bluuebunny/crossref_metadata_2025 huggingface dataset:
Antithrombotic and bleeding effects of glycosaminoglycans with different degrees of sulphation
Authors: Ryn‐McKenna J. Van , Ofosu F. A. , Hirsh J. , Buchanan M. R.
Published: March 2008

Summary We recently reported that the in vitro anticoagulant activities of dermatan sulphate and heparan sulphate were improved with increased sulphation. In this study we determined how the degree of sulphation of glycosaminoglycans influences their antithrombotic and bleeding effects in vivo. We compared the antithrombotic effects of each glycosaminoglycan by measuring their ability to inhibit experimentally‐induced thrombus formation in rabbit jugular veins. The bleeding effect of each glycosaminoglycan was measured by comparing their ability to increase the amount of 51Cr‐blood lost from five standardized cuts in rabbit ears. Increased sulphation only improved the antithrombotic effects of dermatan sulphate and heparan sulphate. In contrast, increased sulphation enhanced the blood loss associated with all the glycosaminoglycans evaluated. We conclude that the antithrombotic effects of heparan sulphate and dermatan sulphate can be enhanced by increased sulphation, but that the improved antithrombotic effects are compromised by the concomitant increase in bleeding side‐effects.
Rabbit serum alpha‐2‐macroglobulin binds to liver ferritin: association causes a heterogeneity of ferritin molecules
Authors: Santambrogio Paolo , Massover William H.
Published: March 2008

Summary Rabbit liver ferritin is unusual since it forms two discrete electrophoretic bands at the beta position of molecular dimers (Santambrogio & Massover, 1987). The present studies have sought to identify the nature of a 170 kDa nonferritin polypeptide that is uniquely present in the larger beta band. Ultrastructural, immunological and biochemical results all indicate that this polypeptide is a subunit of the plasma protein, alpha‐2‐macroglobulin. Experimental results show that rabbit serum alpha‐2‐macroglobulin will bind liver ferritin, and this association induces the de novo formation of the larger beta band. These results thus demonstrate that molecular heterogeneity of ferritin can be caused by its association with a non‐ferritin protein. We conclude that alpha‐2‐macroglobulin is a binder of rabbit tissue ferritin in the circulation; this binding could provide additional means for the receptor‐mediated uptake of circulating ferritin.
A flow cytometric assay for the determination of cell proliferation with a monoclonal antibody directed against DNA‐methyltransferase
Authors: Neubauer Andreas , Serke Stefan , Siegert Wolfgang , Kroll Werner , Musch Reinhard , Huhn Dieter
Published: March 2008

Summary The enzyme DNA‐methyltransferase is responsible for the methylation of a newly synthesized DNA‐strand. A monoclonal antibody directed against DNA‐methyltransferase was used to determine cell proliferation by means of flow cytometry. The reactivity of DNA‐methyltransferase antibody was compared with the known proliferation markers transferrin‐receptor and Ki67. All three methods showed comparable reactivity with the erythroblastic cell line K562 (86%, 81%, 76% respectively). In a second set of experiments peripheral blood mononuclear cells were stimulated with phytohaemagglutinin; in three experiments, a mean of 63% of the cells reacted with DNA‐methyltransferase antibody after 72 h of culture as compared to a mean of 6% in the case of unstimulated control cells. HL60 cells were incubated with DMSO and harvested on day 5 of culture. The results obtained show that in differentiated cells the fraction positive with DNA‐methyltransferase antibody decreased to levels below 10%. It is concluded that the technique described is a fast and easy method for the flow cytometric determination of cellular proliferation.
Platelet specific alloantigens on the platelet glycoprotein Ia/IIa complex
Authors: Smith James W. , Kelton John G. , Horsewood Peter , Brown Claudia , Giles Alan , Meyer Ralph , Woods Virgil , Burrows Robert
Published: March 2008

Summary The majority of platelet alloantigens are located on platelet glycoproteins IIb/IIIa. This report describes a codominant allelic system carried on the glycoprotein la/IIa complex, which we originally designated as Zava/Zavb but which is identical to the Bra/Brb system. Furthermore Zava was found to be identical to Hca. The alloantigens could not be detected using a direct binding enzyme immunoassay (EIA) with intact platelets, but were readily detected using a glycoprotein capture EIA and by radioimmunoprecipitation techniques. The two index cases (designated as homozygous Zava and Zavb) had alloantibodies against the corresponding antigen and did not react with their own platelets. Using these alloantibodies and a monoclonal antibody that reacts with the platelet glycoprotein la/IIa complex (12F1). We demonstrated that all Ia/IIa molecules carry either Zava or Zavb and we found that Zava and Zavb are on discrete populations of Ia/IIa. Following immunodepletion using either anti‐Zava or anti‐Zavb, all detectable Ia/IIa complexes from the respective homozygous platelets were removed. Immunodepletion of heterozygous Zava/Zavb with either anti‐Zava or anti‐Zavb did not reduce the amount of Ia/IIa complexes precipitable using the alternate alloantiserum. Population studies (n= 50) indicated the phenotypic frequency of Zava/Zava is less than 1%; Zava/Zavb is 18% and Zavb/Zavb is 82%. Four different alloantisera that had either anti‐Zava or anti‐Zavb reactivity also carried reactivity against the Baka or Bakb antigens which may suggest an association in the immune response to these alleles.
Acquired Zwa antigen on Zwa negative platelets demonstrated by Western blotting
Authors: Dieleman L. A. , Brand A. , Claas F. H. J. , van de Keur C. , Witvliet M. , Giphart M. J.
Published: March 2008

