bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinicopathological spectrum of haemophagocytic syndrome in Epstein‐Barr virus‐associated peripheral T‐cell lymphoma
Authors: Yao Ming , Cheng Ann‐Lii , Su Ih‐Jen , Lin Ming‐Tseh , Uen Wu‐Ching , Tien Hwei‐Fan , Wang Chiu‐Hwa , Chen Yao‐Chang
Published: March 2008

Haemophagocytic syndrome (HS) is frequently observed in Epstein‐Barr virus‐associated peripheral T‐cell lymphoma (EBV‐PTCL) and represents a major cause of death. In this communication we have further analysed the spectrum of HS in 12 patients with EBV‐PTCL. The patients could be divided into three groups according to the time of onset of HS during the clinical course of PTCL. Group I patients (four cases) had HS as the initial clinical manifestation. All four patients were initially suspected to have malignant histiocytosis (MH) but a MH‐like PTCL was later diagnosed. Group II patients (six cases) developed HS at the time of lymphoma relapse. Four of them belonged to the angioinvasive type PTCL. Group III patients (two cases) developed HS at clinical remission; both were angioinvasive type PTCL. Nine patients had serological evidence suggesting active EBV infection. The clinical course after the onset of HS was generally fulminant in each group with a median survival of only 44 d despite combination chemotherapy and/or empirical therapy with high‐dose immunoglobulin and corticosteroids in six patients. In conclusion, HS represents a severe complication of EBV‐PTCL. Although most patients develop HS at a time of active lymphoma, the syndrome may occur when the lymphoma is in remission. Because of the poor outcome, early diagnosis and a new modality of treatment for HS associated with EBV‐PTCL should be pursued in future.
Inhibition of prothrombin activation by antiphospholipid antibodies and β2‐glycoprotein 1
Authors: Goldsmith G. H. , Pierangeli S. S. , Branch D. W. , Gharavi A. E. , Harris E. N.
Published: March 2008

Summary. Lupus anticoagulants, commonly found in the immunoglobulin fraction of patients with the antiphospholipid syndrome (APS), and the normal plasma protein β2‐glycoprotein 1 (β2GP1) may both contribute to the in vitro impairment of prothrombin activation associated with the APS. We examined the effects upon prothrombin activation supported by phospholipid vesicles of plasma IgG preparations from APS patients in the presence and absence of β2GP1. Using a purified system for measurement of prothrombin activation to thrombin, we demonstrated significant phospholipid concentration‐dependent inhibition of prothrombin activation in the absence of β2GP1 by 11 consecutive patient IgG preparations. The degree of inhibition of prothrombin activation by equivalent concentrations of patient IgG correlated well with the extent of prolongation of the plasma clotting time in lupus anticoagulant assays of whole patient plasma. Additional studies with eight patient IgG preparations indicated that the addition of β2GP1 to patient IgG‐phospholipid vesicle mixtures resulted in either independently additive inhibition by the two protein species (six cases) or potential inhibition of β2GP1 of the IgG inhibitory activity demonstrable in the absence of β2GP1 (two cases). In addition, β2GP1‐independent inhibition of prothrombin activation also occurred with three patient IgG preparations obtained by affinity binding to cardiolipin.
The progression of HCV‐associated liver disease in a cohort of haemophilic patients
Authors: Telfer Paul , Sabin Caroline , Devereux Helen , Scott Fiona , Dusheiko Geoffrey , Lee Christine
Published: March 2008

We have studied morbidity and mortality related to hepatitis C virus infection in haemophilic patients treated at our centre. 11/255 HCV seropositive patients have developed hepatic decompensation. 20 years after first exposure to lyophilized clotting factor concentrate the risk of hepatic decompensation is estimated to be 10.8% (95% CI 3.8‐17.8%). There is a significantly increased risk associated with HIV infection, and also with increased age. For HIV seropositive patients the rates of decline in CD4 lymphocyte count and the development of p24 antigenaemia are significant risk factors for hepatic decompensation. Cirrhosis was seen in 9/19 HIV seropositive patients at post mortem. There was an association of cirrhosis with increased age but not with CD4 count, p24 antigenaemia or AIDS. In conclusion, HCV infection is associated with serious liver disease in haemophilic patients, but so far this has been restricted to a minority of those at risk. HIV coinfection accelerates progression to hepatic decompensation, and we speculate that this is probably due to enhanced HCV replication in the presence of immune deficiency.
An echistatin‐like Arg‐Gly‐Asp (RGD)‐containing sequence in the heavy chain CDR3 of a murine monoclonal antibody that inhibits human platelet glycoprotein IIb/IIIa function
Authors: Deckmyn Hans , Stanssens Patrick , Hoet Bernard , Declerck Paul J. , Lauwereys Marc , Gansemans Yannick , Tornai Istvan , Vermylen Jos
Published: March 2008

We describe the production and biochemical characterization of the first GPIIb/IIIa‐inhibiting monoclonal antibody that contains an RGD sequence in the CDR3 region of the heavy chain. Monoclonal antibodies obtained by immunizing mice with human platelets were screened using consecutive ELISAs based on human platelets and immunoaffinity‐purified glycoprotein (GP) IIb/IIIa coated on microtitre plates. Out of 30 monoclonal antibodies reacting with GPIIb/IIIa, one, MA‐16N7C2, potently inhibited platelet aggregation induced by ADP, thrombin, arachidonic acid, collagen, U46619, adrenaline and platelet‐activating factor, whereas ristocetin‐induced aggregation was unaffected.

