bluuebunny/crossref_metadata_2025 huggingface dataset:
B cells expressing CD5 antigen are markedly increased in peripheral blood and spleen lymphocytes from patients with immune thrombocytopenic purpura
Authors: Mizutani Hajime , Furubayashi Takayasu , Kashiwagi Hirokazu , Honda Shigenori , Take Hironori , Kurata Yoshiyuki , Yonezawa Takeshi , Tarui Seiichiro
Published: March 2008

Summary By two‐colour flow cytometric analysis, we examined the proportion of B lymphocytes bearing CD5 cell surface antigen (CD 5+ B cells), which are capable of producing autoantibodies, both in peripheral blood and spleen from patients with chronic immune thrombocytopenic purpura (ITP). The percentage of CD5+ B cells in peripheral blood lymphocytes (PBLs) was significantly increased (P<0.005) in patients with ITP (3.7 ± 2.2%, n=30) as compared with normal controls (1.7 ± 0.7%, n=28). However, there was no correlation between the percentages of circulating CD5+ B cells and platelet counts: The percentage of splenic CD5+ B cells in ITP patients was much more increased (9.0 ± 4.5%, n=9), P<0.005) compared with that of other disorders (3.2 ± 0.5%, n=5). Furthermore, isolated splenic CD5+ B cells from two out of five ITP patients produced high levels of IgM‐type, platelet‐bindable antibodies (PBIgM) after stimulation with Staphylococcus aureus Cowan I (SAC), while CD5‐ B cells isolated from the same spleen or splenic CD5+ B cells from other non‐autoimmune disorders failed to produce significant amount of PBIgM. In three ITP patients, no increase in PBIgM was detected despite SAC stimulation. The increased proportion of CD5+ B cells in peripheral blood and spleen, and their ability to produce anti‐platelet antibodies indicate that they are directly involved in the autoimmune pathogenesis in ITP.
Megakaryocyte potentiating activity of IL‐1, IL‐6 and GM‐CSF as evaluated by their action on in vitro human megakaryocytic colonies
Authors: Takahashi Takayuki , Tsuyuoka Reiko , Ueda Yasuyo , Suzuki Akira , Ichiba Shigemoto , Okuno Yoshiaki , Nakamura Kishiko , Imura Hiroo
Published: March 2008

Summary We examined whether recombinant cytokines enhance the in vitro platelet production of interleukin‐3 (IL‐3)‐induced human megakaryocytic colonies (Meg‐colony). We classified Meg‐colonies into four categories based on platelet production during in situ observation on day 14: type 0, absence of cytoplasmic processes in a colony; type 1, one to three processes in at least one megakaryocyte in a colony; type 2, four to eight processes; type 3, more than nine processes or division of cytoplasm. Type 3 colonies were considered to be platelet‐producing. In control cultures, type 1 Meg‐colonies were dominant, followed by type 2, type 3 and type 0. Of the cytokines added at the initiation of culture, interleukin‐1 alpha (IL‐1α), interleukin‐6 (IL‐6), and granulocyte/macrophage colony stimulating factor (GM‐CSF) significantly increased the number of colonies. Furthermore, these three cytokines significantly elevated the proportion of type 3 colonies. Interleukin‐4 (IL‐4), granulocyte‐CSF, macrophage‐CSF and erythropoietin did not affect the colony count or distribution of colony type. IL‐1α. IL‐6 and GM‐CSF also significantly elevated the proportion of type 3 colonies, even when added to the culture on days 8 or 11. These results indicate that IL‐1α, IL‐6 and GM‐CSF promote platelet production of in vitro Meg‐colonies.
Rearrangements of the RAR‐α gene in acute promyelocytic leukaemia:
Authors: Coco Francesco Lo , Avvisati Giuseppe , Diverio Daniela , Biondi Andrea , Pandolfi Pier Paolo , Alcalay Myriam , Rossi Giulio De , Petti Maria C. , Cantù‐Rajnoldi Angelo , Pasqualetti Daniela , Nanni Mauro , Fenu Susanna , Frontani Marina , Mandelli Franco
Published: March 2008

Summary We have used genomic probes which specifically recognize DNA rearrangements of the RAR‐α locus on chromosome 17q21 in patients with acute promyelocytic leukaemia (APL) and acute myeloid leukaemia (AML) subtypes. Molecular data were examined in comparison with morphological and immunophenotypic characterization at diagnosis in 20 hypergranular FAB M3 cases, five microgranular APL (M3v), 51 non‐M3 AML and 12 myeloid CML blast crises.

Rearrangements of the RAR‐α locus were only detected in 23/25 APL cases and in none of the other FAB subtypes analysed. Surface marker characterization showed a consistent immunophenotypic profile—HLADR negative. CD9 and CD13/33 positive—in all M3 and M3v cases. Neither HLADR negativity nor CD9 positivity were associated with RAR‐α rearrangements in non M3 AML.