Summary Zwa‐negative platelets that had been incubated in random donor AB plasma for several hours and subsequently cryopreserved, were found to be Zwa‐positive when tested 3 years later by Western blotting. Following this serendipitous observation, we demonstrated that the Zwa antigen could be passively adsorbed onto the membrane of Zwa‐negative platelets incubating for 4 h in the plasma from Zwa‐positive donors. This observation supports the hypothesis that passive acquirement of Zwa antigens is a pathogenic mechanism for inducing post‐transfusion purpura, and suggests that Zwa‐negative platelets should not be stored in Zwa‐ positive plasma before transfusion.
Inhibition of phospholipid and platelet‐dependent prothrombinase activity in the plasma of patients with lupus anticoagulants
Authors: Galli Monica , Béguin Suzette , Lindhout Theo , Hemker Coenraad H.
Published: March 2008

Summary Prothrombinase activity was analysed in the plasma of a series of patients with lupus anticoagulants (LAC). In the presence of purified PS‐PC (20–80%) vesicles the prothrombinase activity triggered by kaolin was retarded by 2–3 min with respect with normal plasma. The maximal values of prothrombinase activity increased by increasing the amount of phospholipid vesicles. However, in the plasma of the patients they were always lower than those of normal plasma at each phospholipid concentration. Platelet‐dependent prothrombinase activity was subsequently investigated. Again, both a delay in appearance and reduced peak values of prothrombinase activity were observed in the plasma of the patients. This inhibition was partially overcome by the addition of an excess of purified phospholipids. Finally, the effect of LAC IgG on platelet rich plasma‐dependent prothrombinase activity was investigated. The main effect observed was a delay of the peak time of prothrombinase activity, while the maximal peaks were affected only by one IgG preparation. We conclude that LAC antibodies can react with both purified negatively‐charged phospholipids and platelet procoagulant phospholipids and inhibit prothrombinase activity in a similar way in both cases.
Complement sensitivity of erythroblasts and erythropoietic precursors in paroxysmal nocturnal haemoglobinuria (PNH)
Authors: Shichishima Tsutomu , Terasawa Takashi , Uchida Tatsumi , Kariyone Shigeo
Published: March 2008

Summary To clarify the regulation of erythropoiesis of complement (C')‐sensitive erythrocytes (E) during erythroid cell maturation and differentiation in paroxysmal nocturnal haemoglobinuria (PNH), the C'sensitivity of (1) erythroblasts from erythropoietic bursts and (2) erythropoietic precursors was examined in seven patients with PNH. For the former, a modified complement lysis sensitivity (CLS) test using the trypan blue dye exclusion method was employed. For the latter, an erythropoietic cell culture following the CLS or sucrose haemolysis (SH) test for mononuclear cells was used. Linear relationships were found between the concentrations of C'and proportions of C'‐mediated haemolysed erythroblasts in both PNH and normal erythropoietic bursts. In addition, PNH erythroblasts showed hypersensitivity to C'; in the C'dilution for 50% lysis was 12·5±8·1 ml in PNH patients and 42·3±4·5 ml in normal controls. There was also a significant correlation (r=0·919; P<0·01) between the proportions of PNH‐III E and haemolysed erythroblasts. Significant hypersensitivity of mature erythroid progenitors (CFU‐E) to C'was found in both the SH (P<0·01 or P<0·05) and CLS (r=‐0·712: P<0·001) tests in PNH patients. However, no hypersensitivity to C'was observed at the primitive erythroid progenitors (BFU‐E) level in either the PNH patients or the normal controls.

These findings suggest that in PNH the property of C'sensitivity appears during differentiation and maturation from BFU‐E to CFU‐E and that the proportions of PNH‐III E are already programmed at the level of erythroblasts.
Immunoglobulin light chains in common acute lymphoblastic leukaemia
Authors: Markey Geraldine M. , Alexander H. D. , Nolan R. L. , Morris T. C. M. , Robertson J. H.
Published: March 2008

A single immunoglobulin light chain λ was identified in the blast cells of two out of 12 patients with common acute lymphoblastic leukaemia (C‐ALL) using the alkaline phosphatase/anti‐alkaline phosphatase (APAAP) technique. Inhibition at the cell surface proved that the reaction was a genuine anti‐λ reaction. Immunoglobulin μ chain was not identified in these patients. Results of immunoglobulin typing in 103 patients with B chronic lymphocytic leukaemia (B‐CLL) are cited to show the increased sensitivity of the APAAP technique as compared to the indirect immunoperoxidase technique for cellular immunoglobulin identification.
PL2‐49, a monoclonal antibody against glycoprotein IIb which is a platelet activator
Authors: Morel M.‐C. , Lecompte T. , Champeix P. , Favier R. , Potevin F. , Samama M. , Salmon C. , Kaplan C.
Published: March 2008

PL2‐49 is a murine monoclonal IgG1 antibody obtained after immunization of Balb/c mice with EDTA washed platelets. Binding could be detected on Zwa(+) as well as Zwa(—) platelets, but not on type I Glanzmann's thrombasthenia platelets using an ELISA screening test. Immunoprecipitation studies showed that PL2‐49 bound to glycoprotein IIb when the glycoprotein IIb/IIIa complex dissociation was performed after the monoclonal antibody binding. Experiments with a human alloantibody against Zwa antigen were run in parallel to control the complex dissociation. Ascitic fluid, as well as the purified antibody, induced activation and aggregation of washed platelets and ATP release. PL2‐49‐induced aggregation did not require exogenous fibrinogen and was inhibited, partially, in the presence of aspirin, apyrase, isosorbide dinitrate. Raising intra‐platelet cyclic AMP with a stable PGI2 analogue, iloprost, and/or a phosphodiesterase inhibitor, RA 233, suppressed the responses to PL2‐49. F(ab')2 fragments did not induce aggregation of normal platelets but inhibited the response to the whole immunoglobulin. Finally PL2‐49 was shown to induce aequorin‐detected elevations in intraplatelet Ca++ levels. Thus PL2‐49 seems to differ from monoclonal antibodies so far described, since it binds to glycoprotein IIb in a complex‐dependent manner at least under our experimental conditions for immunoprecipitation studies, and it induces platelet Ca++ mobilization and platelet aggregation after a lag‐time. These reactions depend both on Fab and Fc domains of the antibody and require neither complement nor exogenous fibrinogen.
A prospective comparison of four techniques for measuring platelet‐associated IgG
Authors: Kelton John G. , Murphy William G. , Lucarelli Anne , Garvey‐Williams Judy , Santos Aurelio , Meyer Ralph , Powers Peter
Published: March 2008

