bluuebunny/crossref_metadata_2025 huggingface dataset:
Antithrombin III Pescara: a defective AT III variant with no alterations of plasma crossed immunoelectrophoresis, but with an abnormal crossed immunoelectrofocusing pattern
Authors: Leone G. , De Stefano V. , Di Donfrancesco A. , Ferrelli R. , Traisci G. , Bizzi B.
Published: July 2010

Summary. An abnormal AT III variant was found in five members from a family where a high incidence of thromboembolism occurred. In all the affected subjects AT III antigen concentration was normal, whereas antithrombin and anti‐factor Xa progressive activities as well as heparin cofactor activities were low. Crossed immunoelectrophoresis performed either in absence or in presence of heparin showed a normal plasma pattern. Further chromatographic investigations showed a normal affinity to heparin. An abnormal plasma pattern was evidentiated by crossed immunoelectrofocusing throughout all the AT III pH range. These data are consistent with the presence of an abnormal AT III variant with a defective binding to serine proteases and clearly identifiable only by crossed immunoelectrofocusing. This variant appeared different from the other qualitative AT III defects so far described and was named ‘Antithrombin III Pescara’.
The bone marrow in human cerebral malaria: parasite sequestration within sinusoids
Authors: Wickramasinghe S. N. , Phillips R. E. , Looareesuwan Sornchai , Warrell D. A. , Hughes Madeleine
Published: July 2010

Bone marrow aspirates from patients with cerebral malaria were studied with the light and electron microscopes. Various abnormalities were found including: (1) an increase in plasma cells and macrophages, sometimes to a marked degree; (2) phagocytosis of parasitized red cells by macrophages and of merozoites by neutrophil metamyelocytes. neutrophil granulocytes and macrophages; (3) an increase in the proportion of eosinophil granulocytes and their precursors; (4) the presence of giant metamyelocytes; and (5) morphological abnormalities of erythroblasts, particularly irregularly‐shaped nuclei and karyorrhexis. A high percentage of the red cells within marrow sinusoids were parasitized and the parasitized cells were attached to the endothelium. Some marrow sinusoids were packed with and completely obstructed by parasitized cells. Strands of electron‐dense material were sometimes found connecting the knobs of parasitized red cells to endothelial cells or to the knobs of adjacent parasitized red cells. A striking finding was a complex interdigitation between cytoplasmic processes developed by some of the parasitized red cells and those developed by the endothelial cells to which they were attached. Occasionally, cytoplasmic processes arising from marginated parasitized red cells completely penetrated the endothelial cell and emerged extravascularly. Several parasitized red cells were also found extravascularly between haemopoietic cells. Sequestration of parasitized red cells within small blood vessels may play a part in the pathogenesis not only of the encephalopathy of cerebral malaria but also of the bone marrow dysfunction in severe malaria.
Cryopreservation disturbs stimulus‐response coupling in a platelet subpopulation
Authors: Dullemond‐Westland A. C. , Van Prooijen H. C. , Rlemens M. I. , Akkerman J. W. N.
Published: July 2010

Cryopreservation of platelet suspensions leads to a severe reduction in the functional responses of these cells. This detect primanly resides in a subpopulation which consists of about half the total platelet mass and shows an increased susceptibility to the Cryopreservation procedure. Following freeze‐thawing this subpopulation failed to aggregate upon stimulation with collagen despite a normal appearance of membrane glycoproteins. Also the contents of α granules was similar to the whole suspension and since changes in α and dense granule contents often occur in parallel, the poor aggregability could not be attributed to a defect in secretion granules. In contrast, the generation of phosphatidyl‐inositol metabolites, the increase in cytosolic Ca2+ content and the phosphorylation of proteins of mol wt 47 000 and 20 000 seen when normal platelets are activated, were completely absent in the subpopulation. These data reveal a severe defect in stimulus‐response coupling mechanisms in about half of the platelets after freeze‐thawing. The other half of the suspension endures this treatment relatively unharmed and may be used for transfusion.
Glycolipids and glycopeptides of red cell membranes in congenital dyserythropoietic anaemia type II (CDA II)
Authors: Zdebska E. , Anselstetter V. , Pacuszka T. , Krauze R. , Chelstowska A. , Heimpel H. , Kosacielak J.
Published: July 2010

The composition and structure of neutral and acidic oligoglycosylceramides, polyglycosylceramides and polyglycosylpeptides were determined in erythrocyte membranes of two patients with congenital dyserythropoietic anaemia type II. In keeping with previous studies we found an elevated accumulation in CDA II erythrocytes of LacCer, Lc3Cer and nLc4Cer. Gb4Cer was elevated in erythrocytes of only one of the two patients tested. In addition we found a significant increase of 6IVNeuAcnLc4Cer ganglioside. Polyglycosylceramides were elevated 6‐fold but they resembled those of cord erythrocytes with respect to complexity and the number of side chains. Polyglycosylpeptides of CDA II erythrocytes were decreased 7‐fold. These glycopeptides were, however, heterogeneous with respect to branching pattern; the minor fraction was highly branched whereas the major one was more linear in structure. Both polyglycosylceramides and polyglycosylpeptides exhibited high I and i antigenicity. We postulate that the accumulation of glycolipids and underglycosylation of glycoproteins in CDA II membranes results from the prolongation of G1 and possibly M phases of the mitotic cycle of the erythroid cells in which glycolipids are preferentially synthesized.
A streptokinase dependent plasma factor (SKDF) induces leucocyte tissue factor activity
Authors: Niemetz Julian , Kang Sungzong
Published: July 2010