Our data indicate that RAR‐α gene rearrangements are relevant diagnostic features of both M3 and M3v, and may prove useful molecular marker for follow‐up analysis in APL patients.
Inactivation of the retinoblastoma susceptibility gene in a human high grade non‐Hodgkin's lymphoma cell line
Authors: Weide R. , Dowding C. , Sucai Bi , Bungey J. , Chase A. , Goldman J. M.
Published: March 2008

Summary We studied the expression of the retinoblastoma (RB) gene product (p105) in a B‐cell line established from a patient with non‐Hodgkin's lymphoma (large cell type). The karyotype of this cell line, named Ri‐1, showed amongst other changes an apparent deletion of one chromosome 13 on band q14. No p105 could be detected by immunoprecipitation analysis and Western blotting in Ri‐1 cells. Northern blotting revealed that RB mRNA is not expressed in Ri‐1. Southern blotting confirmed the loss of one RB allele but showed a normal gross structure of the remaining allele. This suggests that the inactivation of the RB gene in Ri‐1 cells is due to deletion of one allele and point mutations or small deletions in the other, as is often the case in retinoblastomas. Our findings imply that inactivation of the RB gene may play a role in the pathogenesis of high grade malignant lymphomas and that studies of RB in primary lymphoma samples would be of interest.
Heterogenous expression of decay accelerating factor and CD59/membrane attack complex inhibition factor on paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes
Authors: Shichishima Tsutomu , Terasawa Takashi , Hashimoto Chokichi , Ohto Hitoshi , Uchida Tatsumi , Maruyama Yukio
Published: March 2008

Summary In order to clarify the characterization of pheno‐types of paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes (E), we analysed the expression of decay accelerating factor (DAF) and CD59/membrane attack complex inhibition factor (MACIF) on the membrane surface of PNH‐E by means of the flow cytometric method using anti‐DAF and/or CD59/MACIF monoclonal antibodies in nine PNH‐patients. In two‐colour analysis, this expression on PNH‐E was classified into three fractions; negative, intermediate and positive according to intensity. The negative fraction was classified into two groups; one group an exclusively negative population, and the other a negative population having slightly DAF‐positive E. The intermediate fraction was recognized on PNH‐E of cases with PNH II‐E and extremely heterogenous. In the positive fraction, this expression was almost the same as on normal E except for case 8. In single‐colour analysis for DAF or CD59/MACIF, three fractions were classified as well as the definition in two‐colour analysis. In single‐colour analysis, the expression on PNH‐E was also heterogenous in each fraction and among PNH‐patients. However, the intermediate fraction for CD59/MACIF was not found on PNH‐E of cases without PNH II‐E, although intermediate fraction for DAF was recognized on PNH‐E of some cases with PNH III‐E in addition to those with PNH II‐E.

The results suggest that expression of CD59/MACIF and DAF on the membrane surface of PNH‐E phenotypes is heterogenous and varies among PNH patients.
Ankyrin deficiency in dominant hereditary spherocytosis: report of three cases
Authors: Iolascon Achille , Del Giudice Manuele Miraglia , Camaschella Clara , Pinto Luciano , Nobili Bruno , Perrotta Silverio , Cutillo Stefano
Published: March 2008

Summary We describe three italian subjects from two unrelated families affected with isolated hereditary spherocytosis (HS) without other clinical abnormalities, associated with partial spectrin and ankyrin deficiency. In both families the propositus has normal biological parents, and thus appears to be the result of a new mutation; in oneof them the disease is further transmitted in an autosomal dominant fashion. Cytogenetic analysis of the latter family excluded abnormalities of the short arm of chromosome 8. We speculate that in both kindreds ankyrin deficiency is the primary defect related to ankyrin gene mutation. Several pieces of evidence suggest that ankyrin deficiency is probably the most common molecular defect in HS. It is inherited in a dominant manner and its clinical and biochemical expression is heterogenous.
Monosomy 7 in multilineage and acute lymphoblastic leukaemia
Authors: Paietta Elisabeth , Gucalp Rasim , Wiernik Peter H.
Published: March 2008

Summary Monosomy 7 as the sole cytogenetic abnormality was detected in five of 310 consecutive adult patients with acute leukaemia who were characterized by morphological, immunophenotypic and cytogenetic analyses. Morphologically, blast cells were myelomonocytic (FAB M4) in three and lymphoid (FAB L2) in two patients. By immunophenotyping, two M4 patients expressed terminal transferase (TdT) in 15–90% of myelomonoblasts (patients 3 and 1, respectively), and in the third M4 patient (no. 2), a 10% TdT+ component was present distinct from the bulk of myelomonoblasts. In one L2 patient (no. 4), the blast cells had an undifferentiated phenotype only expressing TdT and HLA‐DR but lacking specific lymphoid and myeloid antigens, and patient 5 was typed as CD10+ ALL. Two patients had developed leukaemia following radiotherapy and/or chemotherapy for multiple myeloma or breast cancer. In two patients, induction chemotherapy induced a lineage switch in the immunophenotype without change in karyotype. These observations support the concept that monosomy 7 leukaemia results from the transformation of a multipotential stem cell.
X‐chromosome inactivation patterns using HPRT and PGK polymorphisms in haematologically normal and post‐chemotherapy females
Authors: Gale Rosemary E. , Wheadon Helen , Linch David C.
Published: March 2008

Summary Clonal analysis using X‐chromosome inactivation patterns of the hypoxanthine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK) genes has been used in the assessment of haematological malignancies. In many analyses normal haemopoietic tissue or constitutive DNA from each individual has not been readily available and it has been assumed that when the ratio of the percentage of the two alleles remaining after differential methylation enzyme digestion is >3/1 then an oligoclonal/monoclonal population of cells is present. We report here in a study of 65 haematologically normal females informative for HPRT or PGK that 15 (23%) had a skewed Lyonization pattern with clonal analysis ratios >3/1. No correlation was observed between age and the X‐chromosome inactivation pattern to suggest the detection of pre‐malignant clonal states. The incidence was similar in 23 females who had received conventional combination chemotherapy, with six women (26%) having a ratio >3/1. In the majority of individuals therefore, conventional chemotherapy does not appear to alter the patient's X‐chromosome inactivation pattern. These results indicate that caution must be exercised in interpreting the results of clonal analysis of any given individual.
Detection and quantitation of neoplastic cells in acute lymphoblastic leukaemia, by use of the polymerase chain reaction
Authors: Brisco M. J. , Condon J. , Sykes P. J. , Neoh S. H. , Morley A. A.
Published: March 2008

