bluuebunny/crossref_metadata_2025 huggingface dataset:
The 32·6 kb Indian δβ‐thalassaemia deletion ends in a 3·4 kb L1 element downstream of the β‐globin gene
Authors: Gilman J. G. , Brinson E. C. , Mishima N.
Published: March 2008

Summary. The Indian δβ‐thalassaemia, with elevated fetal γ globin gene expression, was previously found to have a large deletion beginning 1 kb 3’of the Aγ globin gene at GenBank HUMHBB coordinate 42151, and extending into a new L1 sequence. We have now determined the 3’breakpoint of this deletion, and in doing so we have extended the known β‐globin gene cluster DNA sequence from its end at 73326 to projected GenBank coordinate 79016. These data show that the deletion is 32–6 kb long, terminating 11 kb 3’of the β‐globin gene. This 3’breakpoint is at 74772, within a 3.4 kb partial L1 repeat at 74263–77665; the Black (Aγδβ)°‐thalassaemia also terminates in this L1, at 76508. In addition, two Alu sequences were found, at 73692–73816 and 78171–78441. Among the protein‐binding DNA sequence motifs 3’to the Indian δβ‐thalassaemia breakpoint, at 76581/76607 there is a TGATAA/ACACCC pair that binds the erythroid‐specific GATA‐1 and ubiquitous CACCC‐box binding proteins. We hypothesize that elevated fetal haemoglobin may be due to an enhancer or enhancers 3’to the deletion breakpoints and may involve the TGATAA/ ACACCC pair.
Haemoglobin F levels in sudden infant deaths
Authors: Pagan D. G. , Walker A.
Published: March 2008

Summary. Fetal haemoglobin levels have been measured prospectively in 1 35 autopsy cases of sudden, unexpected infant deaths (51 pre‐term. 104 full term) using standard laboratory methods. These results have been compared with Hb F values from a normal control group of 570 living infants (145 pre‐term, 425 full‐term) with a post‐conceptional age < 90 weeks. The gestational age was established for all live controls and sudden infant deaths.

The results show that full‐term (> 38 weeks gestational age) sudden infant death victims as a group have significantly elevated Hb F levels (χ= 25.20. P<0.001) when compared to the gestational age matched control group. Pre‐term (< 38 weeks gestational age) SIDs show no significant differences from the pre‐term control group (χ2= 1.20, n.s.) The division of the controls into pre‐ and full‐term groups demonstrates major differences between the post‐natal Hb F fall in pre‐ and full‐term infants. Use of post‐conceptional age as a growth marker does not produce comparability with full‐term infants.

Extensive controls were carried out to confirm the reliability of post‐mortem Hb F assays, and comparability with in vivo estimation. No significant or systematic differences between pre‐ and post‐mortem samples were identified, nor were any significant differences found on post‐mortem storage up to 72 h.
Serum non‐transferrin‐bound iron in beta‐thalassaemia major patients treated with desferrioxamine and L1
Authors: Al‐Refaie F. N. , Wickens D. G. , Wonke B. , Kontoghiorghes G. J. , Hoffbrand A. V.
Published: March 2008

Summary. Non‐transferrin‐bound iron (NTBI) in plasma is toxic due to its ability to participate in free radical formation with resultant peroxidation and damage to cell membranes and other biomolecules. NTBI concentration was determined in serum in 12 normal volunteers and in 52 patients with β‐thalassaemia major by a modification of the method described by Singh et al (1990). There was no detectable NTBI in normal individuals. In the patients NTBI values ranged from −1.5 to 9.0 μmol/l (mean ± SD: 3.6 ± 2.3). The patients’serum ferritin concentrations ranged from 207 to 11400 μg/l (2674±2538), total serum iron from 20 to 61 μmol/l (39.5 ± 9.6) and transferrin saturation from 44 to 110% (84.5 ± 13.8). The NTBI correlated significantly with serum ferritin (r=0.467, P < 0.001), total serum iron (r=0.608, P<0.001) and transferrin saturation (r=0.481, P<0.005). When patients were grouped according to their compliance with desferrioxamine (DFX) therapy, the good compliers had significantly lower NTBI concentrations compared to the poor compliers (poor: 5.4±1.8 μmol/l v good: 2.7±1.7 μmol/l, P<0.001). There was also a significant difference between the level of NTBI and whether or not the patients had complications of iron overload (5.2±1.7 μmol/l v 2.9±1.6 μmol/l, P <0.001). During this study 10 patients were entered into a trial of the oral iron chelator 1,2‐dimethy 1–3‐hydroxypyrid‐4‐one (L1). Their NTBI values were observed during the first 6 months of the trial and showed a significant fall (paired t‐test: P= 0.007). These results suggest that the level of NTBI may prove helpful in assessing the efficiency of chelation in patients with transfusion dependent anaemia and help to predict organ damage.
Simultaneous establishment of myeloid and B‐lymphoid cell lines with identical chromosome abnormalities from Philadelphia chromosome‐positive chronic myelogenous leukaemia
Authors: Yasukawa Masaki , Yanagisawa Kohsuke , Kohno Hidehisa , Yakushijin Yoshihiro , Kondo Toshifumi , Fujita Shigeru
Published: March 2008