Various inducers endow human leucocytes with a procoagulant activity of tissue factor type. We have observed a novel plasma factor which in combination with streptokinase induces powerful leucocyte procoagulant activity. This streptokinase dependent factor (SKDF) is present in normal plasma or serum albeit quantitatively different in individual donors. The generation of tissue factor activity as a function of streptokinase–plasma complex shows a specific and saturable sigmoidal dose–response curve. The Hill plot shows a straight line with Hill coefficient, H= 2.2, suggesting a strong positive cooperativity for the binding of this streptokinase–plasma complex to the leucocyte surface receptor for the signal transduction leading to the biosynthesis of tissue factor apoprotein. It also suggests that the leucocyte surface receptor for streptokinase–plasma complex differs from that for endotoxin lipopolysaccharides. SKDF is of apparent high molecular weight. It does not appear to be an antibody to streptokinase since its level does not correlate with the level of antibodies to streptokinase, and it does not correlate with the antistreptolysin titre. Furthermore, SKDF does not bind to protein A. It has a narrow pH range of stability, and is destroyed at 56°C, or at freeze‐drying, Urokinase, another plasminogen activator, or plasmin were unable to activate SKDF to induce the leucocyte procoagulant activity. SKDF may play a role in thrombolytic therapy.
A combination of factor Xa and phosphatidylcholine‐phosphatidylserine vesicles bypasses factor VIII in vivo
Authors: Giles Alan R. , Mann Kenneth G. , Nesheim Michael E.
Published: July 2010

A combination of phosphatidylcholine‐phosphatidylserine lipid vesicles (PCPS), as a source of coagulant active phospholipid, when infused with factor Xa bypasses factor VIII in vivo. To demonstrate this, a reproducible model of bleeding in haemophilic dogs was used. Control studies were performed in normal dogs. In initial studies, factor Xa/PCPS at a dose of 6.5 10‐12 and 4.0 × 10‐7 moles/kg respectively failed to correct the abnormal bleeding in the haemophilic animals and initiated a bleeding diathesis in the normal controls. Coagulation studies and immunoblotting demonstrated activation of protein C and an anticoagulant effect resulting from significant falls in the levels of factors V and VIII. Adjustment of the dose of factor Xa/PCPS to 2.6 × 10‐11 and 4.0 × 10‐8 moles/kg respectively produced an immediate haemostatic effect in both haemophilic and normal animals with bleeding stopping within 15–30 s. Despite this observation, protein C activation was again noted. It is concluded that the presence of coagulant active phospholipid and factor Xa in prothrombin complex concentrates may explain the observed factor VIII bypassing activity of these preparations and that the use of a controlled formulation of these two components may provide a more effective approach to the management of patients with factor VIII inhibitors.
In Situ immunophenotyping of lymphocytes in human bone marrow: an immunohistochemical study
Authors: Horny H.‐P. , Engst U. , Walz R. S. , Kaiserling E.
Published: July 2010

Summary Lymphoid cells in human bone marrow are either assembled focally or occur in a diffuse. loosely scattered infiltrate. While the focal lesions are easily detected, the lymphoid cells of the diffuse infiltrate are hardly recognizable with conventional stains. Quantitative immunohistological analysis of 103 trephine biopsies, including cases with reactive disorders (e.g. myeloid hyperplasia, aplastic anaemia) and neoplastic processes (e.g. myeloproliferative disorders, B‐cell non‐Hodgkin's lymphomas) and some specimens with normal architecture yielded the following results: (1) Various antibodies recognizing B cells (L26, 4KB5. MB1, Ki‐B3), T cells (UCHL1, MT1) and NK cells (Leu‐7) are effective in paraffin‐embedded bone marrow sections, thus enabling analysis of the in situ distribution of normal lymphocyte subsets and subtyping of lymphomatous infiltrates. (2) The lymphocytes of the diffuse infiltrate constituted about 1‐5% of all nucleated cells in normal bone marrow. (3) In the diffuse infiltrate, T lymphocytes were regularly observed in higher numbers than B cells, and Leu‐7 + cells were rare or virtually absent, irrespective of the diagnosis. (4) The focally assembled lymphoid cells were mainly B lymphocytes, but many T cells were always intermingled. This was true for both reactive follicles and neoplastic lymphomatous infiltraters, which generally cannot be differentiated on the basis of immunohistological findings alone.
Platelet storage pool deficiency: diagnosis in patients with prolonged bleeding times and normal platelet aggregation
Authors: Israels S. J. , McNicol A. , Robertson C. , Gerrard J. M.
Published: July 2010

We evaluated 46 patients with prolonged bleeding times, and no demonstrable abnormalities of either von Willebrand factor or platelet aggregation, for possible deficiency of platelet storage pool. Studies of ATP release from thrombin‐stimulated platelets and enumeration of dense granules in platelet whole mounts were performed in these patients. Seventeen patients (35%) had both decreased ATP release and decreased numbers of dense granules, suggesting the presence of a storage pool defect. Thus, storage pool deficiency may be present in the absence of the classical aggregation abnormalities. Evidence of storage pool deficiency should be considered in all patients with an isolated unexplained prolongation of the bleeding time. The methods used in this study are readily applicable to most clinical laboratories.
Arachidonic acid metabolism in platelets stored for 5 days
Authors: Cesar J. M. , Navarro J. L.
Published: July 2010