We describe a prospective study comparing four different assays for PAIgG. Platelets from patients with a variety of thrombocytopenic disorders were collected into ACD, washed, and the PAIgG then measured using three assays for surface PAIgG. These included: (a) a direct binding assay using 125I‐monoclonal anti‐IgG (MoAb); (b) a direct binding assay using 125I‐staphylococcal protein A (SPA); and (c) a two‐stage assay. PAIgG also was measured using an assay for ‘total’ PAIgG following platelet lysis. The mean ± SD number of molecules of IgG per platelet on washed platelets from 29 healthy, non‐thrombocytopenic controls was: 86 ± 80 (125I‐MoAb); 94 ± 96 (125I‐SPA); 3520 ± 1890 (two‐stage surface assay); and 10 850 ± 3720 (total PAIgG). A total of 62 different patients with idiopathic thrombocytopenic purpura or thrombocytopenia complicating systematic lupus erythematosus, and 73 different patients with‘non‐immune’ thrombocytopenia, were tested using each of the four assays. These ‘non‐immune’ thrombocytopenic patients included patients with carcinoma, septicaemia, pre‐eclampsia, chronic leukaemia, thrombotic thrombocytopenic purpura, haemolytic uraemic syndrome, acute leukaemia and myelodysplasia. All four essays gave similar results for both the immune and non‐immune thrombocytopenic patients. The sensitivity of the assays for the most severely thrombocytopenic patients with immune thrombocytopenia was: MoAb 60%; SPA 88%; two‐stage 82%; and‘total’ PAIgG 88%. The specificity of the four assays in the non‐immune thrombocytopenic patients was 57%‘total’ PAIgG; 63% two‐stage surface; 25% SPA; 38% MoAb. There was no quantitative cut‐off point that could be used to segregate the non‐immune thrombocytopenic patients from the immune thrombocytopenic patients. These results indicate that all of these assays share a similar high sensitivity but low specificity for ITP.
The action of Russell's viper venom on fibrin formation and fibrinolysis in vivo
Authors: Woodhams B. J. , Than Thein , Than Than , Hutton R. A.
Published: March 2008

Envenoming by Russell's viper caused a marked rise in FPA, B beta 15‐42 fragment and fibrin derived cross‐linked D‐dimer fragment indicative of a consumptive coagulopathy with hyperfibrinolysis. There was no increase in tPA or tPA‐I levels post envenoming, which suggests that the increase in fibrinolytic activity was not due to venom‐induced release of tPA from the vessel walls but may have been attributable to a direct effect of the venom or to a secondary physiological response to fibrin deposition. The effectiveness of the antivenom is demonstrated by its ability to prevent further cleavage of fibrinogen and the return to normal of fibrinogen levels by 24 h. A secondary rise in FPA at this time indicates that the initial dose of antivenom may have been too small. The antivenom alone or in combination with the venom causes the release of tPA, tPA‐I and vWF by the vessel walls. This may be a consequence of the severe anaphylactic reactions seen in some patients.
Recombinant human erythropoietin has little influence on megakaryocytopoiesis in mice
Authors: Grossi Alberto , Vannucchi Alessandro M. , Rafanelli Daniela , Ferrini Pierluigi Rossi
Published: March 2008

Summary. The availability of a preparation of recombinant human erythropoietin (rEp) prompted us to investigate the role of Ep in the regulation of megakaryocytopoiesis in mice, using experimental procedures by which the effects on the mitotic and post‐mitotic compartment of megakaryocytes could be evaluated separately. In agar cultures of murine bone marrow cells, either serum‐depleted or serum‐supplemented, rEp (0.1–2 U/ml) did not stimulate megakaryocyte colony formation and when it was added to suboptimal amount of spleen cell‐conditioned medium (SCM), it failed to show a significant synergistic activity. On the contrary, rEp increased the number of megakaryocytic colonies developed from splenic precursors in the presence of suboptimal amounts of SCM, although it was unable per se to stimulate colony formation.

The effects of rEps on megakaryocyte maturation and platelet production were studied in vivo evaluating the incorporation of 75Se‐selenomethionine into platelets, the platelet count and platelet size, and the number of megakaryocyte precursors (small acethylcholinesterase positive cells, sAchE) in the bone marrow of mice injected with 1–8 U of rEp. No modification of these parameters was found in comparison with control mice. On the other hand, rEp increased the number of recognizable splenic megakaryocytes in a dose‐dependent fashion.