Summary. Two continuously growing cell lines, designated YOS‐M and YOS‐B, were established simultaneously from a patient with Philadelphia (Ph1) chromosome‐positive chronic myelogenous leukaemia (CML) in myeloid blast crisis. Both YOS‐M and YOS‐B had the Ph1 chromosome and identical additional chromosome abnormalities, which were not detected in the chronic phase. Cytochemical analysis showed that YOS‐M was significantly positive for peroxidase, whereas YOS‐B was entirely negative. YOS‐M expressed myeloid‐associated antigens (CD14, CD33) as well as CD4, CD25 and CD34. The surface phenotype of YOS‐M was identical to that of the leukaemic blasts found in the patient. On the other hand, YOS‐B expressed mature B‐cell markers, CD19, CD20, CD21 and surface immunoglobulin, but not myeloid‐associated antigens. These two cell lines showed an identical rearrangement pattern of the break point cluster region on chromosome 22, but rearrangement of the immunoglobulin heavy chain gene was detected only in YOS‐B. These findings provide definite evidence that CML cells still have the capability to differentiate and mature along different haematopoietic cell lineages even after blast crisis.
Diagnosis of acute promyelocytic leukaemia by RT‐PCR: detection of PML‐RARA and RARA‐PML fusion transcripts
Authors: Borrow Julian , Goddard Audrey D. , Gibbons Barbara , Katz Fay , Swirsky David , Fioretos Thoas , Dube Ian , Winfield David A. , Kingston Judith , Hagemeijer Anne , Rees John K. H. , Lister T. Andrew , Solomon Ellen
Published: March 2008

Summary. Acute promyelocytic leukaemia (APL; AML M3) is identified by a unique t(15;17) translocation which fuses the PML gene to the retinoic acid receptor alpha gene (RARA). Reverse transcription coupled with the polymerase chain reaction (RT‐PCR) has been used to develop a diagnostic test for APL based on the PML‐RARA fusion message. Separate PCR assays were designed to amplify either PML‐RARA (15q+ derived) or RARA‐PML (17q‐ derived) chimaeric transcripts. PML‐RARA transcripts were detected in every case from a series of 18 APL patients with cytogenetically confirmed t(15;17) translocations, whereas RARA‐PML messages were detected in only 67% (12/18) of these patients. This suggests that it is the 15q + derivative which mediates leukaemogenesis. Furthermore the PCR approach (or Southern analysis) may be used to identify in which of the alternative PML introns the breakpoint occurs; 52% of cases (15/29 patients) utilize a 5′ PML intron and 48% the 3′ intron (14/29 cases). Neither the choice of PML intron nor the expression of the 17q‐derivative could be correlated with the microgranular variant of APL (M3V), overall survival rate, age, sex or presence of coagulopathy. Finally, the fusion message is undetectable in five remission samples. This indicates a possible use for RT‐PCR in monitoring remission patients for evidence of relapse.
Expression of the IFNα receptor in hairy cell leukaemia
Authors: Platanias Leonidas C. , Pfeffer Lawrence M. , Barton Kevin P. , Vardiman James W. , Golomb Harvey M. , Colamonici Oscar R.
Published: March 2008

Summary. The expression of IFNα receptors on the surface of hairy cell leukaemia cells was studied using affinity crosslinking methods, flow cytometry with a recently produced anti‐IFNα receptor monoclonal antibody (IFNaR3), and binding techniques. IFNα receptors were detected by flow cytometry and affinity crosslinking in eight cases showing a normal receptor structure with 125I‐IFNα2‐receptor complexes with molecular weights of 210, 130 and 110 kD. The hairy cell leukaemia cells from one patient did not express IFNα receptors as determined by flow cytometry, affinity crosslinking and binding studies. Northern blot analysis showed expression of mRNA for the gene coding for the putative IFNα receptor in all cases studied, including the patient with lack of IFNα receptor expression on hairy cells. This patient was refractory to IFNα2 treatment. Our data suggest that lack of expression of IFNα receptors in rare cases may be associated with refractoriness to IFNα2 therapy.
Argyrophilic nucleolar organizer region counts and prognosis in multiple myeloma
Authors: Pich A. , Marmont F. , Chiusa L. , Cappello N. , Resegotti L. , Navone R.
Published: March 2008

The prognostic significance of argyrophilic nucleolar organizer regions (AgNORs) has been evaluated in bone marrow trephine biopsies from 64 patients with multiple myeloma (MM) prior to therapy.

The univariate Kaplan‐Meyer survival analysis showed a significant correlation between survivals and AgNOR counts (median of survival 51.3 months for cases with 4.62 AgNORs per plasma cell (PC) versus 16 months for cases with >4.62 AgNORs per PC;P=0.0000) or AgNOR distribution in PC nucleus (AgNOR configuration) (median of survival 71.67 months for cases with tightly grouped AgNORs. 16.26 for partially grouped and 11.74 for dispersed AgNORs;P=0.001).

Significant prognostic correlations were also found for monoclonal immunoglobulin type (P=0.008), platelet counts (P=0.0078). serum creatinine level (P=0.0001), Durie's clinical stage (P=0.02). percentage of plasma cells in bone marrow biopsies (BMPCX) (P= 0.005), pattern of medullary involvement (P=0.003) and PC atypia (P= 0.009). Borderline result was detected for the percentage of PCs in aspirates (P=0.06). No significant correlation was found between prognosis and patients age, sex, haemoglobin level, serum albumin or calcium level, marrow cellularity and excess of haemosiderin.

Multivariate survival analysis showed that only two variables were significantly correlated with prognosis: AgNOR counts (P=0.003) and AgNOR configuration (P<0.001).

In addition, the analysis of variance showed significant association between AgNOR number and platelet counts, haemoglobin level, calcaemia. creatininaemia, clinical stage, percentage of PCs in aspirates, BMPC%, pattern of medullary involvement, PC atypia, marrow cellularity and configuration of AgNORs.

