bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased serum levels of transforming growth factor β‐1 in patients affected by thrombotic thrombocytopenic purpura (TTP): its implications on bone marrow haematopoiesis
Authors: Zauli Giorgio , Gugliotta Luigi , Catani Lucia , Vianelli Nicola , Borgatti Paola , Belmonte Monica Mattioli , Tura Sante
Published: March 2008

Summary. In this study we evaluated the effect of serum collected from seven thrombotic thrombocytopenic purpura (TTP) patients, either in the acute phase of the disease or in clinical remission, on the in vitro growth of bone marrow haematopoietic progenitor cells, obtained from the same TTP patients in clinical remission and from normal donors.

The addition to the cultures of autologous sera collected from TTP patients in acute phase of the disease showed a clear‐cut dose‐dependent inhibition of immature haematopoietic progenitor cells (BFU‐E, CFU‐meg and 14th day CFU‐GM). On the other hand, no inhibitory effects were observed on more mature 7th day CFU‐GM. Interestingly, also sera collected from TTP patients in clinical remission still maintained some inhibitory activity on the growth of immature progenitor cells. A similar inhibitory activity was noticed when TTP sera were tested on normal bone marrow haematopoietic progenitor cells.

Such inhibitory activity was significantly reduced in blocking experiments by the addition of a polyclonal neutralizing anti‐TGF‐β1 antibody and the presence of increased levels of both bioactive and latent TGF‐β1 in TTP sera was confirmed in a bioassay on CCL64 cells.

These data contribute to explain the lack of a clear compensatory haematopoiesis observed in some patients with active TTP and add further evidence to the notion of the existence of a state of latent platelet activation in TTP patients in clinical remission.
Effect of transfer factor on myelosuppression and related morbidity induced by chemotherapy in acute leukaemias
Authors: Fernández Oscar , Díaz Nixi , Morales Ernesto , Toledo Julio , Hernández Eraida , Rojas Sarai , Madriz Xochilt , Saura Pedro López
Published: March 2008

Summary. The aim of this study is to determine the safety and efficacy of Transfer Factor (TF) in accelerating the haematopoietic recovery in patients with acute leukaemias (AL), following intensive therapy to induce remission of the disease. Twenty‐two patients with different types of AL (16 AML, three BC‐CML and three ALL) were studied. The patients were divided in two groups. Group 1 (eight AML, two BC‐CML and one ALL) received, after myelosuppression induced by chemotherapy, TF (1 unit daily, subcutaneous) until leucocyte count was >2·5 × 109/1 and platelet count > 80 × 109/l. Group 2 was considered the control group and did not receive TF. Treatment with TF accelerated the recovery of neutrophils, leucocytes, platelets (P < 0·001. and haemoglobin (P < 0·01). As a logical consequence, incidence and severity of infection and haemorrhage were lesser in the TF group than in the control group. There was no evidence that TF accelerated the re‐growth of leukaemic cells. It seems that TF is safe in AL, accelerating haematopoietic recovery. However, it should be used with caution until results of additional trials become available.
Immunophenotypic characterization of myelomonocytic cells in patients with myelodysplastic syndrome
Authors: Felzmann Thomas , Gisslinger Heinz , Krieger Otto , Majdic Otto , Ludwig Heinz , Köller Ursula
Published: March 2008

Summary. Infections, an important determinating factor in the clinical course of myelodysplastic syndromes (MDS), result in activation of myelomonocytic cells. In this study we demonstrate activation‐associated immunophenotypic changes of cell surface antigens on monocytes and granulocytes observed in two groups of MDS patients, one with low and another one with high clinical risk, and compared them to healthy individuals. Significantly changed expression of the complement receptors 1 (CD35) and 3 (CD11b), the Fcγ receptor I (CD64), the leucocyte‐homing receptor (CD44) and the activation associated membrane proteins CD67 and M5 were found on monocytes and/or granulocytes of MDS patients. In low‐risk MDS patients we observed activation‐associated phenotypic changes only in monocytes, whereas in high‐risk MDS patients, both monocytes and granulocytes showed such changes. Additionally, we performed respiratory burst experiments and observed an impaired response of monocytes and granulocytes derived from MDS patients. Despite the fact that all patients were free of infection by clinical criteria, cell surface phenotyping as well as the reduced respiratory burst capacity of myelomonocytic cells suggests in vivo preactivation of these cells.
Influence of demographic factors on antithrombin III activity in a healthy population
Authors: Tait R. C. , Walker Isobel D. , Islam S. I. A. M. , McCall F. , Conkie J. A. , Mitchell R. , Davidson J. F.
Published: March 2008

Summary. Antithrombin III (AT III) activity has been measured in 9669 healthy blood donors (5525 male and 4144 female). The distribution of AT III is approximately ‘normal’ with mean 105·6 iu/dl and standard deviation 11·2; however, definite age and sex related variations are evident. Pre‐menopausal females have lower mean AT III compared to their male contemporaries who have remarkably stable mean AT III until 45 years, after which there is a gradual decline. In contrast, post‐menopausal females have higher mean AT III than both males of the same age and younger pre‐menopausal females. Concurrent hormone replacement therapy inhibits this rise. The use of hormonal preparations is associated with a 4 iu/dl reduction of mean AT III in younger females but not in those over 30 years. Smoking may result in a mild increase in AT III of doubtful clinical significance. On‐going genetic and family studies are expected to predict a prevalence rate of congenital AT III deficiency in excess of the previously reported figure of 0·02%. The authors consider these observed variations as minor and recommend the use of a single reference range for AT III activity, but that particular care be taken when interpreting results in pill‐taking females and the elderly.
Inhomogeneity of the circulating BFU‐E regulation in sickle cell anaemia: accessory cells properties and BFU‐E growth factor response pattern
Authors: Croizat Helena , Nagel Ronald L.
Published: March 2008

Summary. Sickle cell anaemia (SS) patients with low (<9%) HbF levels (LFSS) are characterized by an increased number of circulating BFU‐E in active DNA synthesis, and release of burst promoting activity (BPA) by unstimulated low density (LD) adherent cells. In contrast, circulating BFU‐E from SS patients with high (>9%) HbF levels (HFSS) are normal in number, largely in resting phase, and their LD cells do not release BPA‐like activity.