These data suggest that rEp has little influence on megakaryocytopoiesis, at least at the doses we used and which are known to elicit a maximal response of erythropoiesis. However, a subset of megakaryocytes with particular kinetic properties, such as those in the spleen of mice, may be responsive to relatively high doses of rEp. The significance of this observation in the overall regulation of megakaryocytopoiesis remains to be determined.
Enhancement of superoxide anion release by granulocytes harvested from patients receiving granulocyte‐macrophage colony‐stimulating factor
Authors: Sullivan Richard , Fredette Joseph P. , Socinski Mark , Elias Anthony , Antman Karen , Schnipper Lowell , Griffin James D.
Published: March 2008

Summary. We examined the rate and quantity of superoxide anion (O2−) generation by granulocytes harvested from the blood of patients before and after a 12–24 h constant intravenous infusion with recombinant human granulocytemacrophage colony‐stimulating factor (GM‐CSFrh). Seven previously untreated patients with sarcomas who exhibited no bone marrow involvement by their disease were studied. Granulocytes were harvested from the peripheral blood of each patient before and after intravenous infusion with GMCSFrh. The total quantity of O2− produced and the kinetics of its release were then determined following in vitro activation of the cells by the chemotactic peptide N‐formyl methionyl‐leucylphenylalanine (fMLP) or by phorbol myristate acetate (PMA). In all cases, the amount of O2− generated and the rate of its release were significantly increased after GM‐CSFrh infusion. Our findings indicate that intravenous administration of GM‐CSFrh to patients heightens the functional responsiveness of circulating granulocytes.
HRAS1 and INS genes are relocated but not structurally altered as a result of the t(7;11) (p15;p15) in a clone from a patient with acute myeloid leukaemia (M4)
Authors: Morris C. M. , Fitzgerald P. H. , Kennedy M. A. , Hollings P. E. , Garry M. , Corbett G. M.
Published: March 2008

Summary. A patient whose leukaemic cells carried the rare t(7;11)(p15;p15) was diagnosed as having acute myelomonocytic leukaemia (AML‐M4), and supports the association of this specific translocation with forms of acute myeloid leukaemia showing differentiation. Blast phase chronic myeloid leukaemia was excluded by lack of involvement of the ABL and BCR genes.

Chromosome in situ hybridization studies showed that both the HRAS1 and INS genes were present on the terminal part of chromosome 11p which was translocated to chromosome 7p. Neither HRAS1 nor INS were structurally rearranged. Field inversion gel electrophoresis showed that a 400 kb fragment encompassing HRAS1 was structurally entire in leukaemic DNA. Because the INS gene, which was also translocated, is probably located proximal to HRAS1 on chromosome 11p. it is unlikely that HRAS1 was near the chromosome 11 breakpoint or involved in this leukaemia.
Immunophenotypic and enzymatic studies do not support the concept of mixed monocytic‐granulocytic differentiation in acute promyelocytic leukaemia (M3): a study of 44 cases
Authors: Scott Colin Stephen , Patel Divyen , Drexler Hans G. , Master Peter S. , Limbert Howard J. , Roberts Bryon E.
Published: March 2008

Summary. Leukaemic promyelocytes from 30 cases of hypergranular and 14 cases of hypogranular acute promyelocytic leukaemia (M3) were analysed for the presence of monocyte‐associated characteristics to determine whether there was any evidence of mixed (hybrid) granulocytic‐monocytic differentiation. Cytochemically, a high proportion of hypergranular cases showed significant alpha‐naphthyl acetate esterase (ANAE) staining and simultaneous chloroacetate esterase, ANAE expression by single cells was commonly seen. These atypical staining patterns were, however, not a feature of hypogranular cases. Immunophenotypic studies revealed that most hypergranular M3 cases were HLA‐DR‐and that monocyte‐associated membrane CD14 expression was low in all cases tested. In addition, serum lysozyme concentrations (20 cases) were generally within the normal range and thus inconsistent with monocytic involvement in the leukaemic process. The significance of atypical ANAE staining of leukaemic promyelocytes was further examined by analysing ANAE isoenzyme components (defined by isoelectric focusing) in 11 cases. The patterns obtained (G1 and G2) were identical to those found in normal granulocytes and did not show any evidence of monocyte‐associated esterase isoenzyme expression. On the basis of these findings, it is considered that the differentiation process in acute promyelocytic leukaemia is relatively well conserved and that the atypical esterase cytochemistry of hypergranular promyelocytes does not reflect their mixed lineage nature but is simply a consequence of increased granulation.
Risk factors for interstitial pneumonia following bone marrow transplantation for severe aplastic anaemia
Authors: Weiner Roy S. , Horowitz Mary M. , Gale Robert Peter , Dicke Karel A. , van Bekkum Dirk W. , Masaoka Tohru , Ramsay Norma K. C. , Rimm Alfred A. , Rozman Ciril , Bortin Mortimer M.
Published: March 2008

Summary. Data from 547 patients with aplastic anaemia who received bone marrow transplants from HLA‐identical siblings were analysed to determine factors associated with the risk of interstitial pneumonia (IPn). IPn developed in 92 patients (17%). 37% of cases were associated with cytomegalovirus infection and 22% with other organisms: in 41% of cases no organism was identified. The case fatality rate was 64%; the mortality rate due to IPn was 11%. In multivariate analysis, four factors were associated with an increased probability of interstitial pneumonia: use of methotrexate rather than cyclosporine after transplantation (relative risk, 2.8: P<0.0008); occurrence of moderate to severe acute graft‐versus‐host disease (relative risk, 2.2; P<0.002); inclusion of total body radiation in the pretransplant preparative regimen (relative risk 2.2, P<0.004); and patient age >20 (relative risk 1.7, P<0.002). The probability of IPn ranged from 4% for patients with none of these adverse risk factors to 51% (relative risk of 13.4) for patients with all four. The incidence of IPn decreased significantly between 1978 and 1985, paralleling a decrease in the use of total body radiation pretransplant for immune suppression and methotrexate post‐transplant for prophylaxis against graft‐versushost disease.
Effect of polymerization tendency on haematological, rheological and clinical parameters in sickle cell anaemia
Authors: Keidan A. J. , Sowter M. C. , Johnson C. S. , Noguchi C. T. , Girling A. J. , Stevens S. M. E. , Stuart J.
Published: March 2008