Our results indicate that AgNOR counts and configuration have prognostic and diagnostic value and therefore they are useful independent parameters to assess the pretherapeutic aggressiveness of multiple myeloma.
Endogenous platelet fibrinogen: its modulation after surface expression is related to size‐selective access to and conformational changes in the bound fibrinogen
Authors: Gralnick Harvey R. , Williams Sybil B. , McKeown Laurie , Shafer Brenda , Connaghan Gerard D. , Hansmann Kristin , Vail Michael , Magruder Louise
Published: March 2008

Platelet stimulation results in the release of endogenous platelet fibrinogen which binds to the platelet surface. Previous studies have demonstrated that plasma fibrinogen bound to activated platelets becomes inaccessible to a variety of probes. We have studied endogenous platelet fibrinogen binding to activated platelets by employing an immunopurified polyclonal anti‐fibrinogen antibody and F26, a monoclonal anti‐fibrinogen antibody, which recognizes fibrinogen only when it is bound to a surface. Employing the Ig or F(ab')2of the poly‐ or monoclonal antibody we found a marked decrease of fibrinogen accessibility 30–60 min after platelet activation. In contrast, platelet‐bound fibrinogen remains accessible to the Fab fragment of F26 at a constant level for 30 min and increases at 60 min. The reduction of the polyclonal Fab fragment binding at 30 and 60 min is similar to the F26 Ig. These results indicate that the decreased accessibility of bound fibrinogen is related to two mechanisms; (1) that the access route to fibrinogen is size selective for the antibody probes and only small antibody probes, e.g. Fab fragments, can gain access to fibrinogen and (2) fibrinogen undergoes a conformational change(s) after binding which exposes at least one neo‐epitope in the D domain of fibrinogen and which may decrease or mask the reactivity of other fibrinogen domains. Only the F26 Fab probe has full access to and identifies fibrinogen present on the platelet surface 60 min after stimulation.
A patient with von Willebrand's disease characterized by a compound heterozygosity for a substitution of Arg by Gln in the putative factor‐VIII‐binding domain of von Willebrand factor (vWF) on one allele and very low levels of mRNA from the second vWF allele
Authors: Peerlinck Kathelijne , Eikenboom Jeroen C. J. , Ploos Hans K. , Amstel Van , Sangtawesin Wanida , Arnout Jef , Reitsma Pieter H. , Vermylen Jos , Brieut Ernest
Published: March 2008

We describe a patient with a lifelong bleeding disorder previously classified as von Willebrand's disease (vWD) type I. The factor VIII (FVIII) level in this patient was disproportionately low and we showed that this was due to a decreased factor VIII binding capacity of her vWF. To characterize the molecular defect in this type of vWD, a cDNA‐dependent polymerase chain reaction (PCR) amplification was performed using platelet RNA as a template. Direct sequencing of the amplified fragment, which encodes for the FVIII‐binding domain, showed a single nucleotide change in exon 20 at codon 854, resulting in the substitution of CAG glutamine (Gln) for CGG arginine (Arg). At the level of the cDNA only the mutated sequence was found, whereas at genomic DNA level the patient was heterozygous for this mutation. This patient is therefore a compound heterozygote for a point mutation resulting in a FVIII‐binding defect and a vWF allele with low transcript levels.
Interleukin‐4 and nerve growth factor can act as cofactors for interleukin‐3‐induced histamine production in human umbilical cord blood cells in serum‐free culture
Authors: Richard Alain , McColl Shaun R. , Pelletier Guy
Published: March 2008

In order to better define the role of recombinant interleukins and growth factors in supporting histamine production by histamine‐producing‐cells (HPC), namely basophils or mast cells, we have conducted experiments in which we compared the abilities of IL‐3, IL‐4, IL‐6 and nerve growth factor (NGF), either alone or in various combinations, to stimulate histamine production from human umbilical cord blood mononuclear cells cultured in either the presence or absence of serum. Over a period of 15 d, IL‐3 was able to support intracellular histamine production, in both serum‐supplemented and serum‐free medium, although the effectiveness of IL‐3 was lower in the serum‐free cultures. IL‐4, NGF and IL‐6 taken individually, exhibited little, if any, histamine‐promoting activity regardless of whether or not the cells were cultured in serum. Moreover, none of these cytokines significantly enhanced the effect of IL‐3 in serum‐supplemented cultures. In contrast, IL‐3‐promoted histamine production in serum‐free cultures was significantly enhanced by IL‐4 and NGF. These results demonstrate that both IL‐4 and NGF can function as cofactors with IL‐3 in the support of histamine production in human umbilical cord blood cells.
Antibodies for denatured human H‐ferritin stain only reticuloendothelial cells within the bone marrow
Authors: Ruggeri Giuseppina , Santambrogio Paolo , Bonfiglio Francesca , Levi Sonia , Bugari Giovanna , Verardi Rosanna , Cazzola Mario , Invernizzi Rosangela , Zambell Luisa M. , Albertini Alberto , Arosio Paolo
Published: March 2008

Human H‐ferritin homopolymer was denatured in sodium dodecyl sulphate and injected in mice to obtain antibodies for dissociated H‐subunit. The antisera and Moabs obtained were specific for the denatured H‐chain with no cross‐reactivity with assembled ferritins in immunoblotting experiments. In contrast the Moabs for native recombinant H‐ferritin are specific for the assembled ferritin molecules with weak cross‐reactivity with the denatured H‐subunits. The epitope recognized by one of the anti‐denatured H‐chain Moabs was mapped on the C‐terminal helix of ferritin. The antibodies were used to study H‐ferritin conformation in cells. In immunocytochemistry experiments the antibodies for denatured H‐ferritin stained HeLa and K562 cells weakly, with a different intensity and pattern to those obtained with anti‐native H‐ferritin antibody. In human bone marrow smears the anti‐denatured ferritin antibodies stained only reticuloendothelial cells, and did not recognize the H‐ferritin rich immature erythroblasts. It is concluded that assembled and denatured H‐ferritins are immunogenically distinct, and that erythroid and reticuloendothelial cells within the bone marrow contain H‐ferritin in different conformations.
Non‐radioactive in situ hybridization for the detection and monitoring of trisomy 12 in B‐cell chronic lymphocytic leukaemia
Authors: Cuneo A. , Wlodarska I. , Aly M. Sayed , Piva N. , Carli M. G. , Fagioli F. , Tallarico A. , Pazzi I. , Ferrari L. , Cassiman J. J. , Van Den Berghe H. , Castoldi G. L.
Published: March 2008

Summary. Non‐radioactive in situ hybridization (NISII) with a chromosome 12‐specific alpha satellite probe was performed on 20 patients with chronic lymphocytic leukaemia (CLL) with normal karyotype (15 cases) or with inadequate mitotic yield (5 cases) from mitogen‐stimulated cultures.