(C‐14)‐Arachidonic acid [(C‐14)‐AA] metabolism was studied in platelet concentrates (PCs) stored for 5 d. There was a gradual decrease in uptake of radioactivity from day 0 to 3 (P < 0.01). On day 0, distribution of radioactivity in platelet phospholipids (PLs), and formation of phosphatidic acid, HETE and cyclooxygenase products, when platelets were exposed to thrombin (5 U/ml), were similar to that reported for fresh platelets. On day 3 there was a change in the distribution of (C‐14)‐AA in platelet PLs which consisted of an increase in the percentage of radioactivity bound to phosphatidylserine, from 5.3±0.9% on day 0 to 8.8±1.5% on day 3 (P<0.001), and a decrease in (C‐14)‐AA in phosphatidylinositol (PI), from 12.4±1.5% on day 0, to 7.9±0.9% on day 3 (P<0.001). Phosphatidic acid generated by thrombin‐stimulated platelets on day 0, comprised 2.6±0.5% of total radioactivity, but dropped to 1.4±0.3% on day 3 (P<0.001), and 0.9±0.2% on day 5 (P<0.01). These values showed a good correlation with the percentage of (C‐14)‐AA released from PI on the same days (r=0‐9). On day 0, 13.4 ± 4.4% of platelet radioactivity was released from phosphatidylcholine by thrombin, but this amount was reduced to 6.8±3.4% on day 5 (P<0.05). Generation of radioactive 12‐hydroxy‐5,8,10,14‐eicosatetraynoic acid (HETE) also dropped from 7.2±2.9 on day 0. to 2.1 ± 1 on day 5 (P< 0.01). We could not detect changes in cyclooxygenase metabolites. In conclusion, we suggest that various enzymatic pathways implicated in AA metabolism by platelets are impaired by storage.
Establishment of a leukaemic cell line from a patient with acquisition of chromosomal abnormalities during disease progression in myelodysplastic syndrome
Authors: Nakagawa Toshitaro , Matozaki Sachiko , Murayama Tohru , Nishimura Ryuichiro , Tsutsumi Masayoshi , Kawaguchi Ryuji , Yokoyama Yasunobu , Hikiji Kazumasa , Isobe Takashi , Chihara Kazuo
Published: July 2010

Summary. A cell line designated SKM‐1 was newly established from leukaemic cells of a 76‐year‐old Japanese male patient with monoblastic leukaemia following myelodysplastic syndrome (MDS). The cells were obtained from peripheral blood of the patient when he lost multiple point mutations of ras genes with acquisition of chromosomal abnormalities during disease progression in MDS. The cells grew as a single floating cell, and have been continuously growing with the morphological characteristics of immature monoblasts by serial passages during the past 42 months with a doubling time of about 48 h. By cytochemical analysis. the cloned cells were positive for butyrate esterase, but negative for the Epstein‐Barr virus associated nuclear antigen. Phenotypic analysis revealed the expression of myelomonocyte specific antigens such as CD4, CD13, CD33 and HLA‐DR. Cells from the primary peripheral blood and those from SO passages of the SKM‐1 cell line both possessed no activated ras genes but showed karyotype abnormalities with 46.XY, del(9)(q13;q22), der(17) t(17:?)(p13:?). The SKM‐1 cells have two mutations in p53 gene and overexpress the pS3 products. This cell line may contribute to a better understanding of molecular mechanisms in the progression from MDS to myelogenous leukaemia.
Expression of CD45RA (naive) and CD45RO (memory) antigens in T‐acute lymphoblastic leukaemia
Authors: Falcão Roberto P. , Garcia Aglair B.
Published: July 2010

Summary. We have determined the distribution of CD45RO (memory) and CD45RA (naive) antigens in the bone marrow blasts from 25 patients with T‐acute lymphoblastic leukaemia (T‐ALL). Four groups of patients were identified on the basis of reactivity with specific antibodies by flow cytometric analysis: (a) CD45KA‐/CD45K0+ (16 patients): four CD4 ‐ /CD8 +, seven CD4 +/CD8 + and five CD4 ‐ /CU8 ‐: (b) CD45KA +/CD45RO‐ (three patients): three CD4‐/ CD8‐; (c) five CD45RA‐/C1>45RO‐: one CD4 +/CD8‐: one CD4‐/CU8 +: three CD4 +/CD8 +: (d) CD45RA +/ CD45K0+ (one case): CD4+/CD8 ‐. There was no correlation between the expression of the naive and the memory phenotypes and the presence of CD4, CD8 or any other antigen except the CD10 antigen which was expressed by all CD45RA‐/CD45RO‐ patients. The predominance of the CD45KA‐/CD45RO+ phenotype (65%) and the low incidence of the hybrid phenotype CD45KA +/CD45KO+ (5%) in T‐ALL. differs from the results reported by others for chronic or prolymphocytic T‐cell leukaemias, in which the simultaneous expression of these maturational antigens was detected in approximately half of the cases.
Cytogenetic studies in splenic lymphoma with villous lymphocytes
Authors: Oscier D. G. , Matutes E. , Gardiner A. , Glide S. , Mould S. , Brito‐Babapulle V. , Ellis J. , Catovsky D.
Published: July 2010

Summary. We report the cytogenetic findings on 31 cases of splenic lymphoma with villous lymphocytes (SLVL). TPA stimulated cells from peripheral blood (28 cases), spleen (two cases) and lymph node (one case) with SLVL have been analysed. A clonal chromosome abnormality was found in 27/31 patients (87%): this was identified as a simple abnormality in 12 cases and a complex one in 15. Four recurring abnormalities were seen: t(11:14) (q13:q32)in five patients, deletions or translocations involving 7q in seven patients. iso 17q in four patients and translocations involving 2p11 in four patients. The high frequency of clonal chromosome abnormalities in SLVL contrasts with the usually benign clinical course of this disease.

Abnormalities found frequently in patients with chronic lymphocytic leukaemia (CLL) such as trisosmy 12 and deletions or translocations involving 13q14 were each seen in only one patient. No case had the t(14:18) characteristic of follicular lymphoma.