Summary We report a simple and robust method for sensitive quantitation of leukaemic cells in acute lymphocytic leukaemia. Chain determining region 3 (CDR3) of the immunoglobulin heavy chain gene is a precise genetic marker for a patient's leukaemic clone. Quantitation of the leukaemic lymphocytes was achieved by use of the polymerase chain reaction to detect CDR3 at limiting dilution of DNA samples. Five patients were studied and high levels (1 in 1 to 1 in 10) of leukaemic cells were detected at diagnosis or relapse. Leukaemic cells were detected in remission marrows from three patients, at levels of 1 in 1000 to 1 in 100000. All five patients showed a 1000 to 100000‐fold reduction in the levels of leukaemic cells after induction therapy. This technique should prove useful for monitoring therapy and may help predict outcome.
Autoantibodies against platelet glycoprotein Ib/IX: a frequent finding in autoimmune thrombocytopenic purpura
Authors: Kiefel V. , Santoso S. , Kaufmann E. , Mueller‐Eckhardt C.
Published: March 2008

Summary Many autoantibodies involved in the pathogenesis of autoimmune thrombocytopenic purpura (AITP) are directed against epitopes on platelet glycoproteins (GP). These autoantibodies are a specific diagnostic characteristic of patients with AITP. In this study, the relative frequency of antibodies against GPs IIb/IIIa and Ib/IX was assessed in sera from 81 AITP patients with a glycoprotein‐specific enzyme immunoassay (MAIPA assay) using monoclonal antibodies against these platelet GPs. All sera contained platelet‐specific antibodies which had been detected by platelet immunofluorescence. Of the 81 antibodies tested, 58 (72%) reacted with at least one of the platelet GPs studied. Autoantibodies against GPIb/IX were as common as antibodies against the GPIIb/IIIa complex. The same ratio of specificities was observed on autologous platelets of an independent cohort of 29 patients. The epitope of three autoantibodies against GPIb/IX and of mab Gi10, a monoclonal antibody, which inhibits binding of these autoantibodies, was further characterized. Severity of thrombocytopenia was not related to the GP specificity of the autoantibody. The observation that in 23 (28%) of these sera the antigenic determinants could not be assigned to the glycoproteins under investigation suggests that platelet autoantibodies may react with other GPs or other membrane constituents, e.g. glycolipids.
The murine monoclonal antibody, 14A2.H1, identifies a novel platelet surface antigen
Authors: Ashman Leonie K. , Aylett Gabriella W. , Mehrabani Pirooz A. , Bendall Linda J. , Niutta Silvana , Cambareri Antony C. , Cole Stephen R. , Berndt Michael C.
Published: March 2008

Summary A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (103/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on FcγRII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.
Dominant β‐thalassaemia trait in a Portuguese family is caused by a deletion of (G)TGGCTGGTGT(G) and an insertion of (G)GCAG(G) in codons 134, 135, 136 and 137 of the β‐globin gene
Authors: Oner R. , Oner C. , Wilson J. B. , Tamagnini G. P. , Ribeiro L. M. L. , Huisman T. H. J.
Published: March 2008

Summary We have studied a Portuguese family with a dominant β‐thalassaemia trait that was present in one member of each of three generations. It was characterized by a moderate anaemia, microcytosis and hypochromia, anisopoikilocytosis, Heinz body formation in peripheral red cells, splenomegaly, and a blood transfusion requirement during pregnancy. Sequence analyses of amplified DNA detected a deletion of (G) TGGCTGGTGT(G) at codons 134‐137 (ValAlaGlyVal) and the insertion of (G)GCÀ(G) (GlyArg) at the same location. Thus, the resulting β chain has an abnormal structure only at codons 134‐137 and is two residues shorter than the normal 146 residues. This chain could not be detected in circulating red cells and must be degraded rapidly by proteolysis because the Heinz bodies consisted mainly of a chains.
Evidence that exposure to fibrinogen or to antibodies directed against Mac‐1 (CD11b/CD18; CR3) modulates human monocyte effector functions
Authors: Trezzini C. , Schuuepp B. , Maly F. E. , Jungi T. W.
Published: March 2008

Summary.We have recently shown that the treatment of fibrinogen‐coated monocytes (MO) with anti‐fibrinogen as well as the exposure of MO to surface‐bound fibrinogen (Fg) or to albumin haptenized with the Fg C‐gamma‐terminal pentadecapeptide, induces an oxidative burst. Using chemiluminescence (CL) for indicating an oxidative burst, and the ingestion of IgG‐coated erythrocytes as a test of phagocytosis, we have now studied the impact of Fg on MO effector functions. MO that had been either pretreated with Fg and washed, or that were exposed to surface‐adsorbed Fg, exhibited impaired Fc receptor‐mediated phagocytosis. A similar impairment was observed when MO were pretreated with activating agents such as phorbol myristate acetate, n‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) or the calcium ionophore A23187. Moreover, following exposure to Fg or IgG, MO exhibited a reduced oxidative burst upon stimulation with a variety of agents. Similarly, MO pretreated or coincubated with anti‐Mac‐1 exhibited a reduced oxidative burst upon stimulation. Our results raise the possibility that inflammatory mononuclear phagocytes experience a functional modulation upon encountering fibrin by interacting with specific receptors for fibrin(ogen). This type of modulation is analogous to effects induced by the triggering of Fcγ receptors. MO showed a decreased oxidative burst when either pretreated or coincubated with anti‐Mac‐1 antibodies, whereas antibodies directed against other MO surface constitutents had no, or a weak effect only. This is compatible with the suggestion that Mac‐1 acts as a fibrin(ogen) receptor.
High risk of early resistant relapse for leukaemic patients with presence of multidrug resistance associated P‐glycoprotein positive cells in complete remission
Authors: Musto Pellegrino , Melillo Lorella , Lombardi Gina , Matera Rosella , Giorgio Giuseppedi , Carotenuto Mario
Published: March 2008

Summary. The immunocytochemical detection of multidrugresistance (MDR) associated P‐glycoprotein (P‐170) was longitudinally performed on bone marrow smears from 32 responsive patients with acute leukaemia in the different phases of the disease (at diagnosis, in complete remission, at relapse) by means of APAAP technique and monoclonal antibody C219.