All patients had over 70% lymphocytes coexpressing the CD5/CD23 antigens. While less than 1% interphase nuclei showed three fluorescent spots in 16/20 patients, evidence of trisomy 12 in 15–25% interphase cells was detected in four patients. According to the FAB classification the diagnosis in these patients was typical B‐CLL, stage III (Rai's staging system) in one case, CLL/PLL, stage II and III in two cases, PLL, stage III in one case. In order to confirm these results, NTSH was repeated after 1 month in one patient and after 2 years in three patients. All patients had been treated with chemotherapy in the period between the two NISH experiments. In all cases a 1.8–3‐fold increase of percentage of trisomic interphase cells was detected.

These findings suggest that in B‐CLL clones with trisomy 12 may have proliferative advantage over clonal B‐lymphocyte without +12 and possibly, that they may be more resistant to chemotherapy.

We conclude that NISH is a sensitive technique allowing for the detection and monitoring of trisomy 12 in a fraction of B‐CLL patients with normal karyotype or with no analysable mitoses despite employment of polyclonal B‐cell mitogens.
Effects of anti‐TNF monoclonal antibody infusion in patients with hairy cell leukaemia
Authors: Huang David , Reittie Joyce E. , Stephens Susan , Hoffbrand A. Victor , Brenner Malcolm K.
Published: March 2008

Tumour necrosis factor (TNF) can act as an autocrine growth factor for hairy cell leukaemia (HCL) cells. The TNF produced by the malignant clone may also inhibit normal haematopoiesis thereby contributing to the cytopenias observed in patients with the disease. We have studied the effects of infusing a murine monoclonal anti‐TNF antibody in three patients with HCL. In two patients receiving 0.5mg of antibody/kg on alternate days for 12 d, the drug was well tolerated. The third patient received 2 mg/kg on alternate days and developed symptoms of serum sickness by day 9. In two patients with severe B‐lymphocytopenia, circulating CD19 and CD20 positive, B‐cells were restored to normal, the majority of which were negative for the HCL‐associated marker CDllc. B‐lymphocyte recovery was associated with a rise in serum immunoreactive IL‐6 and with an early rise in immunoreactive TNF. These short courses of anti‐TNF MAb treatment had modest effect on the tumour burden, producing a reduction in splenomegaly in one patient. Exploration of the effects of more prolonged administration of higher dose anti‐TNF antibody will only be feasible when less immunogenic MAbs are available.
Clonal lymphocytes are detectable in only some cases of MDS
Authors: Culligan D. J. , Cachia P. , Whittaker J. , Jacobs A. , Padua R. A.
Published: March 2008

Clonal analysis of lymphocytes from patients with myelodysplastic syndrome (MDS) has been carried out using X‐chromosome inactivation patterns detected by the probe M27β, and by polymerase chain reaction amplification of the immunoglobulin heavy chain gene hypervariable region, CDR3. Of 32 female patients heterozygous for M27β only seven (22%) demonstrate monoclonality of peripheral blood lymphocytes. 12 (37%) give unequivocal polyclonal results and the remaining cases give patterns of X‐inactivation which cannot be interpreted either way. A study of 68 MDS patients showed five (7%) with a population of B‐cells with a monoclonal rearrangement of CDR3 compared with none out of 60 normal individuals, none out of 15 with B‐non Hodgkin lymphoma (B‐NHL) in remission and 19 out of 25 (75%) of cases of B‐chronic lymphocytic leukaemia (B‐CLL). Monoclonal lymphocytes were found by both techniques in only two females with MDS. We conclude that the presence of polyclonal lymphocytes is a common finding in patients with MDS.
Blunted rise in intracellular calcium in CD4 ± T cells in response to mitogen following autologous bone marrow transplantation
Authors: Guillaume Thierry , Hamdan Oussama , Staquet Philippe , Sekhavat Maryam , Chatelain Bernard , Bosly Andre , Rubinstein Daniel B. , Humblet Yves , Doyen Chantal , Coiffier Bertrand , Felman Pascale , Symann Michel
Published: March 2008

Summary. Following autologous bone marrow transplantation (ABMT), both impaired T cell activation and defective production of the principal T cell growth factor, interleukin‐2 (IL‐2), has been observed. These processes are dependent on a rise of intracellular calcium ([Ca2±]i), a step which follows binding of T cell receptor (TCR) and transduction of signal via the generation of cytoplasmic second messengers. In order to better understand the nature of defective cellular immunity in ABMT, in the present study we investigated the rise of [Ca2±]i in T cells of recipients of ABMT. By concomitant labelling lymphocytes with anti‐CD4 antibody and addition of fluo‐3 as fluorescent calcium indicator, we have selected for the T cell subset which is the principal source of IL‐2. Short‐term (less than 1 year post‐transplantation) recipients of ABMT show a statistically significant blunted rise in [Ca2±]i in response to concanavalin A as compared to normal controls not accounted for solely by a decreased percentage of CD4± cells in these patients. The [Ca2±]i response of CD4± cells from long‐term (greater than 1 year post‐transplant) recipients was lower than that of the normal group although not to a statistically significant level. These findings suggest that following ABMT is a defect in the early stages of T cell activation involving either T cell receptor binding or early signal transduction ultimately resulting in depressed transcription of IL‐2 mRNA. These defects are analogous to findings in both allogeneic transplantation where factors of histoincompatability and graft‐versus‐host disease (GVHD) come into play, as well as in the defective T cell activation of the normal ageing process.
Immunoglobulin VH4 gene usage in B lymphoid leukaemias
Authors: Deane Miriam , Baker Bartrum W. , Norton John D.
Published: March 2008