Our findings demonstrate the high frequency of clonal and often complex chromosome abnormalities in SLVL. Although a unique chromosome rearrangement has not been identified, a pattern of four recurrent abnormalities has emerged. Our results suggest that SLVL is distinct on cytogenetic grounds from B‐CLL and follicular lymphoma but shows similarity with mantle cell lymphoma, lymphoplasmacytic lymphoma and B‐PLL.
Cytokine gene expression in human multiple myeloma
Authors: Portier Marielle , Zhang Xue‐Guang , Ursule Elisabeth , Lees Delphine , Jourdan Michel , Bataille Regis , Klein Bernard
Published: July 2010

Summary. In the present study the gene expression of cytokines promoting in vitro myeloma‐cell growth was investigated by Northern blot analysis using total RNA of 36 tumour samples of patients with multiple myeloma (MM) or plasma cell leukaemia and poly(A)+ RNA of 10 human myeloma cell lines (HMCL). These cytokines included interleukin (IL)‐1α, IL‐1β, IL‐3, IL‐6, granulocyte‐macrophage (GM)‐colony‐stimulating factor (CSF) and granulocyte (G)‐CSF. IL‐1β, IL‐6 and G‐CSF genes were coexpressed in most patients, although at variable levels. IL‐1α transcripts were detected in 32% of patients in whom coexpression of IL‐1β gene was found. IL‐3 gene was not expressed in patients' cells and GM‐CSF mRNA was detected in only 1/32 patients. No detectable transcripts for the above cytokines were present in HMCL, whereas IL‐6 gene was expressed in 2/10 HMCL.

We also looked for the presence of transcripts for IL‐2, leukaemia inhibitory factor (LIF) and transforming growth factor (TGF)β in cells of tumour samples from the same patients and in HMCL. IL‐2 gene was not expressed in MM patients and HMCL. Weak expression of LIF gene was detected in three patients (9%), and transforming growth factor β (TGFβ) mRNA was observed in 12/12 tumour samples analysed and all HMCL.

These results suggest that, among cytokines shown to control myeloma‐cell growth in vitro, IL‐1, IL‐6 and G‐CSF could play a role in the development of myeloma disease in vivo.
Interaction of hepatitis B and hepatitis C infection in haemophilia
Authors: Hanley J. P. , Dolan G. , Day S. , Skidmore S. J. , Irving W. L.
Published: July 2010

Summary. The management of haemophilia has been greatly complicated by the clinical sequelae of viral infection acquired through contaminated blood products. Many haemophiliacs have been infected by several viruses and the interaction between these viruses may be complex. In a cohort of 42 anti‐HCV positive haemophiliacs, five were also found to be positive for HBsAg. All five were HCV reverse transcriptase/PCR negative compared to the 4/37 (11%) anti‐HCV positive haemophiliacs who were HBsAg negative (P=0.0001). We have identified a striking interaction between hepatitis C (HCV) and hepatitis B (HBV) in haemophiliacs co‐infected by these agents, suggestive of the phenomenon of viral interference.
In vitro inhibition of tumour necrosis factor‐α and interleukin‐6 production by intravenous immunoglobulins
Authors: Toungouz Michel , Denys Chantal H. , Groote Donat De , Dupont Etienne
Published: July 2010

In vitro data about the action of pooled immunoglobulins (Ig) on cytokine (CK) production are controversial. The recent finding of natural antibodies against staphylococcal toxins neutralizing superantigen‐induced activation prompted us to design an assay determining their ability to modulate staphylococcal enterotoxin B (SEB) induced CK production (IL‐6 and TNF‐α). Presence of anti‐SEB antibodies was demonstrated by a dot‐blot assay in the three preparations tested. Preincubation of SEB with pooled Ig prior to addition into the test tube containing PBMCs (neutralizing condition) resulted in a strong inhibition of both TNF‐α and IL‐6 release (TNFα: 59 ± 5% inhibition, P<0.0001; IL‐6: 71 ± 7% inhibition. P<0.0001, n= 15). Anti‐CD3 MoAb‐induced CK production was not modified. During our study it was found that experimental conditions were critical to observe this inhibitory effect. Reversing the previous procedure by adding PBMCs into the test tube containing pooled Ig mixed with SEB resulted in a marked induction of TNF‐α and IL‐6 production. The same observation was made when pooled Ig solely was added (coating condition). F(ab')2 fragments of pooled Ig displayed similar inhibitory capacity when added in neutralizing condition, indicating that the mechanism involved was not Fc dependent. The fragments lost the activating properties of intact Ig when incubated in coating condition, showing that Fc receptor activation occurs in this setting.

The present work demonstrates that inhibition of SEB‐induced CKs release by pooled Ig can be achieved by SEB neutralization, provided that the experimental conditions avoid activation through the Fc receptor. It can be assumed that similar mechanisms take place in some clinical conditions during which pooled Ig are infused.
Phenotypic analysis of functional T‐lymphocyte subtypes and natural killer cells in human cord blood: relevance to umbilical cord blood transplantation
Authors: Han P. , Hodge G. , Story C. , Xu X.
Published: July 2010