The whole group of eight patients with presence of P‐170 positive cells while in complete remission rapidly relapsed with a high proportion of blasts showing MDR phenotype; they were resistant to further treatments.

Twelve out of 24 subjects without cells with MDR phenotype in complete remission remained in this condition, six had a responsive relapse (without significant expression of P‐170 in 5/6 patients) and six a resistant relapse. Four patients of this last group significantly expressed P‐170.

Our data indicate that the detection of scattered P‐170 positive cells during complete remission might identify a subset of leukaemic patients with high risk of early and resistant relapse.
Granulocyte colony‐stimulating factor (G‐CSF) receptors on acute myeloblasts leukaemia cells and their relationship with the proliferative response to G‐CSF in clonogenic assay
Authors: Motoji Toshiko , Watanabe Masahiko , Uzumaki Hiroya , Kusaka Masaru , Fukamachi Hiromi , Shimosaka Akihiro , And Kazuo Oshimi , Mizoguchi Hideaki
Published: March 2008

Summary. The number and the affinity of granulocyte colony‐stimulating factor (G‐CSF) receptors expressed by blast cells in acute myeloblastic leukaemia (AML) were determined using radiolabeled recombinant human G‐CSF (rhG‐CSF). Eighteen of 20 patients demonstrated specific binding, and Scatchard analysis revealed a single class of high affinity (Kd 15‐130 pM) G‐CSF receptors on the AML blasts. The number of G‐CSF receptors varied from 55 to 1200 per cell (mean 278). In the remaining two patients, specific binding was not observed. The number of G‐CSF receptors did not differ significantly between various AML subtypes, but the mean receptor number was the highest on type M2 blasts. A chemical cross‐linking study revealed that the G‐CSF receptor has an approximate molecular weight of 140 000. Autoradiography showed heterogeneity of the distribution of G‐CSF receptors on the AML blasts obtained from a single patient. The number of colonies stimulated by the addition of rhG‐CSF varied from 0 to 566 per dish, and blast colony formation was observed in eight of 20 patients. The population mean of G‐CSF receptor number expressed by blasts that formed colonies on stimulation with rhG‐CSF was significantly higher than that on blasts which did not form colonies. These results suggest that a proliferative response of AML blasts to G‐CSF may be predicted when the blasts express a large number of G‐CSF receptors. Accordingly, it may be safer to restrict the clinical use of G‐CSF to AML patients who have blasts with a low G‐CSF receptor expression and no response to G‐CSF in blast colony assay.
Origin and function of adherent lymphokine activated killer cells in patients with chronic myeloid leukaemia who relapse following bone marrow transplantation
Authors: Mackinnon Stephen , Bungey Julie , Chase Andrew , Paulsen Wendy , Hows Jill M. , Goldman John M.
Published: March 2008

Summary. Adherent lymphokine activated killer (ALAK) cells are a subpopulation of activated natural killer (NK) cells with MHC unrestricted antitumour activity distinguished by their propensity to adhere to plastic in the presence of interleukin‐2 (IL‐2). We generated ALAK cells from seven patients with chronic myeloid leukaemia (CML) following Campath‐1‐depleted bone marrow transplantation (BMT). Five had relapsed and were in chronic phase, one had cytogenetic evidence of relapse and one had prior evidence of cytogenetic relapse but was in complete remission at time of study. Phenotypically the ALAK cells included both CD56 +/ CD3‐ NK cells and CD56‐/CD3+ T cells. The CD3‐subpopulation were studied cytogenetically and their functional activity tested in a 4 h 51Cr release cytotoxicity assay using the pretransplant leukaemia cells as targets. Cytogenetic studies showed that the ALAK cells from six patients were Ph negative, and where donor and recipient were sex mismatched. ALAK cells were exclusively of donor origin. In one patient ALAK cells were Ph positive and of recipient origin in eight of nine metaphases. In the 51Cr release assay the ALAK cells showed significant lysis of the pretransplant leukaemia in five of the seven patients tested. These data indicate that in CML patients who relapse post‐BMT the NK cells are usually of donor origin but may be recipient‐derived. In most patients these ALAK cells have antileukaemic activity in vitro.
Identification of haemophilia B patients with mutations in the two calcium binding domains of factor IX: importance of a β‐OH Asp 64→Asn change
Authors: Winship P. R. , Dragon A. C.
Published: March 2008

Summary. The polymerase chain reaction procedure (PCR) coupled with direct sequencing has been used to screen a panel of haemophilia B patients. This analysis has identified, amongst others, several mutations in the functionally important gla and type B EGF domains of factor IX, both of which are known to bind calcium. Type B EGF domains are widely distributed in proteins; located within these domains are highly conserved amino acid residues important for the formation of a high‐affinity calcium binding site. One prominent feature of these domains is a highly conserved β‐hydroxylated Asp or Asn residue. Of particular interest is the identification of one patient, with a substitution of the β‐hydroxy Asp‐64 residue normally present in factor IX for Asn. This change results in a functionally defective factor IX molecule with altered calcium binding properties. To explain the functional abnormality caused by this substitution of one amino acid residue for another which is commonly found at the equivalent position in other proteins with type B EGF domains, we propose the existence of additional conserved residues within this domain, which are important for calcium binding, and which correlate with whether the β‐hydroxylated residue is Asp or Asn.
Analysis of methylation in the c‐MYC gene in five human myeloma cell lines
Authors: Ohtsuki Takemi , Nishitani Koji , Hatamochi Atsushi , Yavvata Yoshihito , Namba Masayoshi
Published: March 2008