We report now that in LFSS patients, adherent cell depletion decreases BFU‐E growth in culture and apparent BFU‐E cycling. Furthermore, addition of conditioned media (CM) from LD cells of LFSS patients restored cycling BFU‐E expression in culture. Neutralization analysis with anti‐GM‐CSF antibody demonstrated that GM‐CSF is, at least, one factor responsible for BPA activity present in this CM. Thus, GM‐CSF is constitutively produced by unstimulated monocytes in LFSS patients. In contrast, HFSS patients' adherent cell depletion increases cycling of BFU‐E in culture. CM from HFSS patients inhibits BFU‐E expression in culture. Hence, LD adherent cells from HFSS patients may release a yet unknown inhibitor factor(s). In addition, we report a distinct response pattern in SS patients' BFU‐E to growth factor (GM‐CSF, IL‐3): (a) LFSS patients have a BFU‐E population, equally responsive to GM‐CSF and IL‐3; (b) HFSS patients, have a subset of BFU‐E exclusively dependent on IL‐3 (20–40% of the circulating BFU‐E). This pattern is very similar to that of normal BFU‐E.

In conclusion, BFU‐E from LFSS patients represent an actively proliferating population, equally responsive to GM‐CSF and IL‐3, controlled by constitutively produced GM‐CSF, suggesting a unique BFU‐E behaviour in SS patients with low HbF levels and high haemopoietic stress. The heterogeneous regulation of BFU‐E in SS disease seems to be the epiphenomenon of HbF levels, and not vice versa.
Induction of FcγR‐III (CD16) expression on neutrophils affected by paroxysmal nocturnal haemoglobinuria by administration of granulocyte colony‐stimulating factor
Authors: Ninomiya Haruhiko , Muraki Yasushi , Shibuya Kazuko , Nagasawa Toshiro , Abe Tsukasa
Published: March 2008

Summary. The inducibility of glycosyl‐phosphatidylinositol (GPI)‐anchored proteins on affected paroxysmal nocturnal haemoglobinuria (PNH) neutrophils (PMN) after both in vitro and in vivo stimulation was investigated. FcγR‐III (CD16), decay‐accelerating factor (DAF/CD55)and 20 kD homologous restriction factor (HRF20/CD59) were demonstrated to be concurrently deficient on unstimulated defective PNH PMN. Upon in vitro stimulation with either N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), zymosan‐activated serum (ZAS), or recombinant human granulocyte colony‐stimulation factor (G‐CSF), neither CD16 nor CD55 expression was induced on defective PNH PMN. G‐CSF was administered to two patients with PNH when their conditions were complicated by bacterial infections, or to prevent infections associated with the extraction of teeth or cataract surgery. CD16 expression was induced on the defective PNH PMN in both cases during the administration of G‐CSF, but the expression of CD55 and CD59 was not. CD16, induced on the defective PNH PMN during the administration of G‐CSF, was phosphatidylinositol‐specific phospholipase C (PIPLC)‐sensitive, implying that it had GPI‐linkage to the membranes. The patients treated with G‐CSF recovered from infection or evaded infection. These observations suggest that a deficiency of GPI‐anchored proteins is not always seen in defective PNH blood cells, at least under certain stimulation conditions.
Defective and normal haematopoietic stem cells in paroxysmal nocturnal haemoglobinuria
Authors: Terstappen Leon W. M. M. , Nguyen Mai , Huang Shiang , Lazarus Hillard M. , Medof M. Edward
Published: March 2008

Summary. The expression of decay‐accelerating factor (DAF or CD55) and CD59 during haematopoietic cell development in bone marrow aspirates of two patients with paroxysmal nocturnal haemoglobulinuria (PNH) was compared with that in normal bone marrow by five‐dimensional flow cytometry. In contrast to early uncommitted haematopoietic progenitor cells (CD34+, CD38‐) in normal bone marrow which uniformly express DAF and CD59, the majority of CD34+, CD38‐ cells in both patients' marrow exhibited the absence of the two proteins. In both specimens, however, subpopulations of CD34+, CD38‐ cells expressing DAF and CD59 were detectable, indicative of the presence of two lines of haematopoiesis, one abnormal and the other normal. Concurrent abnormal and normal haematopoietic development was further evident by the presence of subpopulations of DAF‐, CD59‐ and DAF+, CD59+ cells along the differentiation and maturation pathways of the myeloid (CD33+, CD15‐ → CD33+→++, CD15+), the erythroid (CD45dim, CD71dim→ CD45‐, CD71++), and the B‐lymphoid cell lineages (CD10++, CD20‐ → CD10‐, CD20++). While the majority of cells differentiating into and maturing along each cell lineage lacked DAF and CD59, the majority of mature B (CD20++, CD10‐) and T‐lymphocytes (CD3+) expressed both proteins suggestive of the presence of lymphocytes with a long life span which were generated from normal haematopoietic progenitors before the onset of the disease. The detection of distinct sets of CD34+, CD38‐ →+ progenitor cells which are DAF+, CD59+ or DAF‐, CD59‐ in marrow of PNH patients has relevance for the treatment of PNH. Cells with the phenotype CD34+, CD38‐, DAF+, CD59+ are capable of self renewal and represent potential candidates for autologous bone marrow transplantation following depletion of CD34+, CD38‐, DAF‐, CD59‐ cells.
An alternative strategy for identification of type IIA VWD mutations
Authors: Winter P. C. , Mayne E. E.
Published: March 2008