Cord blood has been used successfully for stem cell transplantation in several haematological conditions: Fanconi's anaemia, leukaemia and Wiskott‐Aldrich syndrome. On account of the low incidence of GVHD observed following cord blood transplantation, it has been suggested that cord blood be used for HLA‐matched, or perhaps one or two antigens mismatched, and unrelated stem cell transplantation. Based on an extensive immunophenotype‐functional correlation, we determined that cord blood contains mainly immature unprimed T lymphocytes that are predominantly suppressor cells. Recent findings suggest that dysregulated production of cytokines (IL‐1, IL‐2, TNFα) plays a role in GVHD. We showed that T cells in cord blood express receptors for IL‐2, TNFα, but no receptors for IL‐1. Similarly, NK cells, one of the effector cells of GVHD, express receptors for TNFα and γIFN but do not express receptors for IL‐1, nor IL‐2R α‐chain (CD25) although IL‐2R β‐chain is expressed. The potential for activation of T lymphocytes and NK cells therefore exists in the context of bone marrow transplantation. However, the high number of suppressor cells in cord blood most likely modulate the activation of lymphocytes and NK cells thereby minimizing GVHD.
Mediastinal large B‐cell lymphoma: clinical and immunohistological findings in 18 patients treated with different third‐generation regimens
Authors: Falini B. , Venturi S. , Martelli M. , Santucci A. , Pileri S. , Pescarmona E. , Giovannini M. , Mazza P. , Martelli M. F. , Pasqualucci L. , Ballatori E. , Guglielmi C. , Amadori S. , Poggi S. , Sabattini E. , Gherlinzoni F. , Zinzani P. L. , Baroni C. D. , Mandelli F. , Tura S.
Published: July 2010

We report on the immunophenotype, clinical findings and response to aggressive chemotherapy of 18 patients with mediastinal large B‐cell lymphoma (MLCL). Cases were collected from a series of 286 high‐grade non‐Hodgkin's lymphomas (HG‐NHL) which, in the period September 1988 to August 1991, were enrolled in a prospective multicentre trial designed to compare the MACOP‐B and F‐MACHOP regimens. Immunostaining on frozen sections revealed a previously unrecognized phenotype, i.e. co‐expression of B‐cell (CD19, CD20, CD22, Ig‐associated dimer) and activation‐associated antigens (CD30 and CDw70) in about 60% of MLCL cases; in contrast, the activation‐associated antigens CD25 and Ki‐27 (unclustered) were consistently negative. This peculiar phenotype may reflect a derivation of the tumour from a subset of thymic activated B cells. Clinically, the patients (median age 31 years; F/M ratio 2.6) presented with bulky mediastinal mass (72%) associated with mediastinal syndrome in >50% cases; disease was stage IIA in most cases. All 18 patients received aggressive chemotherapy (F‐MACHOP 11; MACOP‐B 7). Complete response (CR) was achieved in 57.1% of cases treated with MACOP‐B. In contrast, the response of the 11 MLCL treated with F‐MACHOP was poor (CR 18.2%) as compared to that of the 135 HG‐NHL treated with the same regimen during the trial (CR 69.6%). This difference was still statistically significant after adjusting for negative prognostic factors (mediastinal mass > 10 cm plus increased LDH) and suggests that F‐MACHOP might not be the most appropriate regimen for this kind of lymphoma.
Establishment of a myeloid leukaemic cell line (SKNO‐1) from a patient with t(8;21) who acquired monosomy 17 during disease progression
Authors: Matozaki Sachiko , Nakagawa Toshitaro , Kawaguchi Ryuji , Aozaki Ryoji , Tsutsumi Masayoshi , Murayama Tohru , Koizumi Tamio , Nishimura Ryuichiro , Isobe Takashi , Chihara Kazuo
Published: July 2010

A novel cell line SKNO‐1 was established from the bone marrow cells of a 22‐year‐old male suffering from acute myeloblastic leukaemia (AML) M2 with t(8;21) whose disease became resistant to chemotherapy after acquisition of 17 monosomy. SKNO‐1 has been maintained for more than 36 months as a granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) dependent line.

Morphologically, SKNO‐1 cells were myeloblasts somewhat maturated. The cells grow in suspension with a doubling time of 48‐72 h. The survival and growth of SKNO‐1 cells was absolutely dependent on granulocyte‐macrophage colony stimulating factor (GM‐CSF). SKNO‐1 cells possessed t(8;21) and monosomy 17 which were observed in original leukaemic cells. We confirmed that the AML1 gene, located on chromosome 21, was rearranged and the AML1‐MTG8 fusion transcript was expressed in SKNO‐1 cells. Over‐expression and mutation of the p53 gene were also detected in SKNO‐1. It is likely that alterations of AML1 or MTG8 gene and p53 gene contribute to a disease progression in this case.

Since t(8;21) translocation is a common chromosome abnormality in AML, and inactivation of the p53 gene may play a crucial role in disease progression in AML, SKNO‐1 would be a useful tool for analysing the molecular mechanisms in myeloid leukaemogenesis.
The use of r‐HuEpo in the treatment of anaemia related to myelodysplasia (MDS)
Authors: Rose Esther H. , Abels Robert I. , Nelson Richard A. , McCullough Doreen M. , Lessin Lawrence , Johnson R. W.
Published: July 2010

One hundred and sixteen (116) anaemic patients with myelodysplastic syndromes (MDS) were treated with recombinant human erythropoietin (r‐HuEpo) in an openlabel, multicentre, compassionate treatment trial; 100 patients received therapy for ≧ 4 weeks and were evaluable for efficacy. The distribution of FAB subtypes was: 44 RA, 40 RARS, eight RAEB, two RAEB‐t, one CMML, and five not specified. Mean baseline haematocrit was 24.5%, and the mean prestudy transfusion requirement in the 12 weeks immediately prior to study entry was 6.5 units. r‐HuEpo treatment was initiated at a dose of 150U/kg three times weekly, with dose escalations of 50U/kg monthly (up to 300U/kg 3 x /week) permitted if the haematocrit failed to rise. Response to therapy was defined as either an increase in haematocrit of ≧ 6 percentage points over baseline, unrelated to transfusion, or a ≧ 50% decrease in transfusion requirement in the last 3 months of study treatment, compared to the baseline period (12 weeks), By these criteria, 28% (28/100) of patients responded to r‐HuEpo treatment. Overall, 86% (24/28) of patients responding to therapy had baseline Epo levels ≧ 100 mU/ml. Response rates by FAB subtype were: RA 39% (17/44), RARS 17.5% (7/40) and RAEB 12.5% (1/8). Additionally, a 54% (15/28) response rate was seen in RA patients with baseline EPo levels ≧ 100 mU/ml. Responses to therapy were durable and generally occurred at r‐HuEpo doses of 150‐200 U/kg t.i.w. There were no reports of thrombosis, seizures or therapy‐related hypertension. The data show that patients with MDS, especially those with the RA and RARS subtypes, can benefit from treatment with r‐HuEpo. Those patients with baseline Epo levels ≧ 100 mU/ml were most likely to respond to therapy.
Acute lymphoblastic transformation of essential thrombocythaemia
Authors: Murphy P. T. , Sivakumaran M. , Rhee F. van , WATMORE A. E. , Mitchell V. E.
Published: July 2010