Summary. VH4 gene rearrangements occur in a similar proportion of cases of B lineage acute lymphoblastic leukaemia (ALL) and B chronic lymphocytic leukaemia (B CLL). However, there may be differences in the pattern of VH4 gene usage between these disorders as is the case for VH1 gene rearrangements. To examine this, we analysed the sequences of 24 PCR‐amplified clonal VH4 gene rearrangements from a series of 15 cases of ALL and nine cases of CLL. Five distinct groups of genes were rearranged, three of which (represented by V2‐1, V71‐2/V71‐4, V4.21) have been described in rearranged form in normal B lymphoid tissues. The most frequently rearranged gene was V4.21 which is strongly associated with autoimmune reactivity. V71‐2, V71‐4 and V2‐1 were more frequently rearranged in CLL than ALL. The remaining two groups (represented by V4.33, V4.35) have not previously been described in rearranged form. One of these, V4.35, was seen only in ALL rearrangements. Both V4.35 and a VH1 gene, 20P3, which is also preferentially rearranged in ALL, are located at the 3’end of the VH locus. The location of these genes suggests that their rearrangement may be developmentally regulated in ALL. The findings in this study confirm restricted repertoires of IgH gene rearrangement in ALL and CLL. Characterization of IgH repertoires provides a means of correlating these transformed B cell populations with normal B cell developmental compartments. Moreover, the distinctive repertoires in ALL and CLL may reflect important differences in the ontogenic timing and microenvironmental milieu of tumourigenesis in these disorders.
A molecular model of the serine protease domain of activated protein C: application to the study of missense mutations causing protein C deficiency
Authors: Wacey A. I. , Pemberton S. , Cooper D. N. , Kakkar V. V. , Tuddenham E. G. D.
Published: March 2008

Summary. A molecular model of the serine protease domain of protein C was constructed by standard comparative methods. Individual missense mutations were inserted into the model and plausible explanations for their interference with protein C structure/function were derived through consideration of location, steric effects and protein stability. A hydrophilic cluster of many Arg and Lys residues, found adjacent to the active site cleft, is proposed to be involved in thrombomodulin and/or protein S interactions. Analysis of comparative binding studies also suggested the presence of an extended substrate binding pocket in the model.
Expression of GPIV and Naka antigen on monocytes in Naka‐negative subjects whose platelets lack GPIV
Authors: Take Hironori , Kashiwagi Hirokazu , Tomiyama Yoshiaki , Honda Shigenori , Honda Yumiko , Mizutani Hajime , Furubayashi Takayasu , Karasuno Takahiro , Nishiura Tetsuo , Kanayama Yoshio , Kurata Yoshiyuki , Matsuzawa Yuji
Published: March 2008

Summary. The platelet antigen Naka was once considered to be a platelet‐specific alloantigen and is carried on platelet membrane glycoprotein (GP) IV. Recent studies suggest that Naka‐negative subjects lack platelet GPIV. GPIV is an important adhesive receptor and expressed on the surface of monocytes as well as of platelets. In the present study, flow cytometry was used to detect GPIV and Naka antigen on the surface of monocytes. Naka antigen was expressed on monocytes as well as on platelets in Naka‐positive subjects (n= 6) (P‐GPIV‐positive subjects). To our surprise, monocytes of Naka‐negative subjects (n= 7) (P‐GPIV‐negative subjects) having no anti‐Naka antibody in their serum expressed GPIV and Naka antigen to almost the same degree as did the monocytes of P‐GPIV‐positive subjects. Competitive experiments using OKM5 (a monoclonal antibody against GPIV) and anti‐Naka antibody showed that the epitope of anti‐Naka antibody on monocytes was very close to that of OKM5. In two P‐GPIV‐negative subjects having anti‐Naka antibody in their serum, GPIV and Naka antigen were not expressed on the surface of either monocytes or platelets. These results indicate that the GPIV molecules and Naka antigen are expressed on the surface of monocytes in the majority of P‐GPIV‐negative subjects, but that in a very few P‐GPIV‐negative subjects neither GPIV nor Naka antigen is expressed on the surface of their monocytes. We hypothesize that P‐GPIV‐negative subjects who carry neither GPIV nor Naka antigen on their monocytes produce anti‐Naka antibody as a result of transfusion or pregnancy.
The presence of messenger RNA for HLA class I in human platelets and its capability for protein biosynthesis
Authors: Santoso Sentot , Kalb Rainer , Kiefel Volker , Mueller‐Eckhardt Christian
Published: March 2008

Summary. In order to determine whether platelets contain specific messenger RNA encoding for HLA class I molecules, polymerase chain reaction (PCR) was performed with RNA from different platelet donors. Two amplified 300 bp and 279 bp cDNA fragments were obtained which encompassed sequences from 321 to 620 and from 795 to 1073. The 300 bp fragment encodes exon 2 and exon 3, the 279 bp encodes a portion of exon 4, exon 5, exon 6 and a portion of exon 7. A 300 bp nested PCR product from one donor, that encoded for the highly polymorphic region α2, was cloned and sequenced. The resulting nucleotide sequences fitted to the expected sequence for HLA B*3801 of this donor. Sequence analysis of the 279 bp PCR product demonstrated the presence of exon 5 encoding for the 117 bp transmembrane domain.

