bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular defects in CRM+ factor VII deficiencies: modelling of missense mutations in the catalytic domain of FVII
Authors: Bernardi F. , Liney D. L. , Patracchini P. , Gemmati D. , Legnani C. , Arcieri P. , Pinotti M. , Redaelli R. , Ballerini G. , Pemberton S. , Wacey A. I. , Mariani G. , Tuddenham E. G. D. , Marchetti G.
Published: March 2008

Summary. The molecular defects causing CRM+ factor VII deficiency were investigated in seven unrelated subjects and several members of their families.

Four missense mutations located in the catalytic domain of factor VII were found. The previously reported 304ArgGln substitution was present in the homozygous and heterozygous forms, with different polymorphic haplotypes, thus demonstrating that it is recurrent and frequent in the Italian population. The 310Cys Phe substitution was found in the homozygous form and in the compound heterozygous condition with the nonsense mutation 356Trpstop. Two missense mutations, 298MetIle and 342GlyArg, were found in the homozygous and in the heterozygous condition respectively.

Molecular heterogeneity was further increased by finding of the 353ArgGln polymorphism in the doubly heterozygous condition with the 304 and 342 mutations.

Plausible explanations for loss of FVII function were found by inspecting a model of the serine protease domain of factor VIIa. Inefficient activation of the catalytic site is predicted for 298MetIle. 342GlyArg would directly distort the geometry of the ‘oxyanion hole’preventing formation of a substrate enzyme intermediate. 310Cyshe is predicted to have an adverse effect on tissue factor interaction. These mutations point to important regions of the factor VII molecule.
α‐Interferon in myelofibrosis: a case report
Authors: Dalla K. P. , Zeigler Z. R. , Shadduck R. K.
Published: March 2008

Summary. A patient with advanced myeloid metaplasia was treated with α‐interferon (29 months) with a remarkable response. He had anaemia, thrombocytopenia and hepatosplenomegaly with infarction. The initial bone marrow showed replacement with fibrosis with no evident haemopoietic cells. Post‐therapy, the patient became asymptomatic, transfusion independent and had normal blood counts. The repeat bone marrow was 30% cellular with 1 + reticulin and no fibrosis. Treatment was well tolerated without appreciable side‐effects. These results indicate that prolonged therapy with α‐interferon can improve haemopoiesis and reverse marrow fibrosis.
Aggressive natural killer cell leukaemia/lymphoma in two patients with lethal midline granuloma
Authors: Soler Jesús , Bordes Ramon , Ortouno Francisco , Montagud Mario , Martorell Jaume , Pons Cristina , Nomdedeu Josep , Lopez‐Lopez Juan Jose , Prat Jaime , Rutllant Miquel
Published: March 2008

Summary. We report two patients with leukaemic proliferations of large granular lymphocytes. The immunophenotype study showed that the leukaemic cells were positive for CD2, CD38, CD56 and anti‐HLA‐DR monoclonal antibodies and negative for other T‐cell (CD3, CD4, CD8) and B‐cell markers (CD19, CD20 and surface immunoglobulins). The clinical course was acute and a diagnosis of aggressive natural killer cell leukaemia/lymphoma was made. No clonal rearrangements of either Cβ T‐cell receptor or JH immunoglobulin genes were found. Functional studies done in one patient demonstrated non‐restricted cytotoxic activity after activation with IL‐2. Lethal midline granuloma had been previously diagnosed in both patients. A possible relationship between this entity and the natural killer cell leukaemia is discussed.
Marked and reproducible increase in trilineage blood cell counts by administration of granulocyte colony‐stimulating factor in a patient with refractory anaemia with excess blasts in transformation
Authors: Chiba Shigeru , Inamori Ken , Mitani Kinuko , Hirai Hisamaru , Yazaki Yoshio
Published: March 2008

Summary. We report a patient with refractory anaemia with excess blasts in transformation (RAEB‐t) who presented with severe pancytopenia and received four intermittent series of granulocyte colony‐stimulating factor (G‐CSF) treatment over 1·5 years. In addition to the increase in mature neutrophils, platelet count and haemoglobin level were dramatically increased. These haematological improvements were dependent on G‐CSF during these treatment series. Bone marrow colony assay revealed that G‐CSF increased both CFU‐E‐ and BFU‐E‐derived colonies in vitro.

Clinical usage of G‐CSF in myelodysplastic syndrome (MDS) is discussed, with particular emphasis on mechanisms of trilineage response.
Recombinant interferon‐γ inhibits the in vitro proliferation of human myeloma cells
Authors: Palumbo Antonio , Battaglio Silvano , Napoli Patrizia , Omedè Paola , Fusaro Antonella , Bruno Benedetto , Boccadoro Mario , Pileri Alessandro
Published: March 2008

Summary. Interferon‐α (IFN‐α), interferon‐γ (IFN‐γ) and dexamethasone (DEX) have shown anti‐tumour effects in multiple myeloma (MM) cells. Bone marrow plasma cells from 39 MM patients were cultured to clarify the intensity and specific activity of each compound on bromo‐deoxyuridine (BrdUrd) uptake and immunoglobulin (Ig) secretion. BrdUrd uptake was inhibited by recombinant human IFN‐γ (100 U/ml) and by DEX (10−6m). The stimulation index (StI), i.e. labelling index (LI) of treated samples/controls, was 0·49 0·009 (mean standard error of the mean, MSEM), P=0·0003, and 0·52·0·07 (MSEM), P<0·0001, respectively. Ig secretion was reduced by IFN‐α (100 U/ml) and DEX. The secretion index (SI), i.e. Ig quantitation of treated samples/controls, was 0·04 (MSEM), P<0·0001, and 0·52·0·04 (MSEM), P<0·0001, respectively. Finally, IFN‐γ inhibits BrdUrd uptake only and IFN‐α secretion only. In 18 patients the simultaneous addition of IFN‐α plus IFN‐γ mainly paralleled the effect of IFN‐γ on BrdUrd uptake and IFN‐α on secretion, but did not result in any additive or synergistic effect, though both BrdUrd uptake and Ig secretion were decreased to about the same extent as with DEX. These data indicate that the combination of IFN‐α plus IFN‐γ and DEX are the strongest inhibitors of both BrdUrd uptake and secretion. Since IFN‐α and IFN‐γ appear to have a different mechanism of action, their combined use could be considered as a possible new treatment strategy.
Effects of ceftazidime, a betalactam antibiotic, on murine haemopoiesis in vitro
Authors: Hauser Simon P. , Udupa Kodetthoor B. , Lipschitz David A.
Published: March 2008