Essential thrombocythaemia is a myelopro‐Iterative disorder which may transform to acute myeloid leukaemia, especially following therapy with alkylating agents or radioactive phosphorus. We describe the rare occurrence of acute lymphoblastic leukaemia transformation in a patient with known essential thrombocythaemia.
PCR‐monitoring of minimal residual leukaemia after conventional chemotherapy and bone marrow transplantation in BCR‐ABL‐positive acute lymphoblastic leukaemia
Authors: Mitterbauer Gerlinde , Födinger Manuela , Scherrer Renate , Knöbl Paul , Jäger Ulrich , Laczika Klaus , Schwarzinger Ilse , Gaiger Alexander , Geissler Klaus , Greinix Hildegard , Kalhs Peter , Linkesch Werner , Lechner Klaus , Mannhalter Christine
Published: July 2010

We report the results of consecutive tests in nine BCR‐ABL‐positive ALL patients by one‐step and two‐step (nested primer) reverse transcriptase‐polymerase chain reaction (RT‐PCR). Six patients could be tested in complete haematological remission (CHR). One patient remained one‐step positive; four patients became one‐step negative, but remained two‐step positive; only one patient became two‐step negative. In five patients the haematological relapse was preceded by one‐step positivity by 1.5‐5 weeks. In two patients who received autologous BMT in CHR, BCR‐ABL was still detectable by two‐step PCR, whereas allogeneic BMT was able to transiently reduce BCR‐ABL below the two‐step detection level. Our results show that one‐step combined with two‐step RT‐PCR analysis gives valuable information about the efficacy of treatment and the dynamics of the leukaemic clone.
Primitive haematopoietic progenitors in the blood of patients with sickle cell disease appear to be endogenously mobilized
Authors: Croizat H. , Ponchio L. , Nicolini F. E. , Nagel R. L. , Eaves C. J.
Published: July 2010

To investigate whether haematopoietic stem cells in patients with sickle cell (SS) disease might be altered, we examined the number and cycling status of 5‐week long‐term culture‐initiating cells (LTC‐ICs) and in vitro multilineage colony‐forming cells (CFCs) present in the blood of a large and clinically diverse group of SS patients. The concentrations of both of these cell types per ml of blood varied over a wide range in individual patients, but, on average, were significantly elevated above normal values (∼sevenfold and 15‐fold respectively) and to an even greater extent than the lineage‐restricted CFCs in the same samples. Wide variations in the concentration of circulating progenitors, particularly the LTC‐ICs, were also seen over time (in concert with changes in the white blood cell count) in SS patients. [3H]‐Thymidine suicide assays showed most of the CFCs and LTC‐ICs in SS blood to be quiescent like their counterparts in normal blood. However, by comparison with historical data, the SS progenitors could be recruited into the cycle more quickly (i.e. within 2 vs. 3 d), thus showing the same kinetics of activation exhibited by ‘mobilized’ progenitors from patients given chemotherapy and exogenous growth factors. Taken together, these findings implicate previously documented increases in endogenous Steel factor, interleukin 3 and granulocyte–macrophage colony‐stimulating factor levels in SS patients in the establishment of a chronically mobilized progenitor phenotype.
Quantitative analysis of circulating cell‐free Epstein–Barr virus (EBV) DNA levels in patients with EBV‐associated lymphoid malignancies
Authors: Lei Kenny I. K. , Chan Lisa Y.S. , Chan Wing Y. , Johnson Philip J. , Lo Y. M. Dennis
Published: July 2010

Cell‐free Epstein–Barr virus (EBV) DNA has recently been detected in the plasma and serum of patients with Hodgkin's disease, post‐transplant lymphoproliferative disease (PTLD) and acquired immunodeficiency syndrome‐related lymphoma. However, no data are available on the temporal variation of plasma/serum EBV DNA levels in patients with EBV‐associated lymphoid malignancies during the course of therapy. Using a real‐time quantitative polymerase chain reaction assay, we studied the plasma EBV DNA levels in 13 patients with EBV‐associated lymphoid malignancies (six patients with Hodgkin's disease, four with nasal natural killer/T‐cell lymphoma, two cases of PTLD and one patient with Burkitt's lymphoma) at presentation and during therapy. Plasma EBV DNA was detected in 12 of the 13 patients (median 2266 copies/ml; interquartile range 181–8379 copies/ml), but not in any of 35 healthy control subjects (P < 0·0001). The EBV status in tumour cells was also examined in 12 of these patients using in situ hybridization for EBV‐encoded small RNAs (EBERs). EBER positivity was observed in 11 patients, all of whom had EBV DNA detectable in plasma. The one patient who had no detectable plasma EBV DNA was also negative for EBERs in tumour tissue. Serial measurements of plasma EBV DNA levels were performed in nine of the patients during the course of therapy. All patients who responded to therapy demonstrated a significant reduction of plasma EBV DNA to low or undetectable levels, whereas in two patients with ineffective therapy, disease progression was associated with a rapid increase in plasma EBV DNA levels. We concluded that plasma EBV DNA is detectable in a wide range of EBV‐associated lymphoid malignancies. As plasma EBV DNA levels correlate well with the therapeutic response, such analysis may be a valuable tool for monitoring clinical progress.
Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures
Authors: Crowther M. , Ford I. , Jeffrey R. R. , Urbaniak S. J. , Greaves M.
Published: July 2010

Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P‐selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re‐infusion of autologous platelets on platelet function. Autologous platelet‐rich plasma (PRP) contained a significantly lower (P < 0·05) percentage of P‐selectin‐positive and fibrinogen‐positive platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P < 0·05) percentage of responsive platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P‐selectin or binding fibrinogen increased significantly after CPB (P < 0·05), while the percentage of platelets responsive to in vitro agonists was decreased (P < 0·05 in autologous transfusion patients), consistent with platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re‐infusion of autologous PRP. P‐selectin expression had returned to pre‐CPB levels 24 h post‐operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.
Anticoagulation management in primary care: a trial‐based economic evaluation
Authors: Parry David , Fitzmaurice David , Raftery James
Published: July 2010

The demand for anticoagulation management is increasing because of a widening of the indications of treatment. A primary care clinic using near‐patient testing and computer decision support software is one model of care to meet this increased demand. The study aimed to determine the cost and cost‐effectiveness of primary care‐based anticoagulation management in comparison with ‘traditional’ hospital care‐based provision by means of a cost‐effectiveness analysis using data from a Birmingham‐based multicentre randomized controlled trial. The costs per patient per year in primary care were £170 [95% confidence interval (CI) £149–190] vs. £69 (95% CI £57–81). Sensitivity analysis demonstrated that the cost in primary care could be reduced to under £100 per patient per year under plausible changes in the variables. Primary care provides similar levels of control to secondary care for patients on anticoagulation therapy. There is an increased cost of managing patients in primary care and at no point did primary care become a lower cost option than secondary care. Local decision‐makers need to assess the increased cost of primary care anticoagulation management in terms of the potential reductions in high‐cost serious adverse events.
Interferon alpha in combination with GM‐CSF induces the differentiation of leukaemic antigen‐presenting cells that have the capacity to stimulate a specific anti‐leukaemic cytotoxic T‐cell response from patients with chronic myeloid leukaemia
Authors: Chen Xiao , Regn Sybille , Raffegerst Silke , Kolb Hans‐Jochem , Roskrow Marie
Published: July 2010

Although interferon α (IFN‐α) is able to induce haematological remission in 60–80% of patients with chronic myeloid leukaemia (CML) in early chronic phase, major cytogenetic remissions are only achievable in 30–40%. Recent clinical data suggest that the addition of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) to IFN‐α therapy can significantly improve the cytogenetic response in some patients, although the mechanism remains unknown. We hypothesized that the combination of GM‐CSF and IFN‐α induces the differentiation of dendritic cells, which subsequently stimulates a specific anti‐leukaemic response. Monocytes from CML patients were cultured in GM‐CSF and interleukin (IL)‐4 (GM/IL‐4)or in GM‐CSF and IFN‐α (GM/IFN‐α). After 7 d, the number of cells exhibiting typical antigen‐presenting cell (APC) morphology was equal in both groups, and fluorescence in situ hybridization (FISH) analysis confirmed that the APCs generated with GM/IFN‐α were of leukaemic origin. Phenotypically, both sets of APCs expressed typical surface markers; however, CD86, CD83, CD11c, HLA‐ABC and HLA‐DR expression was significantly higher in the GM/IFN‐α APCs, whereas CD1a expression was significantly lower. In mixed lymphocyte reactions (MLR), GM/IFN‐α APCs stimulated the proliferation of allogeneic T cells significantly better than GM/IL‐4 APCs. However, both groups of APCs stimulated autologous T‐cell proliferation equally. Finally, we assessed the ability of GM/IFN‐α APCs to induce a leukaemia‐specific cytotoxic T‐cell response. Some samples generated cytotoxic T lymphocytes (CTLs) that specifically lysed bcr‐abl‐positive target cells. These data show that the combination of GM‐CSF and IFN‐α, when used in vitro, induces the differentiation of malignant APCs with potent T‐cell stimulatory capacity. Although there is no in vivo evidence to support these findings, it is possible that, when administered to CML patients, GM‐CSF in combination with IFN‐α results in the generation of highly stimulatory leukaemic APCs.
Encouraging preliminary results in 12 patients with high‐risk haematological malignancies by omitting graft‐versus‐host disease prophylaxis after allogeneic transplantation
Authors: Fassas Athanasios B.‐T. , Rapoport Aaron P. , Cottler‐Fox Michele , Chen Timothy , Tricot Guido
Published: July 2010

Immunosuppressive therapy, routinely given after allogeneic transplantation to modulate graft‐versus‐host disease (GVHD) may have an adverse effect on the graft‐versus‐tumour (GVT) effect. Twelve patients with high‐risk haematological malignancies were given cyclophosphamide, total body irradiation and antithymocyte globulin followed by peripheral blood stem cell grafts from HLA‐identical siblings without prophylactic immunosuppression. At the earliest clinical evidence of GVHD, patients were treated with high‐dose solumedrol and tacrolimus. Prompt haematological recovery [absolute neutrophil count (ANC) > 1·0 × 109/l] was observed (median time 9 d). All patients developed grade III–IV GVHD (median onset 9 d), involving the skin (11), intestine (five) and liver (three). Of nine evaluable patients, seven developed chronic GVHD [extensive (six), limited (one)]. Six patients died 1–6·5 months after transplantation. Three patients died from treatment‐related complications, two from acute GVHD and one from relapsing disease. The remaining six patients are alive 5–26 months after transplantation, five in complete remission and one myeloma patient in very good partial remission. In conclusion, omission of post‐transplantation GVHD prophylaxis is feasible, does not lead to graft failure or a high incidence of uncontrollable GVHD and appears to be associated with encouraging clinical responses in a group of patients with high‐risk disease features.
Venous thromboembolism and hypercoagulability in splenectomized patients with thalassaemia intermedia
Authors: Cappellini M. D. , Robbiolo L. , Bottasso B. M. , Coppola R. , Fiorelli G. , Mannucci And P. M.
Published: July 2010