Summary. A 306 base pair segment of the von Willebrands factor (VWF) gene between nucleotides 4627 and 4932 was amplified by PCR using DNA from a type IIA VWD patient. The amplified DNA was cloned in the plasmid pCRII. Clones of the VWF gene and pseudogene were distinguished by their KpnI restriction patterns. A series of six VWF gene clones was sequenced. A single C to T point mutation at nucleotide 4789 resulting in the substitution of arginine 834 by tryptophan was identified in four clones. This mutation, which destroys a BstEII restriction site, was also detected in amplified DNA of affected relatives of the patient. The method described avoids difficulties associated with strategies employed previously for identification of VWD mutations.
CAMPATH‐1 monoclonal antibody therapy in severe refractory autoimmune thrombocytopenic purpura
Authors: Lim S. H. , Hale G. , Marcus R. E. , Waldmann H. , Baglin T. P.
Published: March 2008

Summary. Six patients with autoimmune thrombocytopenic purpura (three of whom had CLL/NHL and one previous Hodgkin's disease) refractory to conventional therapy were treated with an antilymphocyte monoclonal antibody directed at CDw52 (CAMPATH‐1). We observed response in four of the five evaluable patients; in three patients the response has lasted more than 4–9 months. However. response did not occur in most cases until 4–6 weeks after the commencement of the antibody therapy, suggesting that the mechanism of action is probably that of immune modulation.
Isolation of human herpesvirus 7 from a child with symptoms mimicking chronic Epstein‐Barr virus infection
Authors: Kawa‐Ha Keisei , Tanaka Keiko , Inoue Masami , Sakata Naoki , Okada Shintaro , Kurata Takeshi , Mukai Tetsu , Yamanishi Koichi
Published: March 2008

Summary. Human herpesvirus‐7 (HHV‐7), which is a newly identified human herpesvirus with an unknown pathologic role, was isolated from a 5‐year‐old boy suffering from fever, hepatosplenomegaly and pancytopenia. Although the clinical course was similar to that of chronic active Epstein‐Barr virus infection, no viruses other than HHV‐7 were isolated. This finding raises the possibility that HHV‐7 played a pathogenic role in the present patient.
GM‐CSF: modulation of biochemical and cytotoxic effects of tiazofurin in HL‐60 cells
Authors: Fritzer Monika , Gharehbaghi Kamran , Pillwein Konrad , Chiba Peter , Goldenberg Hans , Szekeres Thomas
Published: March 2008

Summary. Cytokines, such as granulocyte macrophage colony stimulating factor (GM‐CSF) or interleukin‐3 (IL‐3) recruit quiescent cells into the cell cycle and sensitize these cells towards cell cycle specific chemotherapeutic agents. We examined the in vitro effects of GM‐CSF on HL‐60 cells and tested its modulatory influence on biochemical and cytotoxic effects seen with tiazofurin, a potent and specific inhibitor of IMP dehydrogenase. Incubation of HL‐60 cells with 500 U/ml GM‐CSF for 4 d enhanced cell proliferation, which was accompanied by a significant increase in IMP dehydrogenase activity (from 2·22 in control cells to 3·70 nmol/mg/h in cells pretreated with GM‐CSF). When HL‐60 cells were incubated with 100 μm tiazofurin for 2 h, intracellular GTP decreased to 46% of untreated control cells. In HL‐60 cells pretreated with GM‐CSF, GTP pools decreased to 38% of control after incubation with tiazofurin which is 69% of the predicted value for additive effect. The MTT chemosensitivity assay yielded significantly decreased IC50 values for tiazofurin in HL‐60 cells, preincubated with GM‐CSF (IC50 decreased from 13 μm to 10 μm). Therefore our results suggest that combination therapy with GM‐CSF and tiazofurin may be beneficial for the treatment of refractory leukaemia patients.
Subcellular fractionation studies indicate an intracellular localization for human monocyte specific esterase (MSE)
Authors: Patel Divyen , Hooper Nigel M. , Scott Colin Stephen
Published: March 2008

Summary.Human monocyte‐specific esterase (MSE) is one of the few haemopoietic cell enzymes that show absolute lineage restriction. Although the function of MSE has yet to be deduced, its potential role in tumour cell killing has stimulated particular interest. Knowledge of subcellular localization of MSE is fundamental to understanding its function and, in this context, it is widely believed that MSE is expressed as a plasma membrane ectoenzyme; a contention that is largely based upon experiments which examined fixed cells by ultrastructural cytochemistry. However, as recent molecular studies of human MSE indicate, a number of inconsistencies between its structure and a membrane localization, we applied the techniques of phase separation in the non‐ionic detergent Triton X‐114 and differential centrifugation to further investigate whether this particular esterase species is membrane‐bound or associated with an intracellular organelle. These studies provide strong evidence that MSE is in fact a soluble intracellular enzyme that is almost certainly located within the lumen of the endoplasmic reticulum.
Identification of nine novel mutations in type I antithrombin deficiency by heteroduplex screening
Authors: Chowdhury V. , Olds R. J. , Lane D. A. , Conard J. , Pabinger I. , Ryan K. , Bauer K. A. , Bhavnani M. , Abildgaard U. , Finazzi G. , Castaman G. , Mannucci P. M. , Thein S. L.
Published: March 2008