Summary. The polymerization tendency of sickle haemoglobin was estimated as a function of oxygen saturation in 30 patients with homozygous sickle cell anaemia. The deformability of their erythrocytes was also measured, by initial‐flow‐rate filtration at 37°C through pores of 5 μm diameter, and clinical severity was assessed using a visual analogue scale. By means of partial correlation analysis, it was found that correlations between haematological, rheological, and clinical parameters in sickle cell anaemia could be explained on the basis of an association of each variable with polymerization tendency. Patients with the greatest tendency to form polymer had the least deformable erythrocytes and perceived their disease to be more severe as judged by the visual analogue scale. Polymer formation also appeared to be a determinant of the number of dense cells which, in turn, determine haemolytic rate and erythrocyte deformability.
Serum procollagen‐III‐peptide level correlates with disease activity in myelofibrosis with myeloid metaplasia
Authors: Barosi G. , Costa A. , Liberato L. N. , Polino G. , Spriano P. , Magrini† U.
Published: March 2008

Summary. In 78 patients with myelofibrosis with myeloid metaplasia (MMM) the serum procollagen III peptide activity (s‐PIIIP) had a higher mean value than in 22 normal adult volunteers (22.5 v. 10 ng/ml). Nevertheless, 21.8% of the patients had s‐PIIIP within the 95th percentile of the normal control group (16 ng/ml): with respect to those whose concentrations exceeded this limit, patients with normal s‐PIIIP levels were younger (55.8 v. 61.2 years), had a higher Hb value (12.3 v. 10.5 g/l), a lower serum ferritin level (106 v. 464μg/l) and a higher platelet count (390 v. 216.109/l). In the overall patient population, s‐PIIIP was significantly higher in those with symptoms of active disease (fever, sweating, weight loss) than in subjects with non‐active disease (28 v. 16.9 ng/ml). At univariate analysis s‐PIIIP correlated (at the 5% level) with increasing WBC, serum ferritin and number of transfusions and with decreasing Hb and platelet count. At multivariate analysis increasing WBC, serum ferritin and age proved to be independently associated with s‐PIIIP. No relationship was found between the s‐PIIIP level and morphometric grading of bone marrow fibrosis, megakaryocyte number, or lymphoid infiltration. Longitudinal studies showed that s‐PIIIP increased with disease progression. The conclusion of the study is that s‐PIIIP correlates more with overall disease activity than with the extent of bone marrow fibrosis.
Multidrug resistance in haemopoietic cell lines, myelodysplastic syndromes and acute myeloblastic leukaemia
Authors: Holmes Jon , Jacobs Allan , Carter Graham , Janowska‐wieczorek Anna , Padua Rose Ann
Published: March 2008

Summary. Resistance to cytotoxic agents is a common clinical problem encountered in the treatment of human myelodysplastic syndromes (MDS) and acute myeloblastic leukaemia (AML). Cellular acquisition of the multidrug resistance (MDR) phenotype confers loss of sensitivity to a wide range of structurally dissimilar anti‐neoplastic agents. This state can arise through increased expression of the mdrl (P‐glycoprotein) gene. We have used the mdrl gene probe to investigate adriamycin resistant (HL60/AR) and vinblastine resistant (CEM/VLB100) human leukaemic cell lines. In addition, peripheral blood or bone marrow cells from 66 patients with MDS and AML have been screened for gene amplification and 40 cases for increased mRNA expression. P‐glycoprotein gene amplification was observed only in the (CEM/VLB100) and not in the HL60/AR or any other leukaemic cell line. Gene amplification was not found in any patient's cells. Eighteen out of 40 patients showed an increase (2 × 20) of mdrl mRNA expression. These results are not only of significance in understanding the biology of human drug resistance but have practical importance in the design of anti‐leukaemic therapy.
Molecular characterization of β‐globin gene mutations in Malay patients with Hb E‐β‐thalassaemia and thalassaemia major
Authors: Yang K. G. , Kutlar F. , George E. , Wilson J. B. , Kutlar A. , Stoming T. A. , Redondo J. M. Gonzalez , Huisman T. H. J.
Published: March 2008

Summary. This study concerned the identification of the β‐thalassaemia mutations that were present in 27 Malay patients with Hb E‐β‐thalassaemia and seven Malay patients with thalassaemia major who were from West Malaysia. Nearly 50% of all β‐thalassaemia chromosomes carried the G → C substitution at nucleotide 5 of IVS‐I; the commonly occurring Chinese anomalies such as the frameshift at codons 41 and 42, the nonsense mutation A → T at codon 17, the A → G substitution at position −28 of the promoter region, and the C → T substitution at position 654 of the second intron, were rare or absent. Two new thalassaemia mutations were discovered. The first involves a frameshift at codon 35 (‐C) that was found in two patients with Hb E‐β°‐thalassaemia and causes a β°‐thalassaemia because a stop codon is present at codon 60. The second is an AAC → AGC mutation in codon 19 that was present on six chromosomes. This substitution results in the production of an abnormal β chain (β‐Malay) that has an Asn → Ser substitution at position β19. Hb Malay is a‘Hb Knossos‐like’β+‐thalassaemia abnormality; the A → G mutation at codon 19 likely creates an alternate splicing site between codons 17 and 18, reducing the efficiency of the normal donor splice site at IVS‐I to about 60%.
Distribution of a set of idiotopes detected by a monoclonal antibody panel in 42 cases of chronic lymphocytic leukaemia: definition of potential targets for immunotherapy
Authors: Cachia P. G. , Dewar A. E. , Krajewski A. S. , Stockdill G. , Walker L. C. , Habeshaw J. A.
Published: March 2008