Summary. Agranulocytosis has been reported in 5–15% of patients treated with high‐dose betalactam antibiotics (BLA). We investigated the toxic effect of ceftazidime (CEF) as a representative of these antibiotics on colony‐forming unit‐granulocyte/macrophage (CFU‐GM), on burst‐forming unit‐erythroid (BFU‐E) colony growth and on myelopoiesis in murine long‐term bone marrow culture (mLTBMC). The CEF concentration resulting in a 50% inhibition of growth was 146 μg/ml (267 μm) for CFU‐GM, 132 μg/ml (241 μm) for BFU‐E and 180 μg/ml (329 μm) for myeloid cell production in the supernatant of mLTBMC. Following addition of CEF to mLTBMC, CFU‐GM remained low for 1 week and total myeloid cell production remained low for 2 weeks after removal of CEF from culture. Thereafter the values returned to control levels. The myeloid differential counts in the supernatant and adherent layers demonstrated a ‘maturation arrest’, which could be overcome by simultaneously adding all‐trans retinoic acid to culture. These results demonstrate that CEF has reversible inhibitory effects on myelopoiesis and highlight the utility of in vitro haemopoietic assays as models to examine drug‐induced haemopoietic dyscrasias.
Identification of a subgroup of myelodysplastic patients with a neutrophil stimulation‐signalling defect
Authors: Lowe G. M. , Dang Y. , Watson F. , Edwards S. W. , Galvani D. W.
Published: March 2008

Summary. f‐Met‐Leu‐Phe‐stimulated luminol‐enhanced chemiluminescence was found to be repeatedly defective in some MDS patients. This defect was not attributed to myeloperoxidase deficiency, nor to a defect in NADPH oxidase function, because PMA chemiluminescence was found to be normal in these individuals. An arbitrary value of 7 mV (half the mean control value) was chosen to subdivide the group: MDS patients with values <7 mV had a mean f‐Met‐Leu‐Phe chemiluminescence response of 2·5±0·5 compared to MDS patients with values <7 mV who had a mean response of 15·6±1·6mV, P<0·01 (healthy controls 14±2 mV). The characteristics of the f‐Met‐Leu‐Phe receptor and initial calcium flux results suggested that the receptor itself was normal in number and function in low f‐Met‐Leu‐Phe responders. The rate of superoxide generation, which is calcium‐dependent, was also found to be in the normal range in low f‐Met‐Leu‐Phe responders, although total superoxide production was reduced in some of these patients.

When MDS neutrophils with a low f‐Met‐Leu‐Phe response were stimulated with PMA, chemiluminescence was normal, suggesting normal activity of the NADPH‐oxidase complex. Furthermore, myeloperoxidase activity was reduced in only three out of the 11 low f‐Met‐Leu‐Phe responders.

Following priming with GM‐CSF, f‐Met‐Leu‐Phe chemiluminescence was 27·1±6 mV in low f‐Met‐Leu‐Phe responders compared to controls (87·7±11 mV, P<0·005). Thus, although responses were improved, they were not as marked as in control neutrophils.

These data suggest that a subgroup of MDS patients have a low f‐Met‐Leu‐Phe chemiluminescence response which is not due to a defect in the f‐Met‐Leu‐Phe receptor or oxidase activity, and in the majority of cases MPO activity is normal. Initial patient survival data suggest that these patients may have an increased risk of infective mortality. It is proposed that defective f‐Met‐Leu‐Phe chemiluminescence results from a putative defect in cell‐signalling mechanism upstream of PKC, and GM‐CSF priming only partially improves responsiveness.
Point mutations of the N‐ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia
Authors: Vasioukhin Valeri , Anker Philippe , Maurice Pierre , Lyautey Jacqueline , Lederrey Christine , Stroun Maurice
Published: March 2008

Summary. Oncogene mutations are frequently found in several tumour types and, among these, point mutations of the ras gene are particularly significant. A predominance of N‐ras mutations has been found in the bone marrow DNA of patients with myelodysplatic syndrome (MDS) or acute myelogenous leukaemia (AML). On the other hand, increased levels of plasma DNA have previously been observed in patients suffering from various malignant diseases. In the present work we have investigated, by polymerase chain reaction (PCR), point mutations of the N‐ras gene in the DNA of plasma, blood cells and bone marrow of 10 patients suffering from AML or MDS. The different ras mutations detected in five cases were always present in the plasma DNA while sometimes absent in the DNA of peripheral blood cells or bone marrow. This indicates that a bone marrow biopsy or aspiration does not necessarily contain all the malignant clones involved in the disease. Plasma could thus prove to be an easily accessible and useful material for detection and monitoring of myeloid disorders.
Trisomy 8q due to i(8q) or der(8) t(8;8) is a frequent lesion in T‐prolymphocytic leukaemia: four new cases and a review of the literature
Authors: Mossafa Hossain , Brizard André , Huret Jean‐Loup , Brizard Françoise , Lessard Michel , Guilhot François , Tanzer Joseph
Published: March 2008

Summary.Cytogenetic abnormalities found in four cases of T‐cell prolymphocytic leukaemia (T‐PLL) are described. An isochromosome 8q was found in three patients and a t(8;8) in one. In the four cases, karyotypes were complex and showed a high degree of instability. In addition, we reviewed 27 published cases of cytogenetically studied T‐PLL. On the whole, the most frequently recurring anomalies in T‐PLL are 14q lesions with nonrandom breakpoints, inversion (14)(q11q32) or tandem translocations (14;14) (not seen in any of our cases) and trisomy for 8q, mainly due to i(8q), found in more than 40% of patients each.