Summary. We have utilized DNA heteroduplex detection as a method for screening sequences of the antithrombin (AT) gene for the presence of mutations. Affected individuals from 41 kindreds with type Ia antithrombin deficiency were investigated. Heteroduplexes were detected in 12 cases; direct sequencing of the appropriate exons revealed nine cases with novel mutations, and two with previously described mutations. In addition, a new polymorphism in the 5’untranslated region was characterized. The defects included minor insertions and deletions which lead to the removal of intact codons or premature termination, and single base substitutions leading to premature termination or amino acid substitution. In all cases, the affected individuals were heterozygous for the defect and variant AT protein was not detected. In keeping with previous reports the defects associated with type Ia AT deficiency are extremely heterogeneous, the vast majority being point mutations. This study also demonstrates the efficiency of hydrolink gel electrophoresis as a method of screening for unknown mutations by heteroduplex detection.
Heparin‐associated thrombocytopenia: immune complexes are attached to the platelet membrane by the negative charge of highly sulphated oligosaccharides
Authors: Greinacher A. , Michels I. , Liebenhoff U. , Presek P. , Mueller‐Eckhardt C.
Published: March 2008

Summary. The interaction of sulphated oligosaccharides (SO) with platelets and the antibody of heparin‐associated thrombocytopenia (HAT type II) was investigated. 3H‐heparin binding to platelets was inhibited by different SO, depending on their grade of sulphation. Dextran sulphate, pentosan polysulphate, and heparin were more effective than were LMW heparins. De‐N‐sulphated heparin and a LMW heparinoid (Org 10172) had no effect. Platelets preincubated with high‐grade SO and washed, released serotonin in the presence of HAT sera without additional heparin. Platelets preincubated with HAT sera and then washed were not activated when heparin was added. Only high‐grade SO which inhibited heparin binding to platelets caused platelet activation with HAT sera. However, low‐ and high‐grade SO in high concentrations (0·11 g/l) inhibited serotonin release induced by HAT sera and heparin. 32P‐phosphorylation of platelet proteins was enhanced by HAT‐IgG and heparin and by heat‐aggregated IgG, and was inhibited by the moab IV.3. High SO concentrations inhibited only the effect of HAT‐IgG and not that of aggregated IgG. We assume that the antigen in HAT involves a releasable platelet protein with a binding site for SO. This was corroborated by studies with an anti‐platelet factor 4 antibody causing Fc‐receptor dependent platelet activation inhibitable by high SO concentrations.
Induction of TNF‐α in patients with myelodysplastic syndromes undergoing treatment with interleukin‐3
Authors: Seipelt Gernot , Ganser Arnold , Duranceyk Heidi , Maurer Alexander , Ottmann Oliver G. , Hoelzer Dieter
Published: March 2008

Summary. The study was undertaken to analyse whether the presence or the induction of TNF‐α, a potent inhibitor of haemopoiesis, might affect the clinical response to treatment with interleukin‐3 in patients with myelodysplastic syndromes. A total of 15 patients were treated with IL‐3. Baseline serum TNF‐α levels were elevated in MDS patients (14·2 ± 2·4 pg/ml) compared to healthy controls (9·1 ± 1·1 pg/ml). During IL‐3 therapy TNF‐α levels remained unchanged in 3/14 patients in whom platelet counts increased, while in non‐responders TNF‐α levels increased 1·9‐fold (P < 0·025).

These findings indicate that TNF‐α not only is induced during IL‐3 therapy in MDS patients but that this elevation might be associated with a poor platelet response to therapy.
Accelerated erythroid repopulation with no stem‐cell competition effect in children treated with recombinant human erythropoietin after allogeneic bone marrow transplantation
Authors: Locatelli Franco , Zecca Marco , Beguin Yves , Giorgiani Giovanna , Ponchio Luisa , de Stefano Piero , Cazzola Mario
Published: March 2008

Summary. We carried out a pilot study on the use of recombinant human erythropoietin (rHuEPO) to accelerate erythropoietic engraftment in paediatric patients undergoing allogeneic BMT. rHuEPO was administered intravenously at a dose of 75 U/kg per day for 30 d after transplant. Erythroid repopulation, evaluated sequentially through the serum transferrin receptor, was faster in 15 patients receiving rHuEPO than in 16 historical controls (P= 0·0003). This faster erythroid engraftment resulted in a reduction in the total number of red blood cell units required to reach transfusion independence (2·7 ± 1·2 v 4·2 ± 2·3, P= 0·027). No significant difference in leucocyte or platelet regeneration was observed. These findings indicate that rHuEPO administration can accelerate erythroid recovery after allogeneic BMT and reduce red cell transfusion requirements with no stem‐cell competition effect.
Detection of clonal platelet antibodies in immunologically‐mediated thrombocytopenias: association with circulating clonal/oligoclonal B cells
Authors: Christie Douglas J. , Sauro Shari C. , Fairbanks Kyrsten D. , Kay Neil E.
Published: March 2008