Summary. Using a panel of 14 monoclonal V region reactive antibodies generated against a single IgG1λ paraprotein we have identified shared idiotopes in a group of 42 patients with chronic lymphocytic leukaemia (CLL). The specificity of cellular staining by indirect immunoperoxidase was confirmed in the majority of cases by an ELISA assay using secreted idiotypic immunoglobulin. In a few cases weak cellular binding by the panel antibodies could not be confirmed as specific for immunoglobulin. Four monoclonal antibodies specific for lambda chain determinants reacted with 5–29% of lambda expressing CLLs but the significance of this is uncertain as the antibodies may be recognizing one of the commoner Vλ subgroups. Two antibodies, which are only weakly reactive with normal serum immunoglobulin, detected heavy chain associated idiotopes which were expressed by 7% and 14% of CLL cases. One of these antibodies detects an idiotope which was expressed significantly more frequently by CLL associated immunoglobulin than by a panel of paraproteins. Such preferentially expressed idiotopes may be useful in investigating the biology of this disorder as well as providing attractive targets for immunotherapy.
Investigation of the CD10 (cALLA) negative acute lymphoblastic leukaemia: further description of a group with a poor prognosis
Authors: Vannier Jean Pierre , Bene Marie C. , Faure Gilbert C. , Bastard Christian , Garand Richard , Bernard Alain
Published: March 2008

Summary. The absence of CD10 (cALLA) in non‐T non‐B acute lymphoblastic leukaemia (ALL) is usually considered to be of adverse prognostic significance. From a large multi‐centre series of phenotyped ALL, we have identified a group of 23 non‐T non‐B ALL where blast cells were CD 10 negative and CD 19 positive. Class II antigens were present in 80% and CD9 in 70%. Eight patients had successful karyotype analysis at diagnosis, and an additional patient at first relapse. Seven of these karyotypes showed a (4:11) (q21;q23) translocation. Most of the patients (70%) were young females, and they often presented with organomegaly. Six patients were less than 1 year old. The white cell count was over 100 × 109/l in 48% of the cases. The FAB type was L2 in 56% of the patients. The most striking features were the poor response to therapy and survival. Six patients never attained complete remission and nine patients relapsed, most of them during the first year after diagnosis. Allogeneic bone marrow transplantation was performed in three children, of whom two are still alive 2 years after diagnosis. This study emphasizes the prognostic value of immuno‐phenotypic and karyotypic investigations of ALL.
Circulating monotypic B‐cells in multiple myeloma: association with lambda paraproteins
Authors: Bagg Adam , Becker Piet , Bezwoda Werner , Van Rensburg Louise , Mendelow Barry
Published: March 2008

Summary. The peripheral blood and bone marrow mononuclear cell immunophenotype was investigated in a series of 41 patients with plasma cell dyscrasias (29 with multiple myeloma and 12 with monoclonal gammopathy of undetermined significance (MGUS) or solitary plasmacytoma). A statistically significant relationship between the presence of monotypic B‐cells (MBC) in the peripheral blood and the paraprotein light chain isotype was found in the myeloma patients (P= 0·0025). MBCs were documented in 71% of patients with lambda paraproteins, compared with only 13% of patients with kappa paraproteins. The difference in MBC was independent of disease stage as well as of individual prognosticators such as haemoglobin, renal function, paraprotein size and beta‐2‐microglobulin, although lambda producers had significantly higher calcium levels than patients with kappa paraproteins (P=0·03). A similar trend was also noted for MBC to be found more commonly in patients with lambda producing MGUS and solitary plasmacytoma. This phenomenon may account for the worse prognosis in patients with lambda paraproteins. In our hands, immunophenotypic detection of peripheral blood involvement in plasma cell dyscrasias is more sensitive than investigation of karyotype or immunoglobulin gene rearrangements.
Establishment of a human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute megakaryoblastic leukaemia
Authors: Sato Takeyuki , Fuse Akira , Eguchi Mitsuoki , Hayashi Yasuhide , Ryo Ryukichi , Adachi Masashi , Kishimoto Yuji , Teramura Masanao , Mizoguchi Hideaki , Shima Yukichi , Komori Isao , Sunami Shousuke , Okimoto Yuri , Nakajima Hironori
Published: March 2008

Summary. A new megakaryoblastic cell line (CMK), which also exhibits erythroid and myeloid markers, was established from a Down's syndrome patient suffering from acute megakaryoblastic leukaemia. The CMK cells were found to be positive in reactions with anti‐platelet antibodies (anti‐glycoproteins IIb/IIIa and Ib, and Plt‐1). Platelet peroxidase (PPO) reactivity was found to be associated with the nuclear envelope and the endoplasmic reticulum but not with the Golgi apparatus. Some cells possessed cytoplasmic granules with the characteristics of α‐granules and demarcation membranes. Karyotyping revealed near‐tetraploidy (modal chromosome number of 95; ranging 87–98) and a translocation der(17)t(11;17), also found in the original leukaemic cells, confirming that the cells were derived from the patient's malignant blasts. The CMK cells were also found to be positive in reaction with anti‐glycophorin A antibody, as well as with anti‐myeloid antibodies (MY4, MY7 and MY9). Treatment of CMK cells with phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) greatly enhanced the reactivity with anti‐platelet antibodies, increased the number of cells in which cytoplasm was dissociated into numerous segments and suppressed the reactivity with anti‐glycophorin A. The proliferation of CMK cells was stimulated by interleukin‐3 (IL‐3) and granulocyte‐macrophage colony stimulation factor (GM‐CSF). This cell line should be a useful tool for analysing the basis of the afferent association between megakaryoblastic leukaemia and Down's syndrome, as well as for further study of megakaryocytic differentiation.
Subcellular composition of platelet density subpopulations prepared using continuous Percoll gradients
Authors: Chamberlain Kevin G. , Seth Priti , Jones Maxwell K. , Penington David G.
Published: March 2008