Genomic alterations of the human c‐MYC gene were analysed in five human myeloma cell lines established in Kawasaki Medical School and compared with those of normal lymphocytes, Raji cells from Burkitt's lymphoma, and an Epstein‐Barr virus positive lymphoblastoid cell line (LCL). Although no structural chromosome aberrations at 8q24, the c‐MYC locus, were distinct, the mRNA level of c‐MYC in these myeloma cell lines was 30–50‐fold that in normal peripheral blood lymphocytes. Regarding the methylation of c‐MYC, DNAs of the myeloma cell lines were digested with MspI plus EcoRI or HpaII plus EcoRI, and hybridized with three genomic 32P‐labelled probes; the first, second and third exons of the human c‐MYC gene, respectively. The extent of methylation in cytosine at a single CCGG site in the third exon substantially decreased in these myeloma cell lines as compared with that in normal tonsillar B, LCL and Raji cells. No significant differences in hypomethylation between these myeloma, normal B, LCL and Raji cells was detected in the first and second exon of c‐MVC. These results suggest that the hypomethylation in the third exon of c‐MYC might be related to the enhanced expression of c‐MYC in these human myeloma cell lines.
Haemophilia B Leyden arising de novo by point mutation in the putative factor IX promoter region
Authors: Royle G. , Van de Water N. S. , Berry E. , Ockelford P. A. , Browett P. J.
Published: March 2008

Haemophilia B Leyden is characterized by severe factor IX deficiency during childhood with partial resolution at puberty or following the administration of anabolic steroids. The disorder has recently been associated with point mutations in the putative factor IX promoter region, which contains an imperfect direct repeat spanning a possible start site of transcription. We have identified a T to C transition at position +8 in the promoter region of a patient with the haemophilia B Leyden phenotype. A mutation at this site has not been previously reported and occurs within the repeat consensus sequence in the transcribed but untranslated portion of the gene. There is no family history of haemophilia and sequence analysis of his mother and other family members indicates that the mutation has arisen de novo in this patient. This observation provides futher support for a causal relationship between point mutations in the presumptive promoter region of the factor IX gene and the Leyden phenotype.
Sickle cell‐β+ thalassaemia in Orissa State, India
Authors: Kulozik Andreas E. , Bail Sigrid , Kar Bimal C. , Serjeant Beryl E. , Serjeant Graham E.
Published: March 2008

The clinical, haematological, and some molecular genetic features of 17 Orissan Indian patients with sickle cell‐β+ thalassaemia (Sβ+ that) are described and compared with those in 131 Indian patients with homozygous sickle cell (SS) disease. Patients with Sβ+ that had higher Hb A2 levels, and lower mean cell volume (MCV) and mean cell haemoglobin (MCH) compared to SS disease but no other haematological difference of statistical significance. High levels of Hb F occurred in both genotypes and the α+ thalassaemia gene frequency reached 0 · 47 in Sβ+ thai and 0 · 32 in SS disease. Clinically there were no significant differences between the genotypes indicating that the low levels of HbA (3‐5%) in this condition were insufficient to modify the clinical features.

The thalassaemic β globin gene is inactivated by a G → C mutation at position 5 of the first intron of the β globin gene (IVS1–5 G → C) in all cases. This finding should facilitate the introduction of a prenatal diagnosis programme aimed at the prevention of β thalassaemia or Sβ+ thalassaemia in that population.
Productive infection of in vitro generated haemopoietic progenitor cells from normal human adult peripheral blood with parvovirus B19: studies by morphology, immunocytochemistry, flow‐cytometry and DNA‐hybridization
Authors: Serke Stefan , Schwarz Tino Francis , Baurmann Herrad , Kirsch Andreas , Hottenträger Barbara , Brunn Albrecht Von , Roggendorf Michael , Huhn Dieter , Deinhardt Friedrich
Published: March 2008

Parvovirus B19 exerts a highly selective cytopathic effect on erythroid progenitor cells. Studies so far on the pathogenesis of B19‐infection have been performed using bone marrow samples providing large amounts of erythroid progenitor cells. Extensive study, however, has been hampered by the limited access to bone marrow samples. We have designed a liquid culture method allowing the generation of large numbers of erythroid progenitor cells, initiating cultures with CD3‐ and CD14‐poor peripheral blood mononuclear cells. Following a 12 d preincubation in liquid cultures containing recombinant human interleukin 3 (rhll‐3) and recombinant human erythropoietin (rhEpo), cells harvested from the liquid cultures were exposed to B19‐containing plasma, followed by a further cultivation in liquid culture for up to 96 h. Cells expressing the CD13 and the glycophorin A (GlyA) antigens, respectively, were monitored sequentially by flow‐cytometry, demonstrating a selective inhibition of GlyA‐positive cells following B19‐inoculation. Typical morphological changes were observed on cytocentrifuge‐spots, and typical giant‐cells were identified as staining for GlyA. Productive infection by B19 was demonstrable, as B19‐DNA increased by about x 100 after 72 h of culture.