Summary Aware that T and B cells in autoimmune thrombocytopenia are abnormal, including the existence of clonal B cell populations, we sought to characterize this clonal phenomenon in various immunological thrombocytopenias using platelet antibody light chain analysis, flow cytometry, Southern blot analysis, and PCR. Using a monoclonal antibody‐antigen capture ELISA, we analysed sera from 21 of 26 patients with autoimmune, alloimmune. or drug‐induced immunological thrombocytopenia for the light chain phenotypes of their platelet antibodies. Alloantibodies and drug‐dependent antibodies from four and 14 patients, respectively, were found that expressed a predominant type of light chain, suggesting that these platelet‐reactive antibodies were monoclonal or oligoclonal in nature. 14 of the 26 patients were available for light chain B cell phenotyping studies. Of these 14 patients, thrombocytopenia was due to autoimmunity in two, drug‐induced immunity in four, and alloimmunity in eight. We detected clonal populations of B cells in all 14 patients by flow cytometry. Although six of these latter patients possessed platelet antibodies with clonal characteristics, light chain phenotypes of antibodies in five patients were opposite to those of their B cells. Eight of these patients were further examined for immunoglobulin gene rearrangement using Southern and/or polymerase chain reaction analysis. In all eight patients we detected clonal or oligoclonal B cell populations. Only two of these patients had malignancies (chronic lymphocytic leukaemia) that would be expected to have detectable clonal B cells, and thus the mechanism for clonal expansion in the other six patients did not appear to he related to an obvious neoplastic process. Prior to these studies, detection of clonal B cells in thrombocytopenic patients without known malignancies was limited to individuals with autoimmune thrombocytopenia, prompting the speculation that this particular autoimmune disorder arises from B cell dysregulation, rather than from expansion of specific autoantibody producing B cell clones. In contrast, the current studies provide evidence that clonal B cells are common to patients with any form of immunologically‐mediated thrombocytopenia. Moreover, the majority of the platelet antibodies (86%) present in these disorders exhibited monoclonal characteristics in that there was an apparent restriction in light chain usage.
Monitoring of relapse and remission in acute leukaemias by DNA‐fingerprint analysis
Authors: Hübner G. , Battmer K. , Paaz U. , Link H.
Published: March 2008

Summary DNA‐fingerprint (DNA‐F) analysis was successfully performed with DNA from 22 adult patients with acute leukaemia, including 13 patients with acute myeloid leukaemia (AML) and nine patients with acute lymphoblastic leukaemia (ALL). The purpose of this study was to detect differences between the leukaemic phase (at diagnosis or relapse) and remission‐phase DNA. We applied one simple repeat probe (GTG)s and one minisatellite (M13) after DNA‐digestion with different restriction endonucleases (Hinf I and Hae III) and agarose gel electrophoresis. In 7/13 patients with AML and 5/9 patients with ALL it was possible to detect loss of bands. additional bands or band shift with at least one of the probes. Together the probes M13 and (GTG)5 unveiled deviating fingerprint patterns in 54.6% of patients between leukaemic cells and remission‐phase leucocytes. Allogeneic bone marrow transplantation was performed on six patients. In each case the DNA‐F pattern of the donor was different from the relapse and the remission‐phase pattern. We conclude from our studies that the probes M13 and (GTG)5 are useful in the detection of relapse and remission in acute leukaemias after chemotherapy and bone marrow transplantation.
Detection of an epitope specific for the dissociated form of glycoprotein IIIa of platelet membrane glycoprotein IIb‐IIIa complex and its expression on the surface of adherent platelets
Authors: Khaspekova Svetlana G. , Vlasik Tatjana N. , Byzova Tatjana V. , Vinogradov Dimitry V. , Berndt Michael C. , Mazurov Alexey V.
Published: March 2008

Summary Glycoproteins (GPs) IIb and IIIa form a Ca2+‐dependent complex in platelet membrane and change their conformation upon platelet activation and dissociation of the complex. A new anti‐GPIIIa monoclonal antibody (mAb). CRC54, is described which could distinguish different conformational states of GPIIIa. This antibody (i) precipitated GPIIb‐IIIa from platelet Triton X‐100‐lysate. (ii) recognized the GPIIIa band in Western blotting of platelet SDS‐lysate, and (iii) did not react with platelets from a Glanzmann's thrombasthenia patient lacking GPIIb‐IIIa. Immunoblotting of chymotryptic digestion products of purified GPIIb‐IIIa has shown that CRC54 epitope is located within residues 1–100 at the N‐terminus of GPIIIa. CRC54 bound weakly to platelets in the presence of Ca2+ and Mg2+, 2.34 ± 0.28 ± 103 molecules per platelet at saturation. The same level of binding was observed without any divalent cations in the medium. However, binding of CRC54 was increased by several times after treatment of platelets with EDTA, 10.04 ± 0.28 ± 103 molecules per platelet. Increase of CRC54 binding correlated with the dissociation of GPIIb‐IIIa complex which was followed by the decrease of the binding of another mAb, CRC64, directed against complex‐specific epitope of GPIIb‐IIIa. Binding of CRC54 to platelets was changed neither by platelet activation in suspension with thrombin or ADP nor by the occupancy of GPIIb‐IIIa ligand binding site with GRGDSR peptide. However. binding was significantly stimulated by platelet adhesion to polystyrene plastic. As measured using 51Cr‐labelled platelets, binding of l25I‐CRC54 to adherent platelets in the presence of divalent cations was about 4 times higher than to platelets in suspension, 8.68 ± 0.48 ± 103 per platelet. This increase was not due to the dissociation of GPIIb‐IIIa since complex‐specific antibody CRC64 still bound effectively to the surface of adherent platelets. The data obtained indicated that: (1) CRC54 recognized an epitope specific for the dissociated form of GPIIIa: (2) the CRC54‐reactive epitope of GPIIIa is also expressed on the surface of adherent platelets.
Abnormal erythrocyte band 4.1 protein in myelodysplastic syndrome with elliptocytosis
Authors: Ideguchi Hiroshi , Yamada Yumiko , Kondo Seiji , Tamura Kazuo , Makino Shigeyoshi , Hamasaki Naotaka
Published: March 2008

Summary A case of myelodysplastic syndrome with haemolytic anaemia and a marked elliptocytosis is reported. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of erythrocyte membrane proteins revealed that the patient's band 4.1 was decreased to about 50–70% of that of control and contained abnormal molecule migrating in a faster mobility than normal band 4.1. which was confirmed by immunoblotting. The actin/spectrin ratio of the patient's ghosts diminished to about 70% of that of control ghosts. Flowcytometric analysis showed that the glycophorin C content of the patient's erythrocytes was reduced but maintained the level of about 70% of that of normal, indicating that the glycophorin C‐band 4.1 interaction might not be so seriously damaged as to cause elliptocytic shape change. We postulate that the abnormal band 4.1 produced from the abnormal erythroid clone may be the primary molecular defect and result in a dysregulation of spectrinactin interaction to cause erythrocyte shape change and membrane instability.
Gaucher disease mutations in non‐Jewish patients
Authors: Beutler Ernest , Gelbart Terri
Published: March 2008

