF antibody demonstrated G‐CSF production by primary ATL cells in both cases. The neutrophil count fluctuated simultaneously with activity of ATL. Primary ATL cells from one patient were shown to proliferate in response to G‐CSF in vitro. These results suggest autocrine growth stimulation of primary ATL cells in a subgroup of patients.
Cytogenetic abnormalities in PHA‐stimulated lymphocytes from patients with Langerhans cell histiocytosis
Authors: Scappaticci Susi , Danesino Cesare , Rossi Elena , Klersy Catherine , Fiori Gian Mario , Clementi Rita , Russotto Valeria Spica , Bossi Grazia , Aricò Maurizio ,   the AIEOP‐Istiocitosi Group
Published: July 2010

The aetiopathogenesis of Langerhans cell histiocytosis (LCH) is still undefined. Constitutional abnormalities in LCH have rarely been reported. One study showed chromosomal instability in lesional cells from three patients. No chromosomal studies are available on peripheral blood lymphocytes. Peripheral blood lymphocytes were analysed for the presence of chromatid and/or chromosomal breaks and structural rearrangements. A fluorescence in situ hybridization (FISH) painting technique was also applied in two cases. Sixteen patients with multisystem (MS, n = 11) or single system (SS, n = 5) LCH were studied, either at the diagnosis (n = 8), during treatment (n = 2) or during follow‐up, when asymptomatic (n = 6). Thirteen patients had chromosomal abnormalities. Eleven patients (69%) had chromatid and chromosomal breaks in 7–45% of cells. Overall, chromosome and chromatid breaks were significantly more frequent in the 11 patients with MS disease than in the five patients with SS disease: the mean percentage of cells showing chromosome and chromatid breaks was 13·4% in MS patients vs. 6·2% in SS patients (P = 0·003). Chromosomal abnormalities may be found in phytohaemagglutinin (PHA)‐stimulated peripheral blood lymphocytes of LCH patients at diagnosis, during the disease course and even during long‐term follow‐up, more frequently in MS disease. Chromosome instability may be considered as either a basic genetic instability or as a landmark of reaction to an environmental agent (viral?) that, through genome alteration, may play a role in histiocyte proliferation and, in some cases, also in the increased risk of malignancy.
CHO expression of a novel human recombinant IgG1 anti‐RhD antibody isolated by phage display
Authors: Miescher Sylvia , Zahn‐Zabal Monique , De Jesus Maria , Moudry Radmila , Fisch Igor , Vogel Monique , Kobr Michel , Imboden Martin A. , Kragten Eddy , Bichler Johann , Mermod Nicolas , Stadler Beda M. , Amstutz Hanspeter , Wurm Florian
Published: July 2010

Replacement of the hyperimmune anti‐Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti‐RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high‐affinity anti‐RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin‐treated red blood cells enabled the isolation of two high‐affinity Fab‐expressing phage clones, LD‐6‐3 and LD‐6‐33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants DIII, DIVa, DIVb, DVa, DVI types I and II, DVII, Rh33 and DFR. Full‐length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth‐adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti‐RhD antibody adapted to pilot‐scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody‐dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti‐RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.
Fanconi anaemia group A (FANCA) mutations in Israeli non‐Ashkenazi Jewish patients
Authors: Tamary H. , Bar‐Yam R. , Shalmon L. , Rachavi G. , Krostichevsky M. , Elhasid R. , Barak Y. , Kapelushnik J. , Yaniv I. , Auerbach A. D. , Zaizov R.
Published: July 2010