The liquid culture method generating erythroid target cells for effective infection by B19 virus promises to be a useful and easily accessible tool for further research on B19 infection of haemopoietic cells.
Induction of granulocyte and granulocyte‐macrophage colony‐stimulating factors from human monocytes stimulated by Fc fragments of human IgG
Authors: Ishiguro Akira , Nakahata Tatsutoshi , Koike Kenichi , Yoshida Hideaki , Shimbo Toshikazu , Komiyama Atsushi
Published: March 2008

The effect of human IgG on human haemopolesis has been studied in vitro. Dialysed purified IgG stimulated haemopoletic colony growth by bone marrow mononuclear cells (MNC) but not by monocyte‐depleted MNC. Culture media, conditioned by IgG‐stimulated peripheral blood MNC, augmented formation of neutrophil‐macrophage, eosinophil, and megakaryocyte colonies by monocyte‐depleted marrow MNC. Serum‐free IgG‐conditioned media also contained colony‐stimulating activity (CSA). IgG Fc fragments and heat‐aggregated IgG promoted the secretion of CSA, but F(ab′)2 fragments, Fab fragments or ultracentrifuged IgG did not. In the cell‐selection studies, CSA was produced by highly enriched monocytes following stimulation with Fc fragments. The antiserum against human granulocyte colony‐stimulating factor (G‐CSF) and/or granulocyte‐macrophage CSF (GM‐CSF) neutralized the CSA produced by Fc fragment‐activated monocytes. Enzyme immunoassays showed G‐CSF and GM‐CSF in media conditioned by monocytes stimulated with the Fc fragments, heat‐aggregated IgG and anti‐D‐sensitized red blood cells (RBC). Northern hybridization analysis showed mRNA encoding G‐CSF and GM‐CSF in RNA extracted from MNC and monocytes cultured with the Fc fragments, but not in the RNA from unstimulated cells or monocyte‐depleted MNC. These results indicate that IgG Fc fragments, aggregated IgG and antigen‐antibody complexes induce monocytes to produce G‐CSF and GM‐CSF in vitro. The CSFs release induced by IgG may be involved in the in vivo regulatory network in haemopoiesis.
Biological significance of cell cycle kinetics in 128 standard risk newly diagnosed patients with acute myelocytic leukaemia
Authors: Raza Azra , Preisler Harvey , Lampkin Beatrice , Yousuf Naveed , Tucker Christopher , Peters Nancy , White Michael , Kukla Cathy , Gartside Peter , Siegrist Carl , Bismayer John , Barcos Maurice , Bennett John , Browman George , Goldberg Jack , Grunwald Hans , Larson Richard , Vardiman James , Vogler Ralph
Published: March 2008

Bromodeoxyuridine (BrdU) was administered to 128 newly diagnosed patients with standard risk acute myelocytic leukaemia (AML) for cell cycle measurements. Labelling indices (LI) were obtained from both the bone marrow aspirate (BMasp) and biopsies (bx) and durations of S‐phase (Ts) and total cell cycle time (Tc) were measured by double‐labelling the S‐phase cells in vitro with tritiated thymidine. Median LI BMasp was 8% and from BMbx was 25%. The median Ts was 12 h (range 3·1–35 h) and Tc was 48 h (range 11·5–211 h). All patients received induction therapy with a combination of cytosine arabinoside and an anthracycline. Outcome of therapy or FAB type were not related to cell cycle characteristics. Patients with above median LI BMasp, however, had longer remission durations (P= 0·03) as did patients with above median Ts (P= 0·03) and Tc (P= 0·03). Upon longer follow‐ups, even some of the patients with slowly cycling myeloblasts have relapsed (log rank P= 0·453 and 0·203 for Ts and Tc respectively). We conclude that patients with rapidly cycling cells tend to relapse faster; however, slowly cycling nature of myeloblasts is not associated with curability.
Co‐expression of myeloid antigens in childhood acute lymphoblastic leukaemia: relationship with the stage of differentiation and clinical significance
Authors: Cantù‐Rajnoldi A. , Putti C. , Saitta M. , Granchi D. , Foà R. , Schirò R. , Castagni M. , Valeggio C. , Jankovic M. , Miniero R. , Paolucci P. , Basso G.
Published: March 2008

Co‐expression of myeloid antigens on the leukaemic blast cells was evaluated in 532 children with a diagnosis of acute lymphoblastic leukaemia (ALL). Using a panel of monoclonal antibodies belonging to CD11b, CD13, CD14, CD15 and CD33 an overall incidence of 4·3% was found, with values ranging between 1·8% for CD14 and 6·1% for CD15. When the data were further dissected, a significantly higher incidence of co‐expression was noted in null‐ALL (15/70 cases = 21·4%), compared to cases expressing a more mature immunophenotype, i.e. common‐ALL (7/394 cases = 1·7%) and T‐ALL (1/68 cases = 1·4%) (P<0·001). In null‐ALL, 9/15 patients were infants, five of whom with the t(4;11); two further children also had a t(4;11). The clinical outcome of the 23 cases which co‐expressed myeloid antigens was unfavourable. Only two of the 15 null‐ALL, two of the seven common‐ALL and the unique case with T‐ALL are in fact in persistent first remission between 19 and 93 months from diagnosis. Though the overall incidence of childhood ALL expressing myeloid antigens is low, the evidence that this co‐expression may be related to an unfavourable clinical course and that it more frequently occurs in null‐ALL, particularly in the first year of life, suggests that the routine assessment of myeloid antigens may allow to identify a subgroup of childhood ALL with a poor clinical outcome.
A common Vδ2—Dδ2—Dδ3 T cell receptor gene rearrangement in precursor B acute lymphoblastic leukaemia
Authors: Yano Takahiro , Pullman Amy , Andrade Rafael , Uppenkamp Michael , de Villartay Jean Pierre , Reaman Gregory , Crush‐Stanton Sylvia , Cohen David I. , Raffeld Mark , Cossman Jeffrey
Published: March 2008

Despite their apparent commitment to the B lymphocytic lineage, human precursor B cell acute lymphoblastic leukaemias (ALL) frequently rearrange their T cell antigen receptor (TCR) α, β and γ chain genes. Since these three genes are active sites of rearrangement in precursor B cell neoplasms, it seemed that the recently discovered fourth TCR gene, δ, might be similarly rearranged. To investigate this possibility, a series of precursor B cell leukaemias was analysed for rearrangements at the δ chain gene locus, using probes of the variable, joining, and constant regions of the δ chain gene.