Summary The DNA from 64 non‐Jewish patients with Gaucher disease was examined for 29 different glucocerebrosidase mutations that are known to cause the disease. The most common was mutation 1448C and the second most common 1226G. Twenty of the mutations remained unidentified. These results were compared to those obtained from 122 Jewish patients, in whom the most common mutations were 1226G and 84GG. Among the non‐Jewish patients 15.6% of the mutations remained unidentified. while among the Jewish patients only 1.2% remained unidentified, and even screening for the five most common mutations identified all but 2.9% of the Jewish mutations. The greater diversity of mutations among the non‐Jewish patients has important implications with respect to DNA‐based screening. diagnosis and counselling.
Spontaneous remission of acute adult T‐cell leukaemia with chromosomal abnormality infiltrating to skin and liver
Authors: Taniguchi Shuichi , Yamasaki Kazuo , Shibuya Tsunefumi , Asayama Ryokichi
Published: March 2008

Summary Acute type of adult T‐cell leukaemia (ATL) has been recognized as highly‐progressive T‐cell malignancy with a median survival of 7 months. We present a case who had a spontaneous remission from acute type ATL with organ involvement and chromosomal abnormality implying very poor prognosis. Although the mechanism of spontaneous remission remains uncertain, our case was unique with regard to the progression and regression of leukaemia.
Replacement of HLA class II serology by the HLA‐DR microtitre plate oligotyping assay: a one‐year experience in unrelated bone marrow donor selection
Authors: Tiercy J.M. , Roosnek E. , Speiser D. , Cros P. , Allibert P. , Mach B. , Jeannet M.
Published: March 2008

Summary Because the selection of unrelated bone marrow donors requires a more accurate HLA‐typing technique than is used for sibling donors, we have replaced class II serology by a rapid and highly discriminative HLA‐DR DNA oligotyping assay on microtitre plates. We present here our 1‐year experience with class II typing for 112 potential donors identified for 3 3 consecutive leukaemic patients. Although the donors were selected to be HLA‐A, 43, ‐DR serologically identical, the microtitre plate oligotyping assay detected DR incompatibilities in 52.7% of the patient/donor pairs. One third was due to incorrect or incomplete typing of DR11 to DK16, the others to mismatches for the serologically indistinguishable subtypes of DR1, 11, 13 and 15, known to be relevant for T cell recognition.
Overexpression of PRAD1 in a mantle zone lymphoma patient with a t(11;22)(q13;q11) translocation
Authors: Komatsu Hirokazu , Yoshida Kouki , Seto Masao , Iida Shinsuke , Ailawa Tadahiro , Ueda Ryuzo , Mikuni Chikara
Published: March 2008

Summary The PRAD1 gene identified from the chromosome band 11q13 region was previously demonstrated to be overexpressed in cell lines with t(11:14)(q13;q32) trans‐location and was suggested to be a candidate BCL‐1 gene. We report here one case of mantle zone lymphoma with a t(11;22)(q13:q11), a variant translocation at the BCL‐1 locus, having the PRAD1 overexpression. By analogy with the c‐myc gene in Burkitt's lymphoma and the BCL‐2 gene in follicular lymphoma, this case supports strongly the idea that the PRAD1 is the candidate BCL‐1 gene.
Acquired cyclic thrombocytopenia‐thrombocytosis with periodic defect of platelet function
Authors: Balduini Carlo L. , Stella Carmelo Carlo , Rosti Vittorio , Bertolino Giampiera , Noris Patrizia , Ascari Edoardo
Published: March 2008

Summary A periodic fall of platelet number characterizes an acquired pathological condition named cyclic thrombocytopenia. We observed a patient in whom the episodes of thrombocytopenia (platelet number less than 50×109/l) were followed regularly by thrombocytosis (700–2 300 × 109 platelets/l). The period of platelet count fluctuation was about 30 d. Morphological examination of bone marrow showed the cyclic disappearance of mature and immature megakaryocytes: bone marrow cultures revealed a periodic severe defect of both multilineage and single‐lineage progenitor cell growth. When platelet count was falling, a mild defect of platelet aggregation and ATP release was observed. while platelet function was normal when platelet count was rising. Prednisone. thymopentine. high‐dose intravenous γ‐globulin and splenectomy were without effect. After 4 years of cyclic platelet and megakaryocyte fluctuations, stable amegakaryo‐cytic thrombocypenia developed and the patient died of haemorrhagic stroke.
Decreased in vitro megakaryocyte colony formation in chronic idiopathic thrombocytopenic purpura
Authors: Abgrall Jean‐François , Berthou Christian , Sensebé Luc , Niger Catherine Le , Escoffre Martine
Published: March 2008

Summary In vitro megakaryocyte colony formation was studied in 25 patients with chronic idiopathic thrombocytopenic purpura (ITP). and compared to 16 controls. A significantly lower number of megakaryocyte progenitors (CFU‐MK) was observed in patients (10·8 ± 124 CFU‐MK per 105 bone‐marrow non‐adherent mononuclear cells (NAMC), compared to controls (23·5 ± 15·4 CFU‐MK): P<0·002. Only six patients had a normal number of CFU‐MK. The number of CFU‐MK was not correlated to initial platelet count (P= 0·35). These findings indicate that in vitro CFU‐MK could be of interest in the diagnosis of chronic ITP, and also could be predictive for chronicity in patients with acute ITP.
Trisomy 12 in B‐cell chronic lymphocytic leultaemia: an evaluation of 33 patients by direct fluorescence in situ hybridization (FISH)
Authors: Bienz N. , Cardy D. L. N. , Leyland M. J. , Hulten M. A.
Published: March 2008