Fanconi anaemia (FA) is a genetically heterogeneous disease with at least eight complementation groups (A–H). In the present study, we investigated the molecular basis of the disease in 13 unrelated Israeli Jewish (non‐Ashkenazi) patients with FA. All 43 exons of the Fanconi anaemia A (FANCA) gene were amplified from genomic DNA and screened for mutations by single‐strand conformation polymorphism and DNA sequencing. We identified four ethnic‐specific mutations: (1) 2172–2173insG (exon 24), the first ‘Moroccan mutation’; (2) 4275delT (exon 43), the second ‘Moroccan mutation’; (3) 890–893del (exon 10), the ‘Tunisian mutation’; and (4) 2574C > G (S858R), the ‘Indian mutation’. The tetranucleotide CCTG motif, previously identified as a mutation hotspot in FANCA and other human genes, was found in the vicinity of 2172–2173insG and 890–893del. According to our study, the four mutations account for the majority (88%) of the FANCA alleles in the Israeli Jewish (non‐Ashkenazi) FA population. A screening of 300 Moroccan Jews identified three carriers of the first ‘Moroccan mutation’, but we did not find any carrier of the second ‘Moroccan mutation’ among 140 Moroccan Jews, nor any carrier of the ‘Tunisian mutation’ among 50 Tunisian Jews. Two ‘Indian mutation’ carriers were identified among 53 Indian Jews. All carriers within each ethnic group had the same haplotype, suggesting a common founder for each mutation.
Expression and prognostic significance of survivin inde novoacute myeloid leukaemia
Authors: Adida Colette , Recher Christian , Raffoux Emmanuel , Daniel Marie‐Thérèse , Taksin Anne‐Laure , Rousselot Philippe , Sigaux François , Degos Laurent , Altieri Dario C. , Dombret Hervé
Published: July 2010

Survivin is an inhibitor of apoptosis (programmed cell death) overexpressed in various human cancers, but undetectable in normal differentiated tissues. A potential distribution and prognostic significance of survivin in patients withde novoacute myeloid leukaemia (AML) was investigated. By immunofluoresence of bone‐marrow specimens and peripheral blood mononuclear cells, survivin was detected in 75 out of 125 interpretable AML cases (60%), with reactivity in 50–90% of AML cells. Survivin expression correlated with a lower white blood cell count (WBC) (P = 0·008 by the Mann–Whitney test) and was associated, in the 55 cases of FAB M0/M1/M2, with leukaemic granulocytic maturation (one out of five M/L0, 11 out of 22 M/L1 and 23 out of 28M/L2;P = 0·007 by the Fisher test). In 69 patients treated with the Acute Leukaemia French Association (ALFA) 9000 protocol, survivin expression was significantly associated with a lower WBC (P = 0·03 by the Mann–Whitney test) and favourable/intermediate cytogenetics (P = 0·03 by the Fisher test). There was no significant difference in complete remission rate or overall survival between survivin‐positive and survivin‐negative AML patients (P = 0·15 by the log‐rank test). However, survivin expression became an independent negative prognostic factor for survival when adjusted with the Cox model for established prognostic factors in AML (cytogenetics, age and WBC) or for the ALFA 9000 treatment arm (RR = 2·8 andP = 0·026, by the likelihood‐ratio test). These data suggest that survivin expression may be considered as a new unfavourable prognostic factor ofde novoAML and suggest a role for apoptosis inhibition in influencing disease outcome.
Aberrant expression of HLA‐G antigen in interferon γ‐stimulated acute myelogenous leukaemia
Authors: Mizuno Shinichi , Emi Nobuhiko , Kasai Masanobu , Ishitani Akiko , Saito Hidehiko
Published: July 2010

We have analysed the expression of HLA‐G in 40 leukaemia samples of various subtypes [seven cases of acute lymphoblastic leukaemia (ALL), 28 cases of acute myelogenous leukaemia (AML), three cases of chronic myelogenous leukaemia (CML) and two cases of chronic lymphocytic leukaemia (CLL)] by flow cytometry using HLA‐G‐specific monoclonal antibody. No leukaemia samples expressed HLA‐G without incubation with interferon (IFN)‐γ. However, six out of 28 (21%) AML samples expressed HLA‐G upon incubation with IFN‐γ. These six samples derived from one out of seven M2, two out of eight M4 and three out of five M5. The results indicated that AML cells, especially myelomonocytic leukaemia samples, are capable of expressing the HLA‐G molecule.
Abdominal venous thrombosis in neonates and infants: role of prothrombotic risk factors – a multicentre case–control study
Authors: Heller Christine , Schobess Rosemarie , Kurnik Karin , Junker Ralf , Günther Gudrun , Kreuz Wolfhart , Nowak‐Göttl Ulrike ,   for the Childhood Thrombophilia Study Group
Published: July 2010