The majority of precursor B cell ALLs in this series (25/32, 78%) showed rearrangement or deletion of one or more TCR δ genes. This contrasts sharply with a series of 16 mature B cell neoplasms (chronic lymphocytic leukaemia) in which no TCR δ gene rearrangements were detected.

An unusual TCR δ rearrangement, rarely observed in normal or neoplastic T cells, was seen in the majority (14/18) of precursor B cell ALLs with TCR δ rearrangements. In contrast to the utilization ov Vδ1 in T cell ALL, detailed restriction mapping of precursor B ALL revealed an incomplete rearrangement without involvement of Jδ segments. Direct genomic sequencing was performed on one example and demonstrated a nonproductive Vδ2‐Dδ2‐Dδ1 recombination in this precursor B ALL.

We conclude that the TCR δ chain gene is an active locus in precursor B cell neoplasia, involves an unusual type of rearrangement and provides a clonal tumour marker for diagnosis of precursor B ALL.
Prophylactic use of ganciclovir in allogeneic bone marrow transplantation: absence of clinical cytomegalovirus infection
Authors: Atkinson Kerry , Downs Kathryn , Golenia Marie , Biggs James , Marshall Gai , Dodds Anthony , Concannon Alan
Published: March 2008

Ganciclovir was given prophylactically to 25 patients receiving allogeneic bone marrow transplants for haematological malignancy. Patients who were seropositive for cytomegalovirus (CMV) pre‐transplant were given ganciclovir both pre‐ and post‐transplant. Those who were CMV seronegative, but who received marrow from a CMV seropositive donor, received ganciclovir post‐transplant. No non‐haemopoietic toxicity was observed. Toxicity was restricted to late reversible haematological toxicity in four of the 19 evaluable patients (one thrombocytopenia, one pancytopenia, two leucopenia). No CMV interstitial pneumonitis (IP) was observed, nor were any other clinically manifest CMV infections detected. Sixteen patients remain alive at > 84 to > 518 d post‐transplant. In a retrospective comparison of 152 recipients of allogeneic transplants for haematological malignancy not given prophylactic ganciclovir, and in whom either the recipient or the donor or both were CMV seropositive, the incidence of all clinically manifest CMV infections was 23% (P= 0·02) and that of CMV IP 17% (P= 0·05). If only patients in the study group and the control group receiving the same cyclosporin/short methotrexate prophylactic immune suppressive regimen, the same prophylactic acyclovir regimen and the same CMV and leucocyte‐filtered blood product transfusion strategy were considered, the incidence of all clinically manifest CMV infections in the control group was 24% (P= 0·01) and that of CMV IP 13% (P= 0·07). Ganciclovir appears to reduce the incidence of CMV infections in allogeneic marrow transplant recipients even in those given immune suppressive regimens associated with adequate control of acute graft‐versus‐host disease.
Antithrombin Glasgow II: alanine 382 to threonine mutation in the serpin P12 position, resulting in a substrate reaction with thrombin
Authors: Ireland H. , Lane D. A. , Thompson E. , Walker I. D. , Blench I. , Morris H. R. , Freyssinet J. M. , Grunebaum L. , Olds R. , Thein S. L.
Published: March 2008

A female with recurrent thrombosis was found to have a functional abnormality of antithrombin, with a ratio of functional to immunological activity in plasma of ∼ 50%. Crossed immunoelectrophoresis in the presence of heparin was normal, indicating an abnormality of the reactive site, rather than the heparin binding domain. Accordingly, the antithrombin was isolated by heparin‐Sepharose chromatography: this produced a mixture of normal and variant antithrombin, as the patient was heterozygous for the abnormality. To remove the normal component, the antithrombin was passed through a column of thrombin‐Sepharose. On sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE), prior to its application to thrombin‐Sepharose, the antithrombin migrated as a single band with identical mobility to that of normal antithrombin. After thrombin‐Sepharose, the purified variant component was proteolysed, and migrated as two components, one with a reduced and one with enhanced mobility under non‐reducing conditions. This demonstrated that the variant was unable to form stable inhibitor‐thrombin complexes and was cleaved in a substrate reaction with thrombin. One site of cleavage was unambiguously ascertained to be the Arg 393‐ Ser 394 reactive site bond, by NH2 terminal sequencing of the cleaved variant antithrombin: 10 steps beginning at the P 1’position, Ser‐Leu‐Asn‐Pro‐Asn‐Arg…., were clearly identified.

The mutation responsible for this defect was studied by polymerase chain reaction (PCR) amplification of exon 6 of the antithrombin gene and direct sequencing of the amplified product. The presence of both a G and A in the first position of codon 382, identified the mutation GCA to ACA, which results in the substitution of Ala 382 to Thr. This is identical to that reported for antithrombin Hamilton (Devraj‐Kizuk et al, 1988), although antithrombin gene polymorphism analysis suggests that the antithrombin Glasgow II mutation has arisen independently.