Summary. Normal human platelets have been separated by density on continuous Percoll gradients and the subcellular composition of platelets of different density has been analysed. The number and concentration of dense granules increased significantly with platelet density, as did the concentrations of the dense granule constituents calcium and serotonin. The amount of serotonin per granule in the low density (LD) platelets was only two‐thirds of the corresponding amount in the high density (HD) platelets. Platelets of all densities were able to sequester exogenous serotonin and release it in response to thrombin stimulation with similar efficiencies. The concentrations of the α‐granule constituents von Willebrand factor and β‐thromboglobulin increased significantly with platelet density but the concentrations of the lysosomal enzyme β‐N‐acetylglucosaminidase and total sialic acid did not differ significantly in the density subpopulations. The concentrations of the cytosolic enzymes lactate dehydrogenase and glucose‐6‐phosphate dehydrogenase were slightly higher in the LD population than in the other density subpopulations. The concentration of glycogen showed a marked positive relationship with platelet density and calculations suggested that glycogen was an important determinant of platelet density heterogeneity. The findings of the present study are compatible with recent suggestions that LD platelets may be slightly younger than HD platelets in normal human subjects.
Identification of a tissue kallikrein in human polymorphonuclear leucocytes
Authors: Figueroa C. D. , MacIver A. G. , Bhoola K. D.
Published: March 2008

Summary. We have identified a tissue kallikrein in polymorphonuclear (PMN) leucocytes of normal human blood and bone marrow by immunocytochemistry, radioimmunoassay and enzymology. Immunoreactive tissue kallikrein was visualized in the mature neutrophil leucocytes and in immature forms such as metamyelocytes and myelocytes. No tissue kallikrein was detected in eosinophil leucocytes, lymphocytes, macrophages, megakaryocytes and platelets. So far, we have failed to observe immunoreactivity to tissue kallikrein in basophils. The presence of tissue kallikrein in extracts prepared from PMN leucocytes isolated from peripheral blood was demonstrated by immunodiffusion, dot‐blotting and by radioimmunoassay. The kininogenase and amidase activity of the extracts resembled that of tissue kallikrein in being resistant to soya bean trypsin inhibitor and sensitive to trasylol. The amidase activity attributable to tissue kallikrein was completely inhibited by specific antisera.
Platelet‐dependent induction and amplification of polymorphonuclear leucocyte lysosomal enzyme release
Authors: Del Maschio Aldo , Corvazier Elizabeth , Maillet Françoise , Kazatchkine Michel D. , Maclouf Jacques
Published: March 2008

Summary. Degranulation of human polymorphonuclear leucocytes induced by opsonized zymosan (OpZ) was studied in the presence or absence of platelets, either resting or stimulated by thrombin. Lysozyme secretion from polymorphonuclear leucocytes stimulated with OpZ increased in the presence of platelets. A further increase was observed when platelets had been stimulated with thrombin. The effect was dependent on platelet concentration (10–80 platelets/polymorphonuclear leucocytes). Resting polymorphonuclear leucocytes could be activated by platelets that had been stimulated with thrombin with an optimal effect observed at 0·1 U/ml of thrombin. Substitution of platelets with platelet‐released products only resulted in significant stimulation of polymorphonuclear leucocytes at concentrations above those used in the coincubations experiments. Zymosan coated with various opsonins showed a gradient in the relative intensity of polymorphonuclear leucocytes degranulation. Platelet‐dependent enhancement of polymorphonuclear leucocytes degranulation was higher with opsonins that were poorer at inducing lysosomal secretion. The role of platelets as helper cells in the activation of polymorphonuclear leucocytes appears to be dominant when polymorphonuclear leucocytes are challenged in suboptimal conditions. Positive interactions between platelets and polymorphonuclear leucocytes may be relevant in vivo at the site of inflammation where platelets could enhance effector functions of polymorphonuclear leucocytes.
Recombinant human interferon (IFN) alpha‐2b in chronic myelogenous leukaemia: dose dependency of response and frequency of neutralizing anti‐interferon antibodies
Authors: Freund Mathias , von Wussow Peter , Diedrich Helmut , Eisert Roswita , Link Hartmut , Wilke Hansjochen , Buchholz F. , LeBlanc S. , Fonatsch Christa , Deicher Helmut , Poliwoda Hubert
Published: March 2008

Summary. Twenty‐seven patients with Philadelphia chromosome positive chronic myelogenous leukaemia in the chronic phase were treated with low doses of recombinant interferon (IFN) alpha‐2b. Ten patients entered a complete and six a partial haematologic remission with a median duration of 5·8 and 9·1 months respectively. Five minor cytogenetic responses were observed. These results are inferior compared to other studies with higher interferon‐doses. Fever was an acute side effect after injection of IFN, limb pains and fatigue occurred protractedly. Haematologic side effects, nonspecific EEG changes, weight loss, and development of pulmonary infiltrates were observed in later periods of the treatment. Eight patients developed neutralizing anti‐IFN antibodies after 4·2–20·4 months (median 12·8 months). Anti‐IFN antibodies were associated with relapse or refractoriness to IFN treatment: five out of nine patients with rising WBC after initial fall had antibodies, while four did not. Two out of four patients with primary non‐response had IFN‐antibodies. These results may indicate a serious problem in the long‐term treatment of CML with recombinant interferon.
Heterogenous mechanisms of autocrine growth of AML blasts
Authors: Reilly I. A. G. , Kozlowski R. , Russell N. H.
Published: March 2008