The factor V (FV) G1691A mutation, the prothrombin (PT) G20210A variant, the methylenetetrahydrofolate reductase (MTHFR) T677T genotype, together with fasting homocysteine (HCY) concentration, lipoprotein (Lp)(a), anti‐thrombin (AT), protein C (PC), protein S (PS) and anti‐cardiolipin antibodies were investigated in 65 consecutively recruited infants (neonate to < 12 months) with renal venous thrombosis (RVT; n = 31), portal vein thrombosis (PVT; n = 24) or hepatic vein thrombosis (HVT n = 10), and 100 age‐ and sex‐matched healthy controls. FV G1691A was found in 14 babies (heterozygous: RVT n = 9, PVT n = 4; homozygous HVT n = 1) and five controls, the MTHFR TT677 genotype together with increased HCY in four infants with thrombosis (RVT n = 2; PVT n = 1; HVT n = 1) compared with one control, and the PT G20210A variant was present in one control only. PC type I deficiency was diagnosed in three patients (RVT n = 2; PVT n = 1) and AT deficiency in two patients (RVT n = 1; PVT n = 1). Three neonates with spontaneous thrombosis showed FV G1691A combined with Lp(a) and the FV G1691A was combined with the PT G20210A genotype in two infants. Additional triggering factors were reported in 27 patients (41·5%). The overall odds ratios (ORs) and 95% confidence intervals (CIs) with respect to the different thrombosis locations were: RVT (OR/CI: 10·9/3·85–31·1; P < 0·0001), PVT (5·47/1·7–17·6; P < 0·0007) and HVT (3·3/0·58–18·7; P = 0·18). The data presented here suggest that genetic prothrombotic risk factors also play an important role in abdominal venous thrombosis during infancy.
Characterization of osteoclast precursors in human blood
Authors: Shalhoub V. , Elliott G. , Chiu L. , Manoukian R. , Kelley M. , Hawkins N. , Davy E. , Shimamoto G. , Beck J. , Kaufman S. A. , Van G. , Scully S. , Qi M. , Grisanti M. , Dunstan C. , Boyle W. J. , Lacey D. L.
Published: July 2010

Osteoclast precursors (OCPs) circulate in the mononuclear fraction of peripheral blood (PB), but their abundance and surface characteristics are unknown. Previous studies suggest that the receptor activator for NF‐κB (RANK) on cytokine‐treated OCPs in mouse bone marrow interacts with osteoprotegerin ligand (OPGL/TRANCE/RANKL/ODF) to initiate osteoclast differentiation. Hence, we used a fluorescent form of human OPGL (Hu‐OPGL‐F) to identify possible RANK‐expressing OCPs in untreated peripheral blood mononuclear cells (PBMCs) using fluorescence‐activated cell sorting analysis. Monocytes [CD14‐phycoerythrin (PE) antibody (Ab) positive (+) cells, 10–15% of PBMCs] all (98–100%) co‐labelled with Hu‐OPGL‐F (n > 18). T lymphocytes (CD3‐PE Ab+ cells, 66% of PBMCs) did not bind Hu‐OPGL‐F; however, B cells (CD19‐PE Ab+ cells, 9% of PBMCs) were also positive for Hu‐OPGL‐F. All Hu‐OPGL‐F+ monocytes also co‐labelled with CD33, CD61, CD11b, CD38, CD45 and CD54 Abs, but not CD34 or CD56 Abs. Hu‐OPGL‐F binding was dose dependent and competed with excess Hu‐OPGL. When Hu‐OPGL‐F+, CD14‐PE Ab+, CD33‐PE Ab+, Hu‐OPGL‐F+/CD14‐PE Ab+ or Hu‐OPGL‐F+/CD33‐PE Ab+ cells were cultured with OPGL (20 ng/ml) and colony‐stimulating factor (CSF)‐1 (25 ng/ml), OC‐like cells readily developed. Thus, all freshly isolated monocytes demonstrate displaceable Hu‐OPGL‐F binding, suggesting the presence of RANK on OCPs in PB; also, OCPs within a purified PB monocyte population form osteoclast‐like cells in the complete absence of other cell types in OPGL and CSF‐1 containing medium.
The reconstitution of CD45RBhiCD4+ naive T cells is inversely correlated with donor age in murine allogeneic haematopoietic stem cell transplantation
Authors: Hirayama Masahiro , Azuma Eiichi , Jiang Qi , Kobayashi Michihiro , Iwamoto Shotaro , Kumamoto Tadashi , Kisenge Rodrick , Yamamoto Hatsumi , Komada Yoshihiro
Published: July 2010