Similar structural anomalies were found almost as frequently among the 23 cytogenetically studied cases of so‐called T‐chronic lymphocytic leukaemia (T‐CLL) reported prior to 1989. It is now accepted that the T‐cell counterpart of B‐CLL either does not exist or is exceedingly rare and thus previously reported cases of T‐CLL sharing the chromosomal characteristics of T‐PLL may well have been misdiagnosed examples of T‐PLL.

Isochromosomes 8q are exceptionally found in other types of haematological malignancies. However, i(8q) could not be shown to be the primary lesion in any case in T‐PLL and the role of trisomy for 8q, as well of the associated monosomy 8p, is entirely unknown.
Haemophilia A diagnosis by simultaneous analysis of two variable dinucleotide tandem repeats within the factor VIII gene
Authors: Michel R. , Lalloz A. , Schwaab Rainer , McVey John H. , Michaelides Katerina , Tuddenham Edward G. D.
Published: March 2008

Summary. Haemophilia A is a bleeding disorder caused by defects in the gene coding for the co‐factor, factor VIII (FVIII). The few available intragenic restriction fragment length polymorphisms (RFLPs) currently used in carrier detection and prenatal diagnosis of haemophilia A are informative in only about 65% of cases. We earlier reported a multi‐allelic dinucleotide tandem repeat, (CA)n, specific to intron 13, which remains the single most informative marker within the FVIII gene. We here report a second informative dinucleotide repeat of the form (GT)n (AG)n, located to intron 22 of the FVIII gene. The polymerase chain reaction (PCR) method was used to examine the variability of the repeat in 60 individuals (75 X‐chromosomes) and revealed four alleles. The calculated heterozygosity rate is 45%, and family studies showed X‐linked mendelian inheritance. The intron 22 dinucleotide repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. We now show that by simultaneous amplification of the intron 13 and 22 repeats using PCR all alleles for both markers are detectable on a single polyacrylamide gel. The information thus obtained from a single multiplexed analysis is greater than from multiple RFLP analyses. Hence, rapid haplotype determination by simultaneous amplification and detection of two intragenic dinucleotide repeats should supersede less informative RFLP analysis.
The transport of two iron chelators, desferrioxamine B and L1, across Caco‐2 monolayers
Authors: Hamilton Karen O. , Stallibrass Lorraine , Hassan Ian , Jin Yi , Halleux Christine , Mackay Martin
Published: March 2008

Summary. The transport of two iron chelators, desferrioxamine B (DFO) and L1 (1,2‐dimethyl‐3 hydroxypyridin‐4‐one) has been studied in vitro using the human adenocarcinoma cell line, Caco‐2. The transport of DFO and L1 has also been compared with that of their iron‐bound complexes, ferrioxamine (FO) and L13‐Fe, respectively. We report an apparent permeability coefficient (Papp) value for DFO of 0.170 × 10−7± 0.080 cm s−1. The Papp value of L1 was 1.297 × 10−5± 0.133 cm s−1. The Papp values of their iron bound complexes FO and L13‐Fe are 0.230 × 10−7± 0.065 cm s−1 and 2.356 × 10−6 0.365 cm s−1, respectively. We have shown that the transport of DFO and FO is similar in the Caco‐2 cell system. The transport of L1, however, is greatly reduced when complexed to iron. The value for total uptake after 60 min for DFO into the Caco‐2 cells was 1.49 ± 0.09 × 10−3 nmol per filter. The values for total uptake after 60 min for L1 and L13‐Fe were 0.37 0.03 nmol per filter and 0.04 ± 0.01 nmol per filter, respectively. Our results indicate that the poor oral bioavailability of DFO can be attributed to the low epithelial permeability of the molecule coupled with its size (mol wt 656). In contrast, the oral bioavailability observed with L1 is due to the high lipophilicity and low molecular weight (mol wt 139) of the molecule. We believe that these differences between the two molecules account for L1 being better orally absorbed than DFO.
Granulocyte colony‐stimulating factor inhibits the endogenous leukotriene production in tumour patients
Authors: Denzlinger C. , Holler E. , Reisbach G. , Hiller E. , Wilmanns W.
Published: March 2008

Summary. Granulocyte colony‐stimulating factor (G‐CSF) is virtually devoid of inflammatory side‐effects when given to patients in therapeutic doses. This is in contrast to other haemopoietic cytokines such as granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or interleukin‐3 (IL‐3) which may promote inflammatory reactions by increasing the number and/or activity of monocytes, eosinophils, mast cells and basophils. Inflammatory reactions to GM‐CSF and IL‐3 appear to be related to an increased formation of leukotrienes, known as potent mediators of allergy and inflammation. Here we report that, in contrast to GM‐CSF or IL‐3, G‐CSF has the potential to inhibit the leukotriene production in vivo. G‐CSF may thus act as an anti‐inflammatory agent. The differential effects of G‐CSF and other haemopoietins on endogenous leukotrienes may be of major clinical significance.
SLEx expression of normal CD 34 positive bone marrow haemopoietic progenitor cells
Authors: Karakantza Marina , Gibson Frances M. , Cavenagh James D. , Ball Sarah E. , Gordon Myrtle Y. , Gordon‐Smith Edward C.
Published: March 2008