In addition, de novo protein biosynthesis was studied by radioimmunoprecipitation of HLA class I molecules from 35S‐methionine metabolically labelled platelet lysates with a monoclonal antibody (mab) w6/32 specific for a monomorphic epitope on the heavy chain of HLA class I antigens. Analysis of the immunoprecipitates on SDS polyacrylamide gel electrophoresis showed a specific band with apparent molecular weight (Mr) of 44 kD corresponding to integral membrane HLA protein.

On the basis of these results, we conclude that platelets contain specific messenger RNA encoding for HLA class I molecules and have the capability to synthesize the integral HLA membrane protein.
Expression of GpIb on plasma cells in a patient with monoclonal IgG and acquired von Willebrand disease
Authors: Scrobohaci Marie‐Lorraine , Daniel Marie‐Thérèse , Levy Yves , Marolleau Jean‐Pierre , Brouet Jean‐Claude
Published: March 2008

Summary. To get insights into the pathogenesis of acquired von Willebrand disease associated with plasma cell dyspasias, we searched for the expression of the physiological von Willebrand factor receptor, the GpIb/GpIX complex, on bone marrow plasma cells. The monoclonal spike in our patient corresponded to IgGκ molecules; there was no plasma inhibitor to vWF:Ag or vWF:RiCoF. The bone marrow contained 1–2% plasma cells. Fresh bone marrow cells or plasma cells enriched bone marrow cells after a 48 h in vitro culture in the presence of interleukin 6 were stained by an immuno alkaline phosphatase technique using monoclonal antibodies (mAb) to von Willebrand factor, GpIb α and β chain, GpIIb/IIIa and Gp IX. Two different mAb to GpIb α chains reacted with the majority (75%) of plasma cells whereas all other reagents yielded no staining. Malignant plasma cells from patients with multiple myeloma without haemostatic disorder were unreactive with anti‐GpIb mAb. These data suggest that in some patients with acquired von Willebrand syndrome there is a GpIb mediated selective adsorption of von Willebrand factor on clonal plasma cells.
A tumour‐associated antigen expression in human haematological malignancies
Authors: Chambost H. , Brasseur F. , Coulie P. , De Plaen E. , Stoppa A. M. , Baume D. , Mannoni P. , Boon T. , Maraninchi D. , Olive D.
Published: March 2008

Summary. Objective responses obtained with high‐dose in vivo recombinant interleukin 2 (r‐IL2) in some leukaemic patients suggest among other hypotheses that blasts might express tumour rejection antigens potentially recognized by cytolytic T lymphocytes. Such antigens have been described in human melanomas and the MAGE‐1 gene, coding for a tumour rejection antigen was recently identified. This gene is expressed in various solid tumours, but not in normal cells. We have screened a panel of haematological malignancies by reverse transcription and PCR and we report that MAGE‐1 is not expressed in the blasts from 48 patients whereas three cell lines derived from leukaemias express this gene.
Parvovirus B19‐associated haemophagocytosis in Evans syndrome: aplastic crisis accompanied by severe thrombocytopenia
Authors: Uike Naokuni , Miyamura Tomoya , Obama Kohsuke , Takahira Hiroyuki , Sato Hiroyuki , Kozuru Mitsuo
Published: March 2008

Summary. A 36‐year‐old man who had been treated for Evans syndrome (ES) developed an aplastic crisis caused by acute human parvovirus B19 (HPV) infection. Profound thrombocytopenia (8·0 × 109/l) followed with a sudden increase in platelet‐associated IgG (PAIgG) (1376·9 ng/107 plts). Bone marrow examination revealed a considerable number of haemophagocytic histiocytes without any disturbance of megakaryopoiesis. To our knowledge this is the first case of aplastic crisis with virus‐associated haemophagocytosis in a patient with ES, which provides an interesting insight into the mechanisms for thrombocytopenia in HPV infection.
The bone marrow in murine AIDS
Authors: Magnani Mauro , Brandi Giorgio , Rossi Luigia , Carnevali Andrea , Fraternale Alessandra , Albano Amedeo
Published: March 2008

Summary. In order to increase the understanding of blood cytopenias in HIV infection we have investigated the bone marrow in murine AIDS. C57BL/6 mice infected with the LP‐BM5 retrovirus show a decrease in cellularity, numerous haemophagocytic histiocytes, a reduction of all erythroid precursor cells, an increase in eosinophil number and an increase in lymphocytes. Immunostaining with an anti‐Pr60gag antibody shows that the majority of bone marrow cells express the viral protein. Thus, the bone marrow in MAIDS has many similarities with the bone marrow from patients with advanced AIDS and may prove useful as a model for therapy aimed at treating blood cytopenias.
Haematological disorders and occupational hazards: a British Society for Haematology/Health and Safety Executive study
Authors: Jacobs A. , Geary C. , Osman J.
Published: March 2008

Summary. During a 3‐year period 229 patients in the U.K. with haematological disorders thought to be associated with occupational hazards were notified to the British Society for Haematology/Health and Safety Executive office. Most were suffering from malignant or premalignant states or aplastic anaemia. Benzene and ionizing radiation were the most common agents recorded. While no estimate can be made of the incidence of chemical or radiation induced disease at the present time, the data suggests that routine documentation of exposure at work is an important step in allowing more specifically focused studies.
Granulocyte colony‐stimulating factor down‐regulates the surface expression of the human leucocyte adhesion molecule‐1 on human neutrophils in vitro and in vivo
Authors: Ohsaka Akimichi , Saionji Katsu , Sato Naotake , Mori Takeshi , Ishimoto Koichi , Inamatsu Takashi
Published: March 2008