A high incidence of opportunistic infections after unrelated bone marrow transplantation has been reported. Delayed lymphocyte recovery may be associated with opportunistic infections. Immune reconstitution is influenced by recipient age and graft‐vs‐host disease (GVHD). In fact, children develop GVHD less frequently than adults. However, the role of donor age is largely unknown. We examined the effect of donor age on lymphocyte reconstitution after transplant. Three‐month‐old BALB/c recipient mice were lethally irradiated and transplanted with allogeneic haematopoietic stem cells from A/J donor mice of different ages, ranging from 0 d to 12 months. The recovery of absolute lymphocyte counts and those of CD3+ T cells, CD4+ T cells and CD45RBhi CD4+ naive T cells in the early post‐transplant period correlated inversely with donor age. Recipient mice transplanted with haematopoietic stem cells from younger donors showed significantly higher survival rates and mitogenic responses than adult donors. As T cells, especially CD4+ naive T cells, play an important role in host defence, faster recovery of CD4+ naive T cells in younger donors may contribute to reduced mortality in the early post‐transplant period. The results suggest that it could be better to choose a younger donor if sufficient cell dose is available.
A factor XI deficiency associated with a nonsense mutation (Trp501stop) in the catalytic domain
Authors: Iijima Kenji , Udagawa Akihide , Kawasaki Hironaka , Murakami Fumiyo , Shimomura Tokio , Ikawa Shiro
Published: July 2010

We identified a novel mutation in an asymptomatic 65‐year‐old Japanese man with severe factor XI deficiency. Sequence analysis after polymerase chain reaction single‐stranded conformation polymorphism (PCR‐SSCP) analysis of his factor XI gene revealed a G→A transition in codon 501 of exon 13, resulting in a substitution of Trp501 (TGG) by a stop codon (TAG) in the catalytic domain. This mutation abolished a FokI restriction site. The PCR product from normal subjects was digested with FokI and yielded two fragments, one of 223 bp and one of 47 bp. The PCR product from the patient gave a single 270‐bp fragment, demonstrating possible homozygosity.
A randomized comparison of once versus twice daily recombinant human granulocyte colony‐stimulating factor (filgrastim) for stem cell mobilization in healthy donors for allogeneic transplantation
Authors: Kröger Nicolaus , Renges Helmut , Krüger William , Gutensohn Kai , Löliger Cornelius , Carrero Isabel , Cortes Lourdes , Zander Axel R.
Published: July 2010