Thromboembolic phenomena have been described in patients with thalassaemia intermedia and major, although there are relatively few epidemiological data on the overall frequency of these complications. To obtain more insight into the risk and mechanism of venous thromboembolism in thalassaemia, the aims of this study were: (i) to establish retrospectively the prevalence of thromboembolic events in a large group of adults with thalassaemia intermedia and major during a follow up period of 10 years; (ii) to measure in subgroups of these patients sensitive markers of activation of coagulation and fibrinolysis enzymes; and (iii) to look for possible procoagulant mechanisms. A high prevalence of thromboembolic events was found, particularly in splenectomized patients with thalassaemia intermedia (29%). These patients had high plasma levels of markers of coagulation and fibrinolysis activation. Furthermore, thalassaemic red cells and erythroid precursors from splenectomized patients with thalassaemia intermedia had an enhanced capacity to generate thrombin. To evaluate the role of splenectomy per se on procoagulant activity, we evaluated the capacity to form thrombin in healthy individuals who had been splenectomized for trauma. They produced the same amount of thrombin as non‐splenectomized controls. In conclusion, the results of this study show the existence of a hypercoagulable state in splenectomized patients with thalassaemia intermedia and that their red and erythroid cells are capable of acting as activated platelets in thrombin generation.
Osteoporosis in β‐thalassaemia major patients: analysis of the genetic background
Authors: Perrotta Silverio , Cappellini Maria Domenica , Bertoldo Francesco , Servedio Veronica , Iolascon Giovanni , D'agruma Leonardo , Gasparini Paolo , Siciliani Maria Carmen , Iolascon Achille
Published: July 2010

Regular blood transfusions from infancy until adulthood in β‐thalassaemia major patients have substituted severe bone deformities with less marked skeletal lesions as osteoporosis. Osteoporosis is characterized by low bone mass and disruption of bone architecture, resulting in reduced bone strength and increased risk of fractures. Genetic factors have an important role in determining bone mineral density (BMD). We have investigated the possible association between BMD and two polymorphisms in 135 β‐thalassaemic patients: (i) a substitution G→Τ in a regulatory region of the COLIA1 gene encoding for the major protein of bone (type 1 collagen), and (ii) a one‐base deletion in intron 4 (713–8del C) of transforming growth factor beta 1 (TGF‐β1) gene. We have found a remarkable incidence (90%) of osteopenia and osteoporosis among regularly transfused patients. Bone mass was lower in men than in women (P = 0·0023), with a more prevalent osteopenia/osteoporosis of the spine in men than in women (P = 0·001). The sample was stratified on the basis of BMD expressed as Z‐score, i.e. normal, osteopenic and osteoporotic patients, and genotype frequencies of each group were evaluated. TGF‐β1 polymorphism failed to demonstrate a statistical difference in BMD groups. However, subjects with heterozygous or homozygous polymorphism of the COLIA1 gene showed a lower BMD than subjects without the sequence variation (P = 0·012). The differences among genotypes were still present when the BMD was analysed as adjusted Z‐score and when men and women were analysed separately (P = 0·022 and 0·004 respectively), with men more severely affected. Analysis of COLIA1 polymorphism could help to identify those thalassaemic patients at risk of osteoporosis and fractures.
Extended routine polymerase chain reaction surveillance and pre‐emptive antiviral therapy for cytomegalovirus after allogeneic transplantation
Authors: Peggs Karl S. , Preiser Wolfgang , Kottaridis Panagiotis D. , McKeag Nikki , Brink Nicola S. , Tedder Richard S. , Goldstone Anthony H. , Linch David C. , Mackinnon Stephen
Published: July 2010

Pre‐emptive treatment strategies based on sensitive screening for cytomegalovirus (CMV) infection up to day +100 after allogeneic transplantation have been shown to reduce the incidence of CMV disease during the period of surveillance. However, the use of ganciclovir has been associated with delays in immune reconstitution and an increased incidence of late CMV disease after day +100. In the present study, 81 patients undergoing allogeneic transplantation received polymerase chain reaction (PCR)‐guided pre‐emptive therapy based on detection of CMV DNA by PCR on 2 consecutive weeks up to day +180. Thirty‐three of the 52 high‐risk patients (CMV‐seropositive donor or recipient) received a total of 45 treatment episodes up to day +100. Three of these patients (5·7%) developed CMV disease, with one fatality. Twelve of the surviving 44 high‐risk patients (27%) required pre‐emptive treatment between days +101 and +192, but none of these patients developed late CMV disease with a median follow‐up of 402 d (range 117–952 d). Antiviral therapy was stopped after a single negative PCR result with no subsequent episodes of CMV disease while patients remained off antiviral treatment. As all initial episodes of CMV DNA detection occurred within 60 d of transplantation, it may be possible to discontinue monitoring beyond day +100 in patients who have remained CMV PCR negative before this. Thus, we have confirmed that PCR‐guided pre‐emptive therapy results in a low incidence of CMV disease before day +100 and that discontinuing treatment on the basis of viral clearance as determined by CMV PCR appears to be safe practice. In addition, we have observed no episodes of late CMV disease with an extension of surveillance to 26 weeks.