We have recently shown (Caso et al, 1991) that mutation at a nearby position, Ala 384 to Pro, also transforms another variant, antithrombin Vicenza/Charleville, into a substrate for thrombin. The present results with antithrombin Glasgow II suggest that all the alanine residues at the base of the reactive site loop in positions P12–10 may be important for the formation of a stabilized inhibitor‐thrombin complex.
Role of spleen in hereditary spherocytosis: evidence for increased in vitro proteolysis of red cell membrane
Authors: De Matteis M. C. , Angelis V. De , Sorrentino F. , Bonollo E. , Vettore L.
Published: March 2008

In vitro proteolysis of red cell membranes has been studied by means of electrophoretic separation on SDS‐polyacrylamide gel of solubilized ghost proteins and subsequent densitometry of separated, stained bands; the amounts of major membrane proteins were measured in ghosts either with inhibited or with allowed proteolysis in the following cases: 15 patients suffering from hereditary spherocytosis (HS) with variable degree of spleen enlargement, eight cirrhotic patients with spleen enlargement and 12 healthy blood donors as control group.

Proteolysis was present to a greater extent in HS patients with larger splenomegaly, lesser in HS with smaller splenomegaly, and was comparable to healthy controls both in splenectomized HS and in patients with spleen enlargement due to liver cirrhosis.

The results suggest the involvement of splenomegaly in the enhancement of in vitro proteolysis in HS red cell membrane; it is probably attributable to joint effects of the damage induced in red cells by prolonged retention within haemolysing spleen together with the abnormalities genetically affecting the structure of HS red cell membrane.
Recipient origin of bone marrow‐derived fibroblastic stromal cells during all periods following bone marrow transplantation in humans
Authors: Agematsu Kazunaga , Nakahori Yutaka
Published: March 2008

The bone marrow microenvironment, which is composed of fibroblasts, endothelial cells, adipocytes and macrophages, plays an important role in the haematopoiesis and lymphopoiesis by producing various cytokines. Therefore, investigation of the origin of these cells following allogeneic bone marrow transplantation (BMT) is very significant, in terms of the haematological and immunological reconstitution after BMT. We have investigated the origin of fibroblastic stromal cells in long‐term cultures in seven of the sex‐mismatched cases. This was carried out by in situ hybridization using a Y‐chromosome specific cDNA probe (PHY10), conserving the morphology of the cells. In situ hybridization analysis showed that bone marrow fibroblasts (BMF) in long‐term cultures in all the sex‐mismatched cases originated from the recipients. We have also performed Southern blot analysis using a PHY10 probe in the sex‐mismatched cases and using a variable number of tandem repeats (VNTR) probe, which can detect DNA polymorphisms, in two of the sex‐matched cases. In addition, we have employed polymerase chain reaction (PCR) using the VNTR marker (MCT118). Although all the patients showed haemopoietic engraftment with donor cells, their BMF were found, by Southern blot analysis and PCR method, to be of the recipient origin. These data indicate that bone marrow‐derived fibroblastic stromal cells which proliferate in long‐term cultures are not transplantable in the conditioning regimens used for allogeneic BMT in humans.
The treatment of patients with newly diagnosed poor prognosis acute myelogenous leukaemia: response to treatment and treatment failure
Authors: Preisler Harvey D. , Larson Richard A. , Raza Azra , Browman George , Goldberg Jack , Vogler Ralph , Day Roger , Gottlieb Arlan , Vardiman James W. , Bennett John , Kukla Cathi , Grunwald Hans
Published: March 2008

The failure of poor prognosis patients with newly diagnosed AML to enter remission is usually due to two phenomena: a high mortality rate and resistance of the leukaemia to chemotherapy. We conducted a pilot study of a regimen designed to overcome these two types of treatment failure. Patients were carefully selected for therapy on the basis of their likelihood of surviving. Chemotherapy consisted of high dose cytosine arabinoside (HDaraC) with the doses modified on the basis of patient age so as to reduce the risk of toxicity. Finally, daunorubicin was administered only to those patients for whom HDaraC was not likely to produce sufficient antileukaemia effects to produce a remission.

The median patient age was 67 years and 69/88 (78%) patients had a history of preleukaemia and/or toxic exposure. Only 11/84 (13%) patients died during remission induction therapy and 40/84 (48%) entered CR. Patients with only one risk factor had a higher CR rate than those with more than one risk factor (56%v. 30%, P= 0·02) and also had longer durations (222 d v. 113·5 d, P= 0·035). Two types of resistance to chemotherapy were observed: ‘classical resistance’(the failure of chemotherapy to produce substantial killing of leukaemia cells) and the rapid regrowth of leukaemia cells subsequent to a level of cytoreduction which otherwise would have been sufficient to produce a CR.
Thrombocytopenia in acute leukaemia patients treated with IL2: cytolytic effect of LAK cells on megakaryocytic progenitors
Authors: Guarini Anna , Sanavio Fiorella , Novarino Anna , Tos Anna Gillio , Aglietta Massimo , Foa Robert
Published: March 2008

In vivo administration of recombinant interleukin 2 (IL2) has been associated, in acute leukaemia as well as in other tumours, with a variable degree of thrombocytopenia. In two patients with acute myeloid leukaemia who showed a progressive and severe fall in platelet count during daily continuous i. v. infusion of IL2, we assessed whether peripheral blood IL2‐generated lymphokine activated killer (LAK) lymphocytes could affect growth of the autologous bone marrow megakaryocytic progenitor cell compartment (CFU‐MK) in vitro. Following overnight pre‐incubation in liquid culture of the marrow cells with autologous LAK effectors, there was an almost complete abrogation of the CFU‐MK colony growth (97% and 89% inhibition). Pre‐incubation in the presence of a monoclonal antibody to tumour necrosis factor alpha (TNF) completely reversed the inhibitory effect. The role played by TNF was confirmed by the finding that recombinant TNF caused a dose‐dependent inhibition of the growth of CFU‐MK. IL2 alone was ineffective. These results suggest that the often severe thrombocytopenia observed in patients with acute leukaemia treated with IL2 is at least partly due to autologous LAK cells activated in vivo following the administration of IL2.