To evaluate the schedule dependency of granulocyte colony‐stimulating factor (G‐CSF) (filgrastim) for stem cell mobilization, we conducted a randomized comparison in 50 healthy donors, with one subcutaneous daily injection of 10 µg/kg G‐CSF (n = 25) compared with twice injections daily of 5 µg/kg G‐CSF (n = 25). The two groups were well balanced for age, body weight and sex. G‐CSF application was performed on an out‐patient basis and leukapheresis was started in all donors on day 5. The most frequent side‐effects of G‐CSF were mild to moderate bone pain (88%), mild headache (72%), mild fatigue (48–60%) and nausea (8%) without differences between the two groups. The CD34+ cell count in the first apheresis was 5·4 × 106/kg donor weight (range 2·8–13·3) in the 2 × 5 µg/kg group compared with 4·0 × 106/kg (range 0·4–8·8) in the 1 × 10µg/kg group (P = 0·007). The target of collecting > 3·0 × 106 CD34+ cells/kg donor weight with one apheresis procedure was achieved in 24/25 (96%) donors in the 2 × 5 µg/kg group and in 17/25 (68%) donors in the 1 × 10 µg/kg group. The target of collecting > 5·0 × 106 CD34+ cells/kg in the first apheresis was achieved in 64% in the 2 × 5µg/kg group, but in only 36% in the 1 × 10 µg/kg group. The progenitor cell assay for granulocyte–macrophage colony‐forming units (CFU‐GM) and erythroid burst‐forming units (BFU‐E) was higher in the 2 × 5 µg/kg group than in the 1 × 10 µg/kg group (7·0 vs. 3·5 × 105/kg, P = 0·01; 6·6 vs. 5·0 × 105/kg; P = 0·1). Administering G‐CSF (filgrastim) at a dosage of 5 µg/kg twice daily rather than 10 µg/kg once daily is recommended; this leads to a higher CD34+ cell yield and requires fewer apheresis procedures without increasing toxicity or cost.
Expression and production of interleukin 10 in human myeloma cell lines
Authors: Otsuki Takemi , Yamada Osamu , Yata Kenichiro , Sakaguchi Haruko , Kurebayashi Junichi , Yawata Yoshihito , Ueki Ayako
Published: July 2010

Recent investigations of the cytokine network surrounding myeloma cells have disclosed the importance of gp130‐related cytokines including interleukin (IL)‐6 for myeloma cell survival and proliferation, identification of IL‐10 as a growth factor for myeloma cells, the close relationship between IL‐10 and the receptors for gp130‐related cytokines, and the growth enhancement effect of IL‐11 and IL‐7 on myeloma cells. In this study, IL‐10 production was observed in three out of seven human myeloma cell lines examined and five (including three producing lines) out of 10 lines exhibited mRNA expression of IL‐10. The IL‐10 mRNA expression was also enhanced in approximately one third of primary specimens, whereas the IL‐10 receptor (R) expression was not changed compared with that of normal component marrow controls. However, reverse transcription‐polymerase chain reaction (RT‐PCR) assay of various cytokines and their receptors showed no particular association with IL‐10‐producing myeloma lines compared with non‐producing lines. Supplementing exogenous IL‐10 or neutralization of the IL‐10 signal by anti‐IL‐10 monoclonal antibody (mAb) in a culture conditions did not significantly affect myeloma cell growth regardless of expression of IL‐10 or its receptor (IL‐10R). However, supplement of anti‐IL‐10 mAb caused upregulation of certain genes such as IL‐11, leukaemia inhibitory factor receptor (LIFR) and syndecan‐1 in IL‐10R‐expressing cell lines. These findings indicate that the cytokine network surrounding myeloma cells is complicated and variable. In addition, IL‐10 may modify this network and the cellular biological properties of myeloma cells rather than cell proliferation.
Minimal residual disease studies are beneficial in the follow‐up of TEL/AML1 patients with B‐precursor acute lymphoblastic leukaemia
Authors: De Haas V. , Oosten L. , Dee R. , Verhagen O. J. H. M. , Kroes W. , Van Den Berg H. , Van Der Schoot C. E.
Published: July 2010

The t(12;21)(p13;q22) translocation has been identified as the most common chromosomal abnormality in childhood acute lymphoblastic leukaemia (ALL). Initially, several investigators reported an excellent prognosis in paediatric leukaemias with this translocation, but other studies showed a 20% incidence in relapsed ALL. We performed an extensive analysis of 90 ALL patients. In 17 (19%) cases a TEL/AML1 fusion was found. However, this group was not representative as it included a high number of relapsed patients compared with the normal incidence in B‐precursor ALL [54 in continuous complete remission (CCR) and 36 relapsed patients] and only a slightly better prognosis for TEL/AML1‐positive patients was found (not significant) (four relapses in 17 TEL/AML1‐postive patients vs. 32 relapses in 73 TEL/AML1‐negative patients). Comparison of known prognostic factors (age, sex, ploidy, white blood cell count and immunophenotype) between relapsed TEL/AML1‐positive and TEL/AML1‐positive patients in CCR did not reveal differences, except that the white blood cell count was significantly higher in the relapsed group (P = 0·001). Time between diagnosis and relapse was not different for the relapsed TEL/AML1‐positive group vs. the relapsed TEL/AML1‐negative group. In 11 TEL/AML1‐positive patients, the minimal residual disease (MRD) level at the end of induction therapy was quantified in a limiting dilution assay using IGH or TCRD junctional regions as polymerase chain reaction (PCR) targets. In all four relapsed patients, the level of MRD at the end of induction therapy was high (range 0·24–1·2%), whereas in all seven CCR patients, the MRD level was extremely low (0·02 to < 0·001%). In agreement with previous studies in which MRD levels at the end of induction therapy were found to be the strongest risk factor independent of other risk factors, in the present study we show that the MRD level remains a risk factor independent of the presence of a TEL/AML1 fusion gene.
Late outcomes after bone marrow transplant for aplastic anaemia
Authors: Eapen Mary , Ramsay Norma K. C. , Mertens Ann C. , Robison Leslie L. , DeFor Todd , Davies Stella M.
Published: July 2010