Summary. We investigated sialylated Lewis x (sLex) antigen expression on CD 34 positive (CD 34+) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD 34+ bone marrow progenitors strongly expressed the sLex antigen. This contradicts previous publications which reported sLex expression on malignant blast cells but not on normal CD 34+ progenitor cells.
Trisomy 12 in B‐cell chronic lymphocytic leukaemia: assessment of lineage restriction by simultaneous analysis of immunophenotype and genotype in interphase cells by fluorescence in situ hybridization
Authors: Garcíga‐Marco J. , Matutes E. , Morilla R. , Ellis J. , Oscier D. , Fantes J. , Catovsky D. , Price Cathy M.
Published: March 2008

Summary. We have studied the lineage restriction of trisomy 12 in six patients with B‐cell chronic lymphocytic leukaemia (CLL) by simultaneous analysis of immunophenotype and fluorescence in situ hybridization (FISH) signals in single interphase cells. Fresh uncultured cells from each patient were immunophenotyped by the alkaline phosphatase anti‐alkaline phosphatase method (APAAP) using monoclonal or polyclonal antibodies and hybridized with a chromosome 12 specific alpha‐satellite DNA probe. In all cases trisomy 12 was restricted to the clonal B‐cells, kappa positive or lambda positive, whereas T‐cells (CD3 positive) and non clonal B‐cells had only two chromosome 12 signals. Within the clonal B‐cell population a large proportion of cells were disomic for chromosome 12, whilst trisomic cells ranged from 21% to 37%. The absence of trisomy 12 in T‐cells and the mosaicism demonstrated in the clonal B‐cells suggests that this abnormality is a secondary event during the leukaemic transformation of CLL and develops in an already established neoplastic B‐cell population.
Prevention of primary transfusion‐associated cytomegalovirus infection in bone marrow transplant recipients by the removal of white cells from blood components with high‐affinity filters
Authors: Prooijen H. C. van , Visser J. J. , Oostendorp W. R. van , Gast G. C. de , Verdonck L. F.
Published: March 2008

Summary. A prospective study was carried out to determine whether use of cytomegalovirus (CMV) unscreened red blood cells and platelet concentrates, white blood cell (WBC) depleted with high‐efficiency filters, would prevent transfusion‐associated (TA) CMV infection in CMV seronegative bone marrow transplant recipients. Blood components were filtered in the bloodcentre under quality control and after filtration residual WBC counts were always below 5 × 106 cells/U. Since 1990, 23 consecutive allogeneic and 37 autologous CMV seronegative marrow transplant recipients, have been transfused with filtered blood components and followed for 6 months for evidence of CMV infection by monitoring culture and CMV serology. None of the patients showed clinical symptoms of CMV infection, and CMV cultures during episodes of fever were always negative. IgM anti‐CMV antibodies were negative during the study in all patients. Low titres of IgG anti‐CMV antibodies (5‐12 relative ELISA units) were found in 24/60 patients during the first month after bone marrow transplantation (BMT), probably due to passive transfer of IgG administered with the platelet transfusions. 3 and 6 months after BMT, 56 and 48 patients respectively were still alive; and CMV serology was negative in all patients. The results show that TA‐CMV infection is preventable by filtration of blood through high‐efficiency filters in patients undergoing autologous and allogeneic BMT.
Substantial expression of glycoproteins IX and V on the platelet surface from a patient with Bernard‐Soulier syndrome
Authors: Arai Morio , Yamamoto Naomasa , Akamatsu Noriko , Suzuki Hidenori , Yamaguchi Atsumi , Nishida Yasuharu , Fukutake Katsuyuki , Tanoue Kenjiro
Published: March 2008

Summary. Flow cytometric studies demonstrated that the platelets from a patient with Bernard‐Soulier syndrome expressed substantial amounts of both GPIX and GPV, although in reduced amounts.
Rapid onset of chronic granulocytic leukaemia after treatment for Hodgkin's disease
Authors: Jackson G. H. , Latham J. A. , Lennard A. L. , Taylor P. R. A. , Proctor S. J.
Published: March 2008

Summary. A 17‐year‐old white female presented with stage IVB Hodgkin's disease. After chemotherapy and radiotherapy she achieved a complete clinical remission and underwent an autologous bone marrow transplant (ABMT). 22 months later she developed chronic granulocytic leukaemia (CGL).

Polymerase chain reaction (PCR) analysis of bone marrow harvested at the time of ABMT did not show any evidence of the bcr‐abl sequence that was detectable at the diagnosis of CGL. This case provides further information on the kinetics of the development of CGL and adds to the small pool of data on CGL developing after treatment for Hodgkin's disease.
Establishment and characterization of a novel myeloid cell line from the bone marrow of a patient with the myelodysplastic syndrome
Authors: Tohyama Kaoru , Tsutani Hiroshi , Ueda Takanori , Nakamura Toru , Yoshida Yataro
Published: March 2008

SUMMARY. A novel long‐term cultured myeloid cell line was established from the bone marrow of a patient with myelodysplastic syndrome (MDS). This cell line, designated MDS92. proliferated in the presence of interleukin‐3 or granulocyte‐macrophage colony‐stimulating factor and transiently in the presence of Steel factor, with a tendency for gradual maturation, and formed myeloid colonies in the semi‐solid culture condition.

In addition, the MDS92 cell line represented rather complicated karyotypic abnormalities including the deletion of fifth and seventh chromosomes and a point mutation at codon 12 of the N‐ras oncogene.