Summary An adaptation of the standard fluorescence in situ hybridization (FISH) technique allowing rapid analysis (4 h) has been used to study the prevalence of trisomy 12 in 33 patients with B‐CLL, of whom 54% have been shown to have this abnormality. The presence of trisomy 12 has been compared with clinical parameters, and there may be a relationship between the prevalence of trisomy 12 in B‐CLL and duration of disease.
Reduced responsiveness of bone marrow megakaryocyte progenitors to platelet‐derived transforming growth factor β1, produced in normal amount, in patients with essential thrombocythaemia
Authors: Zauli Giorgio , Visani Giuseppe , Catani Lucia , Vianelli Nicola , Gugliotta Luigi , Capitani Silvano
Published: March 2008

Summary In this study we evaluated the amount of transforming growth factor‐β1 (TGF‐β1) in platelet lysates obtained from 12 patients affected by essential thrombocythaemia (ET) in comparison with five patients affected by myelofibrosis with myeloid metaplasia (MMM) and 15 healthy donors.

The levels of both bioactive and latent TGF‐β1, evaluated in a bioassay on CCL64 cells, before and after transient acidification, were similar in platelet lysates from ET patients and normal donors and significantly (P < 0.01) elevated in platelet lysates from MMM patients. Moreover, platelet lysates from ET patients and normal controls, showed a similar degree of colony suppression when tested on haematopoietic progenitor (CD34+) cells, purified from normal bone marrows, whereas platelet lysates from MMM patients showed a higher (P < 0.01) inhibitory activity on normal CFU‐meg and BFU‐E growth.

In parallel, platelet lysates form ET patients and normal controls were tested on CD34+ cells, purified from ET bone marrows. ET bone marrow BFU‐E, similarly to normal bone marrow BFU‐E, were markedly inhibited by platelet lysates, whereas ET bone marrow CFU‐meg were significantly (P < 0.05) less responsive to the inhibitory activity of platelet lysates than normal bone marrow CFU‐meg. The main factor responsible for the inhibitory activity contained in platelet lysates was transforming growth factor‐β1 (TGF‐β1), as demonstrated by the ability of a polyclonal neutralizing anti‐TGF‐β1 antibody to almost completely reverse the suppressive effect of platelet lysates on CFU‐meg and BFU‐E growth.

Our data demonstrate that the amount of intraplatelet TGF‐β1 is similar in ET patients and normal controls, whereas it is increased in platelets from MMM patients. Moreover, megakaryocyte progenitors in ET show a reduced sensitivity to platelet‐derived inhibitors and, in particular, to TGF‐β1.
Antibody By 114 is selective for the 90 kD PI‐linked component of the CD66 antigen: a new reagent for the study of paroxysmal nocturnal haemoglobinuria
Authors: Mayne K. M. , Pulford K. , Jones M. , Micklem K. , Nagel G. , van der Schoot C. E. , Mason D. Y.
Published: March 2008

Summary This paper describes a monoclonal antibody which reacts with transfected cells carrying a gene (NCA‐50/90) which has been shown to encode the human CD66 antigen. However, antibody By 114 recognizes only a single 90 kD polypeptide from human neutrophils, whereas the antibodies which originally defined the CD66 antigen also recognize a larger 180‐200 kD protein. We conclude that antibody By 114 is selective for the smaller of the two CD66 gene products. which is a surface membrane phosphatidylinositol (PI)‐linked molecule. The reactivity of antibody By 114 on peripheral blood cells (positive on neutrophils, weak or negative on eosinophils, and negative on basophils, monocytes and lymphocytes) and myeloid precursor cells was identical to those of a reference CD66 antibody, as was the staining of leukaemic cells. However, the reactions of the two antibodies differed on kidney, liver and pancreas, and in cases of myeloma, Waldenström's macroglobulinaemia and lymphoma, indicating that By 114 represents a new CD66 sub‐specificity. Granulocytes from a case of paroxysmal nocturnal haemoglobinuria (PNH) were negative with antibody By114, indicating that it may be of value in detecting the defect in PI‐linked surface molecules characteristic of this condition. Antibody By114 also stained formalin fixed paraffin embedded tissues and may therefore be of use in routine diagnostic histopathology.
Quantification of leukaemic cells based on heteroduplex formation of tal‐1 gene sequences after PCR coamplification
Authors: Borkhardt A. , Repp R. , Harbott J. , Kreuder J. , Lampert F.
Published: March 2008

Summary Based on the high sensitivity of the polymerase chain reaction (PCR) several assays have already been described which can be applied to monitor minimal residual disease in patients with leukaemia. However, most of these approaches are only qualitative or semiquantitative at best. Moreover, the semiquantitative assays require rather largescale procedures such as oligonucleotide hybridization or sampling of aliquots during the exponential phase of PCR amplification, which is time consuming and may be error prone.