Allogeneic transplantation is effective in reconstituting haemopoiesis in severe aplastic anaemia (SAA). We report long‐term health‐related outcomes in 37 children and young adults with SAA transplanted between 1975 and 1996. The median length of follow‐up was 17 years (range, 4–25 years). Using a case–control design, late social and medical outcomes in transplant recipients were compared with 146 control subjects matched for gender and age. The majority of patients received an irradiation‐containing preparative regimen. There were no significant differences in the self‐rating of health status between transplant recipients and controls (P = 0·8), with 71% reporting their health status as excellent and 29% as good compared with 74% and 26% of controls. They demonstrate the same normal psychosexual function as their peers and have similar educational achievements and employment history. Transplant recipients and controls are equally likely to have held a job or be currently employed and there are no significant differences in their personal income (OR = 0·60, 95% CI = 0·11–3·37). Although transplant recipients have had problems related to health insurance policies, the majority have adequate health insurance coverage. There were no differences in chronic health problems between transplant recipients and control subjects, except for expected increases in cataracts, short stature in men, hypothyroidism and gonadal dysfunction. Using self‐assessment, these transplant recipients indicated an excellent level of satisfaction and social integration, showing transplantation to be an effective long‐term therapy for SAA.
Genetic linkage of Kozak sequence polymorphism of the platelet glycoprotein Ibα with human platelet antigen‐2 and variable number of tandem repeats polymorphism, and its relationship with coronary artery disease
Authors: Ishida Fumihiro , Ito Toshiro , Takei Manabu , Shimodaira Shigetaka , Kitano Kiyoshi , Kiyosawa Kendo
Published: July 2010

The −5 C/T polymorphism of platelet glycoprotein (GP) Ibα is a major determinant of the level of GP Ib/V/IX complex surface expression. We investigated the frequency of this polymorphism among Asian populations. The gene frequencies of cytosine (C) in this polymorphism were 0·283 and 0·219 in Japanese and Korean populations respectively. The C allele is linked with human platelet antigen (HPA)‐2a and smaller types of variable number of tandem repeats (VNTR). A novel allele, C‐HPA‐2a‐D of VNTR, was found. No association was observed between these alleles and coronary artery disease in this case–control study. The clinical relevance of this polymorphism in the thrombotic status remains undetermined.
Identification of a new CA dinucleotide repeat in the human factor VIII gene
Authors: Lin Shu‐Rung , Lin Shu‐Wha , Hsu Ya‐Chu , Shen Ming‐Ching
Published: July 2010

We describe the identification of a new CA dinucleotide repeat marker for the diagnosis of haemophilia A carriers. The marker (CA‐6) is present in intron 6 as a single copy 5 kb upstream of exon 7. Of 195 and 118 X chromosomes from normal individuals and haemophilia A patients, respectively, we observed three alleles of CA‐6 with 12–14 repetitions [(CA)12−14]. The frequencies were 0·5% and 0% for (CA)12, 99% and 95·8% for (CA)13, and 0·5% and 4·2% for (CA)14 in normals and patients respectively. We conclude that the low polymorphism of the CA‐6 marker renders it less useful for the diagnosis of Chinese haemophilia A carriers.