These characteristics of the MDS92 cell line are exclusively compatible with the property of preleukaemia.
Involvement of interferon‐γ and macrophage colony‐stimulating factor in pathogenesis of haemophagocytic lymphohistiocytosis in adults
Authors: Akashi Koichi , Hayashi Shin , Gondo Hisashi , Mizuno Shin‐ichi , Harada Mine , Tamura Kazuo , Yamasaki Kazuo , Shibuya Tsunefumi , Uike Naokuni , Okamura Takashi , Miyamoto Toshihiro , Niho Yoshiyuki
Published: March 2008

Summary We investigated the role of monocyte/macrophage‐activating cytokines in pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in 21 adult patients. Sera from patients with active HLH contained extremely high levels of macrophage colony‐stimulating factor (M‐CSF) and of interferon‐γ (IFN‐γ). These levels returned to almost normal during remission. Neither interleukin‐4 nor granulocyte/macrophage colony‐stimulating factor could be detected. Active HLH sera also contained high concentrations of inflammatory monokines, such as interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). Serum concentrations of soluble CD8 and soluble interleukin‐2 receptor were extremely high during active HLH, and returned to virtually normal levels during remission. Circulating CD2+ T‐cells obtained from patients with active HLH spontaneously secreted M‐CSF and IFN‐γin vitro, whereas circulating monocytes did not produce detectable levels of both M‐CSF and IFN‐γ, but produced high levels of IL‐6 and TNF‐α. These findings suggest that IFN‐γ and M‐CSF at least partly from T‐cells, such as CD8+ T‐cells, might contribute to activation of monocytes or histiocytes, resulting in the up‐regulated monokine production and haemophago‐cytosis in HLH.
Analysis of cytoplasmic and surface antigens in childhood T‐cell acute lymphoblastic leukaemias: clinical relevance of cytoplasmic TCRβ chain expression
Authors: Inukai Takeshi , Saito Midori , Mori Taijiro , Nishino Kazuyoshi , Abe Takashi , Kinoshita Akitoshi , Suzuki Toshio , Kurosawa Yoshihiro , Okazaki Toshiko , Sugita Kanji , Nakazawa Shinpei
Published: March 2008

SUMMARY. Expression of surface and cytoplasmic antigens on the blasts from 42 cases of childhood T‐cell acute lymphoblastic leukaemia (T‐ALL) were analysed. All with childhood T‐ALL, except for one case expressing cytoplasmic TCR δ chain, were classified on the basis of differential expression of cytoplasmic CD3 (cCD3), TCRβ chain (cTCR β) and surface CD3 (sCD3) into the following three groups: group I (cCD3+, cTCRβ‐, sCD3‐), eight cases (19.5%); group II (cCD3+, cTCRβ+, sCD3‐), 23 cases (56.1%); group III (cCD3+, cTCRβ+, sCD3+), 10 cases (24.4%). Each group defines the stepwise maturational stage of the CD3/TCR complex along the intrathymic T‐cell differentiation. Group I had the lowest initial WBC count among the three groups (P < 0.05) and showed significantly (P < 0.05) a higher event‐free survival (0.75) than those of group II (0.33). There was no significant difference in both the initial WBC count and the event‐free survival between groups II and III. Thus, the absence of cTCRβ in sCD3‐negative T‐ALL appears to be a good prognostic factor, suggesting that this classification provides a useful tool to predict the prognosis of childhood T‐ALL. This is the first report, to our knowledge, studying the relationship between the expression of cytoplasmic CD3/TCR antigens and the clinical features in T‐ALL.
PCR assessment of bone marrow status in ‘isolated’extramedullary relapse of childhood B‐precursor acute lymphoblastic leukaemia
Authors: Goulden Nicholas , Langlands Kenneth , Steward Colin , Katz Fay , Potter Michael , Chessells Judith , Oakhill Anthony
Published: March 2008

SUMMARY. Approximately one‐third of first relapses of childhood ALL occur at an extramedullary site without morphological evidence of bone marrow disease. However, the high incidence of subsequent medullary relapse in these cases strongly suggests that leukaemia is present at submicroscopic levels at the time of ‘isolated’relapse. PCR analysis of immunoglobulin heavy chain (IgH) and T‐cell receptor (TCR) gene rearrangements now allows detection of leukaemia at levels as low as 0.001%. We have therefore used this technique to reassess bone marrow status at morphologically isolated relapse in 13 children with B‐lineage ALL (11 with off‐treatment relapses, two on treatment). In 12 of these 13 patients marrow disease was detectable by PCR at the time of this relapse—in all cases at levels below the threshold of light microscopy. Where relapse occurred off‐therapy this indicated re‐emergence of disease, since MRD has never been detected by PCR at this stage in patients remaining in long‐term remission. In both patients who relapsed on‐therapy the level of MRD at the time of relapse represented an increase on that seen in their previous marrow sample. We conclude that re‐emerging bone marrow disease can be detected in most cases of ‘isolated’relapse when investigated by this highly sensitive technique. Our findings at a molecular level confirm a long‐held clinical suspicion and indicate that full systemic re‐induction as well as local therapy is obligatory for these children.
Retroviral mediated gene transfer in chronic myelogenous leukaemia
Authors: Cornetta Kenneth , Moore Ann , Leemhuis Thomas , Moen Robert C. , Tricot Guido , Leibowitz David , Hoffman Ronald
Published: March 2008

SUMMARY. Gene therapy for chronic myelogenous leukaemia (CML) may provide a therapeutic option for patients who are ineligible for bone marrow transplantation. To determine the feasibility of such an approach we evaluated the transduction efficiency of CML progenitor colonies from seven patients in chronic phase. Vector transduction was optimized using the CML‐derived K562 cell line and applied to CML mononuclear cells. After vector exposure, optimal gene transfer was noted when CML mononuclear cell cultures contained stem cell factor, IL‐3, GM‐CSF and erythropoietin. The additon of IL‐6 to this combination decreased transduction efficiency. Using these conditions, 20.4%±2.4 (SE) of erythroid colonies (CFU‐GEMM and BFU‐E) and 20.2%±4.7 of CFU‐GM colonies were G418 resistant. This compares with a transduction efficiency of 5.9%± 1.1 and 6.4%± 1.5, respectively, for erythroid and CFU‐GM colonies using marrow obtained from normal donors. Only a modest increase in gene transfer was noted when CML cells were stimulated with cytokines for the 24 h preceding vector exposure. Vector DNA in colonies expressing the BCR/ABL transcript was documented by performing PCR analysis on individual colonies. The relatively high gene transfer rate in CML suggests that this disease might be very suitable for gene therapy.
Defective natural killer (NK) cell‐mediated cytotoxicity does not imply clonal involvement of NK cells in myelodysplastic syndromes
Authors: Ogata K. , Fuju H. , Yokose N. , An E. , Tamura H. , Kamikubo K. , Dan K. , Hamaguchi H. , Sakamaki H. , Onozawa Y. , Nomura T.
Published: March 2008