Summary. The leucocyte adhesion molecule‐1 (LAM‐1) is the human homologue of the murine peripheral lymph node homing receptor, MEL‐14, and might play a crucial role in neutrophil localization at inflammatory sites. We have reported previously that recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) stimulates or enhances several neutrophil functions in vivo, as well as in vitro. To further explore the possible role of G‐CSF in inflammation we studied the effect of rhG‐CSF on the surface expression of LAM‐1 on human neutrophils, both in vitro and in vivo. The expression of LAM‐1 by human neutrophils was investigated by indirect immunofluorescence using flow cytometry and monoclonal antibodies anti‐Leu‐8 and TQ1. A whole blood analysis was performed to minimize in vitro manipulation. Most circulating human neutrophils expressed LAM‐1 on the cell surface. Brief exposure of neutrophils to rhG‐CSF in vitro decreased the surface expression of LAM‐1. rhG‐CSF down‐regulated neutrophil LAM‐1 expression in a time‐ and dose‐dependent manner. Neutrophils from healthy volunteers and from patients who were receiving rhG‐CSF exhibited a decreased expression of LAM‐1 after rhG‐CSF administration, and the expression thereafter returned or overshot the pretreatment level after stopping rhG‐CSF administration. These findings indicate that rhG‐CSF down‐regulates the surface expression of LAM‐1 on human neutrophils in vivo, as well as in vitro, and G‐CSF might participate in neutrophil‐endothelial cell interaction in inflamed tissue.
Evaluation of late cardiotoxicity with pulsed Doppler echocardiography in patients treated for Hodgkin's disease
Authors: Kreuser Ernst‐Dietrich , Völler Heinz , Behles Christian , Schröder Klaus , Uhrig Alexander , Besserer Angela , Thiel Eckhard
Published: March 2008

Summary. The impact of valvular, myocardial and pericardial abnormalities on cardiac haemodynamics in patients treated for Hodgkin's disease with COPP/ABVD with and without mediastinal irradiation was determined in 49 patients 2–10 years after induction therapy. Diagnostic procedures to evaluate cardiac function consisted of history, physical examination, exercise bicycle stress test, M‐mode two‐dimensional and pulsed Doppler echocardiography. No patient reported symptoms related to cardiomyopathy, and only one of the 49 had evidence of coronary heart disease. Pericardial thickening was seen on echocardiograms in 19/49 patients (38.8%), valvular thickening in 21/49 (42·9%), and reduced fractional shortening in 9/49 (18·4%). The Doppler‐derived mean E and A (±SD) of transmitral flow were 0·75 ± 0·14 m/s and 0·56 ± 0·09 m/s, respectively, in patients receiving chemotherapy and 0·81 ± 0.19 m/s and 0·63 ± 0·20 m/s in those with additional mediastinal irradiation. There was no statistically significant difference between mean E and A in transmitral flow in patients treated for Hodgkin's disease and control subjects. Furthermore, the transtricuspid and hepatic vein flow velocities did not differ significantly. Although the present study demonstrates high frequencies of pericardial and valvular thickening in patients treated for Hodgkin's disease with the COPP/ABVD regimen with or without mediastinal irradiation, it showed no impact on cardiac flow velocities. The abnormalities might thus be of minor clinical relevance in these patients.
Acute myeloid leukaemia blast cells bind to human endothelium in vitro utilizing E‐selectin and vascular cell adhesion molecule‐1 (VCAM‐1)
Authors: Cavenagh J. D. , Gordon‐Smith E. C. , Gibson F. M. , Gordon M. Y.
Published: March 2008

Summary The adhesion of acute myeloid leukaemia (AML) blast cells to human umbilical vein endothelial cells (HUVECs) was investigated in vitro. Adhesion of blast cells from 10 cases of AML to unstimulated and interleukin‐1β (IL‐1) stimulated HUVECs was similar to or greater than that of control neutrophils. The extent to which endothelial E‐selectin and vascular cell adhesion molecule‐1 (VCAM‐1) were involved in this adhesive process was investigated using blocking monoclonal antibodies to these proteins. In the majority of cases studied (7/8), anti‐E‐selectin significantly inhibited adhesion to IL‐1 stimulated endothelium (26–65% inhibition) and in 5/8 cases so did anti‐VCAM‐1 (maximum of 31% inhibition). All cases were found to express the sialylated Lewis x antigen and very late activation antigen‐4, ligands for E‐selectin and VCAM‐1 respectively. Our results indicate that leukaemic blast cells adhere to human endothelium and that there are E‐selectin and, to a lesser extent. VCAM‐1‐dependent components to this process. Such adhesive interactions are likely to confer on AML blast cells the ability to migrate across the vascular wall and so to establish extravascular disease.
Antibodies against terminal galactosyl α(1–3) galactose epitopes in patients with idiopathic myelofibrosis
Authors: Leoni Pietko , Rupolj Serena , Salvi Aldo , Sambo Paola , Cinciripini Adriana , Gabrielli Armando
Published: March 2008

Summary Sera from patients with myelofibrosis were analysed for circulating antibodies against an antigenic determinant characterized by two molecules of galactose in α 1–3 linkage (anti‐Gal antibodies).

500% of patients were found to have values above the 90th percentile of the values of control sera chosen as a cut‐off. The median level of the antibodies was significantly higher than the value detected in normal controls, but no difference coulti be found between patients with idiopathic myelofibrosis and those with myelofibrosis associated to a chronic myeloproliferative disorder.

Anti‐Gal antibodies were found to correlate with disease activity and with platelet count whereas no correlation was detected with other haematological parameters.

Furthermore, for evaluation of disease activity, determination of serum anti‐Gal antibodies was a sensitive and specific parameter.