Using the deletion in the tal‐1 gene as a clonal marker of malignant cells, which can be found in about 10–25% of T‐cell acute lymphoblastic leukaemia, we have developed a rapid and simple PCR assay for the quantification of residual leukaemic cells. The assay is based on heteroduplex formation between standard and template DNA sequences after coamplification of both DNA sequences in the same PCR reaction. The sensitivity of this PCR approach allows the detection of neoplastic cells at frequency of at least 10‐4. Application of such an assay needs not to be restricted to patients harbouring a tal‐1 gene deletion. It may easily be adapted to other clonal DNA markers of blast cells and therefore facilitate monitoring of minimal residual disease in many different kinds of leukaemia.
Characterization of an acute micromegakaryocytic leukaemia: evidence for the pathogenesis of myelofibrosis
Authors: Reilly J. T. , Barnett D. , Dolan G. , Forrest P. , Eastham J. , Smith A.
Published: March 2008

Summary The current hypothesis for the pathogenesis of myelofibrosis involves the intramedullary release of growth factors from defective or abnormal megakaryocytes. We describe a case of an acute micromegakaryocytic leukaemia, in a patient with chronic myelofibrosis, that provides additional evidence for this concept. The micromegakaryocytes, which reached 223 × 109/1, were characterized morphologically by both light and electron microscopy, immunocytochemically and by platelet peroxidase activity. The cells were shown to have a mature cytoplasm, containing alpha granules and the associated proteins: vWF:Ag, fibrinogen, fibronectin and protein S. DNA analysis, by both a Seescan Solitaire Plus image analysis system and flow cytometry, revealed nuclear immaturity, with 92% of cells being diploid.

Serum markers of connective tissue synthesis, namely carboxy terminal peptide of procollagen I (PICP), procollagen terminal peptide III (PIIIP) and laminin all increased significantly following transformation and were associated with an increase in platelet‐derived growth factor (PDGF) and transforming growth factor‐β (TGF‐β). These observations support the current hypothesis for bone marrow fibrosis formation and provide, for the first time, a link between in vivo growth factor release, bone marrow stromal turnover and megakaryocyte mass. In addition, the release of biologically active TGF‐β may explain both the increased fibronectin and angiogenesis characteristic of myelofibrotic bone marrow.
Expressions of DNA topoisomerase I and II gene and the genes possibly related to drug resistance in human myeloma cells
Authors: Ishikawa Hideaki , Kawano Michio M. , Okada Kosuke , Tanaka Hideo , Tanabe Osamu , Sakai Akira , Asaoku Hideki , Iwato Koji , Nobuyoshi Masaharu , Kuramoto Atsushi
Published: March 2008

Summary In order to clarify the mechanism of drug resistance in human myeloma cells, we investigated the expressions of DNA topoisomerase I and topoisomerase II gene and the genes possibly related to drug resistance; multi‐drug resistant gene 1 (MDR‐1), glutathione S‐transferase class π gene (GST‐π), by Northern blotting. Myeloma cells in eight of 15 cases prior to chemotherapy expressed topoisomerase I mRNA considerably, while the expression of topoisomerase II mRNA was detected weakly in only one of 16 myeloma patients. There was not any correlation between expression of topoisomerase I mRNA and clinical drug resistance. Significant expression of MDR‐1 mRNA and P‐glycoprotein was not detected in 25 cases of multiple myeloma prior to chemotherapy and even after several courses of VAD (vincristine, adriamycin and dexamethasone) therapy by Northern blotting and immunostaining using monoclonal anti‐P‐glycoprotein antibody (MRK‐16), respectively. On the other hand, 16 of 21 myeloma cases showed significant expression of GST‐π protein and GST‐π mRNA with the various strengths, but there was no apparent correlation between GST‐π mRNA expression and clinical response. Therefore these data suggest that expression of the genes we tested may not determine the level of drug resistance in multiple myeloma, but lower or no significant expression of topoisomerase II mRNA in most myeloma cells indicates the possibility that topoisomerase II inhibitors such as VP‐16 and topoisomerase II‐mediated cytotoxic drugs such as adriamycin, are not so effective for the treatment of multiple myeloma.
The effects of leukaemia inhibitory factor on platelet function
Authors: Waring Paul , Wall Dominic , Dauer Ray , Parkin Des , Metcalf Donald
Published: March 2008

Summary Leukaemia inhibitory factor (LIF) is able to promote megakaryocytopoiesis in vitro and elevate platelet counts in vivo, and is a potential new therapeutic agent for the treatment of thrombocytopenia. To determine whether platelets released under conditions of LIF‐stimulated megakaryocytopoiesis have intact function, we compared aggregation responses of platelets from mice with constitutively elevated LIF levels (FD/LIF mice) and mice injected with recombinant murine LIF (rmLIF mice) with their respective control mice. We report that ex vivo platelet aggregability and thromboxane B2 release were intact in the LIF‐treated mice, and were significantly enhanced in some situations. LIF‐treated mice also had significantly increased platelet counts (FD/LIF mice: 1302 ± 173 × 109/1 compared to 1012 ± 99 × 109/1 for FD mice; rmLIF mice: 1460 ± 193 × 109/1 compared to 985 ± 67 × 109/1 for FCS/NS mice), increased platelet volumes and elevated plasma fibrinogen and calcium levels. The platelet hyperreactivity seen in the LIF‐treated mice is likely to reflect the larger platelet volumes and/or the effect of plasma components such as fibrinogen, elevated levels of which were due to the concomitant action of LIF as a stimulant of acute phase protein synthesis.
Acquired sideroblastic anaemia during treatment of Wilson's disease with triethylene tetramine dihydrochloride
Authors: Condamine Luce , Hermine Oliver , Alvin Patrick , Levine Martine , Rey Caroline , Courtecuisse Victor
Published: March 2008

Summary We report one case of acquired sideroblastic anaemia in a patient treated for Wilson's disease with triethylene tetramine dihydrochloride (TTH). No other cause of acquired sideroblastic anaemia was found, and neither iron nor pyridoxine therapy could correct this anaemia. In contrast, decreasing the dose of TTH led to disappearance of ringed sideroblasts.

Thus TTH should be added as a further cause of secondary acquired sideroblastic anaemia. The pathophysiology of this finding, probably linked to an abnormality of mitochondrial iron metabolism, is briefly discussed.