SUMMARY. The clonality of purified cells was examined in 10 myelodysplastic syndromes (MDS) patients by analysing the restriction fragment length polymorphism and methylation pattern of the phosphoglycerate‐kinase gene. Natural killer (NK) cell‐mediated cytotoxicity was also examined. The granulocytes and monocytes were monoclonal or oligoclonal in all cases, except for the monocytes in one case. Conversely, the NK and T cells had a polyclonal pattern in most cases, including all cases who had defective NK cellmediated cytotoxicty. The hypothesis that reduced NK cellmediated cytotoxicity in MDS is caused by a clonal involvement of NK cells was not supported by the present study.
Acquired factor VIII inhibitor associated with chronic interferon‐alpha therapy in a patient with haemophilia A
Authors: Castenskiold E. C. , Colvin B. T. , Kelsey S. M.
Published: March 2008

SUMMARY. Both the development of factor VIII inhibitors and infection by hepatitis C virus are serious complications of haemophilia A. We describe the first reported case of the subsequent development of a factor VIII inhibitor in a patient with haemophilia A after treatment with interferon‐alpha for chronic active hepatitis C.
Influence of combined thrombin stimulation, surface activation, and receptor occupancy on organization of GPIb/IX receptors on human platelets
Authors: White James G. , Krumwiede Marlys , Cocking‐Johnson Debra , Escolar Gines
Published: March 2008

Summary. Down‐regulation and clearance of as many as 60–80% of GPIb/IX receptors from exposed surfaces on thrombin‐activated platelets to channels of the open canalicular system (OCS) is considered to be a fundamental mechanism regulating platelet adhesivity in vitro and in vivo. The present study has combined thrombin stimulation in suspension, surface activation on formvar grids, receptor occupancy by von Willebrand factor (vWF) and exposure to anti‐vWF antibody in an effort to demonstrate the removal of GPIb/IX receptors from activated cells. Individually the stimuli failed to cause any change in the frequency of GPIb/ IX receptors. Combined, the stimuli were no more effective than when each was used alone. The only way to cause GPIb/IX to move was to add anti‐vWF to thrombin‐activated platelets allowed to spread on formvar grids and covered with multimers of ristocetin‐activated human or bovine vWF. Translocation of GPIb/IX‐vWF‐anti‐vWF complexes from peripheral margins into caps over cell centres, however, did not clear the peripheral zone of vWF binding capacity. Exposure of capped platelets after fixation to a second incubation with vWF demonstrated as many multimers extending from the central cap to the peripheral margins as were seen on platelets exposed a single time to vWF. Antibodies to GPIb, but not to GPIIb/IIIA, prevented the second labelling by vWF. Down‐regulation or clearance of GPIb/IX, in light of this study, does not appear to be a fundamental mechanism modulating platelet adhesivity.
Dissociation between the phospholipases C and A2 activities in stimulated platelets and their involvement in the arachidonic acid liberation
Authors: Faili Ahmad , Emadi Sharareh , Vargaftig B. Boris , Hatmi Mohamed
Published: March 2008

Summary. In previous work we have demonstrated that platelets depleted from secretory phospholipase A2 (sPLA2) produced similar amounts of thromboxane (Tx)B2 as control platelets upon stimulation by thrombin. However, since depletion of sPLA2 was not total, this sole finding only suggested the non‐involvement of sPLA2 in arachidonic acid release.

In the present study we provide further evidence for the non‐involvement of sPLA2 in arachidonic acid liberation during platelet activation. Thus, rabbit platelets exposed to thrombin secreted sPLA2, released free arachidonic acid and formed TxB2 and inositol phosphates. In contrast, U46619, a stable prostaglandin (PG)H2 analogue, activates phospholipase C (PLC) and induces release of sPLA2 without TXB2 generation nor arachidonic acid liberation. At each concentration tested of both agonists, stimulation of sPLA2 activity paralleled the production of inositol phosphates. These data suggest that sPLA2 is dependent on phosphoinositide hydrolysis and on the release reaction and that it is not involved in the liberation of arachidonic acid from stimulated platelets. In addition, a dissociation was observed between sPLA2 and the enzyme involved in the arachidonic acid mobilization, suggesting that the liberation of this fatty acid from membrane phospholipids was mediated by cytosolic phospholipase A2 (cPLA2). Finally, PLC does not play a major role in arachidonic acid liberation, since U46619, which induced the breakdown of inositol phospholipids, failed to release arachidonic acid. In confirmation, neomycin, which inhibits PLC activity, failed to inhibit ATP, sPLA2 and arachidonic acid release upon stimulation of platelets by fluoroaluminate. These data demonstrate that sPLA2 is not involved in the arachidonic acid release by stimulated platelets and indicate that the activations of PLC, sPLA2 and cPLA2 are independent events.
Factor IX Bm Kiryu: a Val‐313‐to‐Asp substitution in the catalytic domain results in loss of function due to a conformational change of the surface loop: evidence obtained by chimaeric modelling
Authors: Miyata Toshiyuki , Kuze Kuninori , Matsusue Tomokazu , Komooka Hitoshi , Kamiya Kenshu , Umeyama Hideaki , Matsui Akira , Kato Hisao , Yoshioka Akira
Published: March 2008