We conclude that humoral immunity against Galα1–3Gal may provide a sensitive tool to detect disease activity in patients with idiopathic myelofibrosis and may be important for understanding its pathogenesis.
Leukaemia arising in donor cells following allogeneic bone marrow transplantation for β thalassaemia demonstrated by immunological, DNA and molecular cytogenetic analysis
Authors: Katz Fay , Reeves B. R. , Alexander Shirley , Kearney Lyndal , Chessells Judith
Published: March 2008

Summary Combined DNA analysis, molecular cytogenetic and immunological techniques have been used to identify the donor origin of common acute lymphoblastic leukaemia (cALL) which arose in a male patient with β thalassaemia major, 5 years after an allogeneic bone marrow transplant from his HLA‐matched sister. The necessity of using multiple techniques in this and similar cases is emphasized and the possible mechanisms for the development of donor leukaemia and the leukaemic transformation of donor cells are discussed.
The inter‐relationship of factor VII and its activity state with plasma lipids in healthy male adults
Authors: Wright D. , Poller L. , Thomson J. M. , Gowland E. , Burrows G. E.
Published: March 2008

Summary A close inter‐relationship between raised factor VII clotting activity and elevated blood lipids, particularly serum triglycerides, is well established. A study of factor VII, its activation state and of plasma lipids has been undertaken in two groups of healthy middle‐aged males to elucidate this mechanism. A control group with normal factor VII levels were closely matched for age and body‐mass index with a second group with elevated levels. Factor VII assays, using rabbit and bovine thromboplastin and a factor VII Ag method, were employed. Triglycerides correlated with the rabbit factor VII thromboplastin assay and factor VII Ag (P<0.05) but not with the bovine thromboplastin method. Higher HDL‐cholesterol and apolipoprotein A‐I levels were found in subjects with increased factor VII (P<0.001) and appeared to be due to differences in alcohol consumption. Cholesterol levels were significantly higher with elevated factor VII. Differential testing suggests that higher factor VII is predominantly mediated through a rise in total VII, rather than an increase in its activity state.
Diagnosis of paroxysmal nocturnal haemoglobinuria by phenotypLc analysis of erythrocytes using two‐colour flow cytometry with monoclonal antibodies to DAF and CD59/MACIF
Authors: Shichishima Tsutomu , Terasawa Takashi , Saitoh Yurie , Hashimoto Chokichi , Ohto Hitoshi , Maruyama Yukio
Published: March 2008

Summary We investigated the relationship between the complement lysis sensitivity test and two‐colour flow cyto‐metric analysis using monoclonal antibodies to decay accelerating factor (DAF) and CD59/membrane attack complex inhibitory factor (MACIF) in patients with paroxysmal nocturnal haemoglobinuria (PNH) and other haematological diseases.

Flow cytometry showed that all 59 PNH patients had two or three erythrocyte populations, while all 74 patients with other haematological diseases and all 31 healthy volunteers had a single erythrocyte population. We compared the percentage of PNH III erythrocytes in the lysis test with the percentage of negative cells shown by flow cytometry in 52 PNH patients, and found a significant correlation (r=0–960, P<0.001). However, in 13 patients the erythrocyte pheno‐types did not correspond in both tests. This was generally related to difficulty of detecting PNH II erythrocytes in the lysis test.

In the PNH patients the ranges of mean fluorescence intensity for the negative, intermediate and positive erythrocyte populations were respectively 1.1–2.5. 2.2–29, and 61–600 for CD59IMACIF positivity and 1.9–7.2, 3.6–22, and 31–350 for DAF positivity. In contrast, the mean intensities in healthy volunteers ranged from 190 to 720 for CD59/ MACIF and from 150 to 350 for DAF.

These findings suggest that PNH can be diagnosed and phenotypic analysis of PNH erythrocytes can be performed by respectively assessing the fluorescence profiles and mean fluorescence intensities of both proteins using flow cytometry. Flow cytometry may provide a superior diagnostic method to the traditional tests for PNH.
Hairy cell leukaemia: descriptive epidemiology and a case‐control study
Authors: Staines A. , Cartwright R. A.
Published: March 2008

Summary The descriptive epidemiology of hairy cell leukaemia is reported from a specialized register of haematological malignancies covering approximately one‐third of England and Wales. The overall incidence of hairy cell leukaemia at 2.9 per million persons per year is similar to that recorded in America. There is a marked male preponderance (4.0 compared with 1.7 per million per year for females).

A case‐control study on hairy cell leukaemia was conducted in the Yorkshire and Trent Regional Health Authority areas. 50 cases and 95 controls were identified, and interviewed using a structured questionnaire. Previous results on the aetiology of hairy cell leukaemia were not supported by this study, with the exception of an association between hairy cell leukaemia and exposure to organic solvents, petrochemicals and related products.
Factor XIII ABristol 1: detection of a nonsense mutation (Arg171→+stop codon) in factor XIII A subunit deficiency
Authors: Standen G. R. , Bowen D. J.
Published: March 2008

Summary Molecular analysis has been performed on a patient with coagulation factor XIII A subunit deficiency. A previously published genomic sequence indicates that exon 3 of the factor XIII A subunit gene contains two TaqI restriction sites within which arginine (CGA)→stop (TGA) nonsense mutations are possible. Oligonucleotide primers were therefore used to amplify exon 4 by the polymerase chain reaction. TaqI digestion of the 326 base pair (bp) product derived from normal genomic DNA yielded expected fragments of 244, 73 and 9 bp in size. In the case of the patient, however, an additional fragment of 253 bp was present. Direct sequence analysis showed that the 5′ TaqI site had been lost from one allele by a C→T transition at nucleotide 598. Family studies demonstrated the mutation in the patient's father but no other first‐degree relatives. This is the third independent mutation described in the factor XIII A subunit gene and the first to be identified in a patient compound heterozygous for the disorder.