Summary. The ability of blast cells to grow autonomously and to produce autostimulatory growth factors has been investigated in 25 consecutive patients with AML. An autostimulatory index (ASI) was calculated (no. of colonies without CSF ÷ no. of colonies with CSF) and patients classified into four groups: Group 1 (n= 3): non‐growers; Group 2 (n= 4): CSF‐dependent (ASI < 0·1); Group 3 (n= 11): partially autonomous (ASI 0·1–0·8); and Group 4 (n= 7): fully autonomous/CSF‐unresponsive (ASI > 0·8). In Group 3 patients colony formation and DNA synthesis were significantly (P < 0·01) augmented by CSFs but at high cell concentrations became CSF‐independent. Blast cell‐conditioned medium (BCCM) from these patients exhibited potent autostimulatory activity, increasing DNA synthesis by ≤ 5‐fold, and also stimulated CSF‐dependent homologous blasts by ≤20‐fold. In 5/5 this activity was neutralized by anti‐GM‐CSF, which also inhibited autonomous proliferation of their blast cells. Group 4 blasts also secreted GM‐CSF but their BCCM possessed no autostimulatory activity, and anti GM‐CSF failed to inhibit their autonomous growth. No membrane‐associated CSF activity was found, however purified cytosolic fractions stimulated proliferation of CSF‐dependent homologous blasts, consistent with production and secretion of CSF which is present in active form in the cytosol but does not autostimulate via membrane receptors. These results suggest that autocrine mechanisms are important in regulating blast cell proliferation, but that the mechanisms are heterogeneous.
Bone marrow histopathology of patients with severe aplastic anaemia before treatment and at follow‐up
Authors: de Planque Mariet M. , van Krieken Johan H. J. M. , Kluin‐Nelemans Hanneke C. , Colla Leny P. J. M. , van der Burgh Fred , Brand Anneke , Kluin Philip M.
Published: March 2008

Summary. Pretreatment bone marrow biopsies of 63 patients with severe aplastic anaemia (SAA) who were not transplanted and of whom 55 received ATG, were evaluated according to the amount and character of residual haematopoiesis (‘genuine’ aplasia/intermediate/hypoplastic myelodysplasia (MD)), inflammatory infiltrate (Te Velde & Haak, 1977, grade I/II/III), and number of mast cells (normal or slightly increased/increased). Of 61 evaluable biopsies, 47 were ‘genuine’ aplastic, 11 intermediate and three hypoplastic MD. Inflammatory infiltrates were graded as III in 36/60 evaluable biopsies, as II in 21 and I in three. A moderate to marked increase of mast cells was seen in 19/61. Of grade III patients, 86% had a <90 d interval between diagnosis and administration of ATG, versus 50% of grade I/II patients (P <0·01). No other correlations with pretreatment characteristics were found. No significant prognostic value for survival or response to ATG of any of these three criteria has been identified. More patients with grade III inflammation tended to show adequate recovery at 4 and 6 months after ATG. Stem cell damage, not identifiable morphologically, and/or impairment of accessory cells might play a major role in eventual outcome of SAA patients. Thirty‐five patients are currently alive, median 3·8 years (up to 12·4) after ATG. Follow‐up bone marrow aspirates and biopsies of 32 patients were evaluable and none showed normal haematopoiesis. One patient revealed persistent aplasia. Of the remaining 31, haematopoiesis was decreased in 14 and increased in eight. All had dyserythropoiesis, 28 dysplastic myelopoiesis and in 16/29 with evaluable megakaryocytes, dysmegakaryopoiesis was found. Sixteen patients had normo‐ to hypercellular bone marrows with two dysplastic cell lines (consistent with myelodysplastic syndrome (MDS) according to the FAB‐group). The prognostic impact of the dysplastic abnormalities found in these patients needs longer follow‐up. Close observation is indicated in view of the previously recognized, albeit uncommon, evolution of SAA to MDS/acute non‐lymphocytic leukaemia.
On the diagnosis of erythrocyte enzyme defects in the presence of high reticulocyte counts
Authors: Lakomek M. , Schröter W. , De Maeyer G. , Winkler H.
Published: March 2008

Summary. The separation of red blood cells into reticulocytes and young and old erythrocytes enables investigations of fractions with different contents of reticulocytes. Activities of hexokinase, glucose phosphate isomerase, phosphofructokinase, pyruvate kinase and glucose‐6‐phosphate dehydrogenase showed a linear relationship to reticulocyte counts. The dependence of these enzyme activities on the age of the red blood cells exhibited a strong decline from the reticulocyte to the young erythrocyte stage followed by only little further loss of activity, thus leading to a biphasic decay of enzyme activities.

By linear regression analysis enzyme activities in erythrocytes (AE) and reticulocytes (AR) could be evaluated. The activity of a given enzyme in the reticulocyte exceeded that of the erythrocyte; the quotient AR/AE represents the decline of enzyme activity from the reticulocyte to the erythrocyte stage. This value AR/AE is 16·7 for pyruvate kinase and 9·4 for hexokinase and thus considerably higher than that for the other enzymes investigated (glucose phosphate isomerase: 2·9, phosphofructokinase: 4·3, glucose‐6‐phosphate dehydrogenase: 4·5). In patients suffering from erythrocyte enzymopathies, the AR/AE for pyruvate kinase was 16·2 and thus almost identical to the normal enzyme.

Calibration curves where the enzyme activity is plotted versus the fraction of reticulocytes enable the determination of normal activity of a given erythrocyte enzyme depending on the content of reticulocytes in red blood cell suspensions. Thus an unambiguous diagnosis of enzyme defects irrespective of reticulocyte counts becomes possible.