Summary. Factor IX Kiryu is a naturally occurring mutant of factor IX that has 2.5% coagulant activity, even though normal plasma levels of factor IX antigen are detected. Factor IX Kiryu was purified from a patient's plasma by immunoaffinity chromatography with a calcium‐dependent anti‐factor IX monoclonal antibody column. It was cleaved normally by factor XIa in the presence of Ca2+, yielding a two‐chain factor IXa. However, the resulting factor IXa showed only 1.5% of the normal factor IXa in terms of factor X activation in the presence of factor VIII, phospholipids, and Ca2+, and had 20% of the normal esterase activity for Z‐Arg‐p‐nitrobenzyl ester. Therefore factor IXa Kiryu showed the defect of the catalytic triad or primary substrate binding site as well as defective interaction with factors VIII/X. Single‐strand conformational polymorphism analysis and DNA sequencing of the amplified DNA revealed a missense point mutation, a T‐to‐A substitution at nucleotide number 31 059 of the factor IX Kiryu gene. This mutation resulted in the amino acid substitution of Val‐313 by Asp in the catalytic domain. Restriction enzyme analysis of the amplified DNA showed that the mutation was inherited from the patient's mother. The chimaeric method was employed to construct a model of the serine protease domain of factor IXa, and the resultant model suggested that the Val‐313 to Asp substitution altered the conformation of the substrate‐binding site. These data combined with our previous findings on a Gly‐311‐to‐Glu mutant of factor IX suggest that the loop conformation from Gly‐311 to Arg‐318 is important for the expression of coagulant activity.
Established IL‐2‐dependent double‐negative (CD4‐ CD8‐) TCRαβ/CD3+ ATL cells: induction of CD4 expression
Authors: Yamada Yasuaki , Fujita Masatoshi , Suzuki Haruhiko , Atogami Sunao , Sohda Hisashi , Murata Ken , Tsukasaki Kunihiro , Momita Saburo , Kohno Tomoko , Maeda Takahiro , Joh Tatsuroh , Kamihira Shimeru , Shiku Hiroshi , Tomonaga Masao
Published: March 2008

Summary. We established IL‐2‐dependent T cells from an adult T‐cell leukaemia (ATL) patient whose leukaemic cells changed from CD4 single‐p‐positive in the initial phase to double‐negative (CD4‐ CD8‐) at the time of exacerbation. The cells termed SO‐4 were of ATL cell origin and showed the double‐negative TCRαβ/CD3+ T‐cell phenotype. SO‐4 cells acquired CD4 antigen expression following stimulation with concanavlin A (ConA) or immobilized anti‐CD3 antibody. The induction was inhibited by herbimycin A, an inhibitor of protein tyrosine kinase (PTK) activity. No CD4 mRNA was detectable in unstimulated SO‐4 cells but a 3.0 kb signal specific for CD4 mRNA was detected after stimulation. These findings indicate that SO‐4 cells return to their original phenotype (CD4 single‐positive) by stimulation involving PTK. The results indicate that there is a pathway of phenotypic cycling between CD4 single‐positive and double‐negative T cells.
Aplasia after donor lymphocyte infusion (DLI) for CML in relapse after sex‐mismatched BMT: recovery of donor‐type haemopoiesis predicted by non‐isotopic in situ hybridization (ISH)
Authors: Garicochea B. , Rhee F. van , Spencer A. , Chase A. , Lin F. , Cross N. C. P. , Goldman J. M.
Published: March 2008

Summary. We report a patient with CML in relapse after a sex‐mismatch bone marrow transplant who was treated with donor lymphocyte infusions and developed severe marrow aplasia 3 months later. As cytogenetic analysis at this point was not possible because of the very low number of marrow cells available, we used in situ hybridization (ISH) for sex chromosomes and the BCR/ABL gene together with quantitative PCR to monitor the patient's response. The results suggest that recovering haemopoiesis was derived from donor cells and contributed to the decision not to transfuse donor marrow cells on a second occasion.
Over‐expression of the MDM2 gene is found in some cases of haematological malignancies
Authors: Quesnel Bruno , Preudhomme Claude , Oscier David , Lepelley Pascale , Hooghe Maud Colly , Facon Thierry , Zandecki Marc , Fenaux Pierre
Published: March 2008

Summary. We looked for MDM2 gene amplification and over‐expression by Southern and Northern blot analysis in 135 and 66 cases of haematological malignancies, including ALL, AML, CML in chronic phase, CLL, MDS, PLL, non‐Hodgkin's lymphoma (NHL) and myeloma. No amplification of the gene was found. An over‐expression of MDM2 RNA was seen in 9/66 (14%) patients tested, including 3/9 ALL, 3/24 AML, 2/4 myelomas, 1/1 PLL, but 0/2 CML, 0/2 NHL and 0/21 MDS. None of the patients over‐expressing MDM2 had modifications of P53 gene transcript or p53 mutations. Most of the patients over‐expressing MDM2 gene had poor prognostic features (including ‘unfavourable’ cytogenetic abnormalities), poor response to chemotherapy and short survival, Our finding suggest that over‐expression of MDM2 is seen in a relatively small number of haematological malignancies, and is associated with poor prognosis.
Management of pregnancy when maternal blood has a very high level of fetal haemoglobin
Authors: Kaeda Jaspal S. , Prasad Keshava , Howard Richard J. , Mehta Atul , Vulliamy Thomas , Luzzatto Lucio
Published: March 2008

Summary. Fetal blood normally has a higher oxygen affinity than maternal blood because of the predominance of haemoglobin (Hb) F in the former and of Hb A in the latter; this predominance facilitates the transfer of oxygen from maternal to fetal blood. We report two patients who had exclusively or predominantly Hb F in their blood and were managed differently. When patient 1 became pregnant she had regular exchange blood transfusions in order to reduce her Hb F from 80% to below 50%; patient 2, who had 100% Hb F, was not transfused before, during or after her pregnancy. Each patient delivered a normal healthy baby. We conclude that the differential oxygen affinity produced by the combination of Hb A in the maternal blood and Hb F in the fetal blood is not indispensable to ensure an oxygen supply adequate for normal fetal development and growth.
RNA transcripts of the β‐thalassaemia allele IVS‐2–654 C → T: a small amount of normally processed β‐globin mRNA is still produced from the mutant gene
Authors: Huang Shu‐zhen , Zeng Fan‐yi , Ren Zhao‐rui , Lu Zhi‐hong , Rodgers Griffin P. , Schechter Alan N. , Zeng Yi‐tao
Published: March 2008

Summary. IVS‐2–654 C → T is a common Chines β‐thalassaemia mutation. Previous studies report that this mutation resulted in the formation of an abnormally spliced mRNA and the absence of detectable normal β‐globin mRNA, hence the mutation was considered to cause βd́; ‐ thalassaemia. We recently used the method of PCR amplified cDNA copies of circulating erythroid cell mRNA to analyse the mutant gene transcripts and found that this IVS‐2–654 mutation does not abolish normal RNA processing entirely, but that a significant amount (over 15%) of normally processed β‐globin mRNA is produced. Microglobin chain biosynthetic analysis using the HPLC method showed that β‐globin chain was also present in the blood of patients with IVS‐2–654 C → T mutation. Accordingly, this mutant allele leads to a β+‐thalassaemia. Further, the methodology described in this paper provides a new approach towards the detection of RNA transcripts in β‐thalassaemia and other genetic diseases.
Thrombin binding to platelets and their activation in plasma
Authors: Liu Longbin , Freedman John , Hornstein Adriana , Fenton John W. , Ofosu Frederick A.
Published: March 2008

Summary. The interactions of α‐thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of α‐thrombin to platelets and some of its consequences in platelet‐rich plasma. The binding of α‐thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human α‐thrombin and a monoclonal antibody to GMP‐140, respectively. Dose‐dependent binding of α‐thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10 s after → 1.0 nmα‐thrombin was added to recalcified PRP containing 1 μm recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet‐rich plasma. α‐Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail‐like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for α‐thrombin binding to platelets suspended in plasma. d‐Phe‐Pro‐ArgCH2‐α‐thrombin only bound to platelets when its concentration was → 100 nm, and it did so without inhibiting platelet activation by α‐thrombin. Whereas concentrations of hirudin equimolar to those of α‐thrombin failed to abrogate α‐thrombin‐mediated activation of platelets, a 10‐fold molar excesses of hirudin over α‐thrombin abrogated α‐thrombin binding to platelets. The demonstration that → 1.0 nmα‐thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo.
Recombinant human erythropoietin may correct erythropoietin‐deficient hyporegenerative anaemia in children given cardiac transplantation
Authors: Locatelli Franco , Zecca Marco , Mamprin Filippo , Gamba Amando , Giorgiani Giovanna , Stefano Piero De
Published: March 2008

Summary. Cyclosporin‐A reduces erythropoietin production and, together with the inhibitory effect of cytokines on erythropoiesis, may be potentially responsible for the anaemia observed in some patients after heart transplantation. Two children given cardiac transplantation and receiving cyclosporin‐A developed transfusion‐dependent hyporegenerative anaemia. Erythropoietin production was inappropriately low for the degree of anaemia, with an observed/predicted log(serum EPO) ratio of 0.54 and 0.49, respectively. The children were treated with rHuEPO at a dose of 75U/kg three times weekly for 1 month and then twice weekly via subcutaneous injection. No further transfusion was necessary and restoration of normal erythroid activity was obtained, with normal haemoglobin values. No adverse effects were observed. Our experience suggests that recombinant human erythropoietin may be useful in treating the anaemia associated with cardiac transplantation.
Use of G‐CSF alone to mobilize peripheral blood stem cells for collection from children
Authors: Kanold J. , Rapatel Ch. , Berger M. , Chassagne J. , Lutz P. , Lumley L. de , Plantaz D. , Vannier J. P. , Malpuech G. , Demécocq F.
Published: March 2008

Summary We report the data of 19 children with neuroblastoma (NB) or Ewing's sarcoma (EW) who had peripheral blood stem cells (PBSCs) harvested after mobilization by: (1) cyclophosphamide (CY) + etoposide + G‐CSF, (2) CY+GM‐CSF, or (3) G‐CSF alon. There were no consistent differences in the number of PBSCs collected following these three different mobilization regimens as assessed by CFU‐GM. 17 patients were reinfused with PBSCs after myeloablative therapy and had successful haemopoietic recovery. These results show that in children with solid tumours such as NB or EW a sufficient number of PBSCs can be collected after G‐CSF alone, and that PBSCs collected following stimulation by G‐CSF alone are as effective in reconstituting haemopoiesis as those collected after mobilizing chemotherapy + HGFs.
Kinetics of serum cytokines in adults undergoing peripheral blood progenitor cell transplantation
Authors: UCHIYAMA HITOJI , SHIMAZAKI CHIHIRO , FUJITA NAOHISA , TATSUMI TETSUYA , YAMAGATA NOBORU , HIRATA TOSHIYUKI , ASHIHARA EISHI , OKU NARITOSHI , GOTO HIDEO , INABA TOHRU , HARUYAMA HARUE , NAKAGAWA MASAO
Published: March 2008

Summary. We investigated the serum cytokine levels (G‐CSF, GM‐CSF, IL‐l/?, IL‐3 and IL‐6) using an ELISA in 14 patients with haematological malignancies undergoing peripheral blood progenitor cell transplantation (PBPCT). Serum G‐CSF levels in all patients rose immediately after PBPCT, then gradually decreased as the neutrophil counts began to rise. No detectable serum levels of GM‐CSF or IL‐lp were observed, but serum levels of IL‐3 rose transiently immediately following PBPCT. Serum levels of JL‐6 rose transiently during a fever in four patients. These observations suggest that G‐CSF and L 3 may contribute to the early haemopoietic reconstitution in PBPCT.