MA‐16N7C2 (IgG2a) bound ˜ 4 times faster to activated than to resting platelets, with a Kdcalc of 6.6 nm and of 17.5 nm, respectively. Equilibrium binding studies to non‐activated platelets showed a Kd of 18.2 nm with 41 x 103 binding sites per platelet. The antibody recognized GPIIb/IIIa only as a Ca2+ ‐dependent complex. MA‐16N7C2 blocked fibrinogen and von Willebrand factor binding to GPIIb/IIIa in a competitive manner with a Ki of 8.5 nm and 13.2 nm, respectively. Sequence analysis revealed a RGD‐containing sequence with homology to disintegrins, in the CDR3 region of the heavy chain.

That this RGD‐containing sequence could be involved in the interaction of the antibody to GPIIb/IIIa was finally indicated by showing that the binding is completely and competitively inhibited by echistatin.
Circulating cytokines in sickle cell patients during steady state
Authors: Croizat Helena
Published: March 2008

Plasma levels of GM‐CSF, IL‐3, IL‐6 and IL‐1(α,β) were investigated in steady‐state sickle cell patients. We find that SS patients with low levels of HbF (< 9% [LFSS]) are characterized by elevated plasma GM‐CSF, whereas in patients with high levels of HbF (HFSS), GM‐CSF is not detectable. In contrast, HFSS patients exhibited increased plasma IL‐3 (m = 84.8 ± 57pg/ml), whereas LFSS patients had lower or no detectable plasma IL‐3. IL‐1(α,β) and IL‐6 were also detected in plasma from some SS patients, but there was no correlation with HbF levels. Normal controls tested negative for all cytokines, except for one individual positive for IL‐3. These results are compatible with a model in which the level of haemopoietic stress determines the level of participation of GM‐CSF or IL‐3 in the regulation of SS circulating BFU‐E.
Prevention of MHC‐alloimmunization by UV‐B irradiation in a murine model: effects of UV dose and number of transfused cells
Authors: Grijzenhout M. A. , Aarts‐Riemens M. I. , Claas F. H. J. , Prooijen H. C. van
Published: March 2008

The optimal dose of UV‐B radiation for prevention of in vivo alloimmunization (AI) against major histocompatibility complex (MHC) antigens was investigated in a murine transfusion model. Two groups with five C57BL/6 mice (H2b) each were transfused at weekly intervals with 1 x 105 or 1 x 106 DBA/2 (H‐2d) leucocytes. Both suspensions induced anti‐H‐2d antibodies in all mice after the second transfusion. The minimal UV‐B dose required for abolition of alloreactivity in the mixed leucocyte reaction (MLR) was 0.6 J/cm2. This dose completely prevented the onset of MHC‐AI in all five mice transfused with six suspensions containing 1 x 105 leucocytes. In contrast, suspensions with 1 x 106 leucocytes and exposed to 0.6 J/cm2 induced immunization in 4/5 mice. Further increase of the dose to 1.8 or 5.4 J/cm2 did not prevent the onset of MHC‐AI. The use of UV radiation for prevention of secondary MHC‐AI was investigated in five mice with a primed immune system. Transfusion of suspensions with 1 x 105 leucocytes and irradiated at a dose of 1.8J/cm2 did not prevent booster reactions. We conclude that the number of leucocytes per transfusion determines the efficacy of UV irradiation for the prevention of MHC‐AI. For UV irradiation of human platelet concentrates (PCs) we propose to reduce the number of leucocytes by centrifugation prior to UV exposure. UV‐B irradiation of PCs with high numbers of leucocytes may not be effective for prevention of alloimmunization.
Lymphoid blast crisis at the onset of chronic myelogenous leukaemia: molecular evidence
Authors: Roman Jose , Andres Pilar , Jimenez Maria A. , Torres Antonio
Published: March 2008

A patient with a typical haematological pattern of acute lymphoblastic leukaemia with BCR and IgH rearrangements was brought into complete remission by treatment. A few weeks later she developed the typical peripheral and bone marrow pattern of chronic myelogenous leukaemia (CML) with persistence of the BCR rearrangement and disappearance of the IgH rearrangement, suggesting that this case is an example of CML presenting in blast crisis without a detectable chronic phase.
Sequential relapses of blastic crisis may involve different clones of cells with different molecular abnormalities
Authors: And A. Foti , Cline M. J.
Published: March 2008

A patient with typical Ph1‐positive CML was studied during sequential phases: (1) initial chronic phase, (2) first myeloid blast crisis, (3) second chronic phase, and (4) accelerated disease leading to a second blast crisis. A point mutation in codon 239 of the p53 gene and a novel chromosome 17 alteration appeared concomitantly with the first blast crisis and then disappeared with re‐establishment of a second chronic phase. They did not reappear with the second acute phase, indicating that the clone responsible for the original blast crisis had been suppressed and supplanted by another clone of malignant cells. This observation suggests that in at least some CML patients drug therapy can suppress or eliminate an aggressive malignant cell clone, but that the underlying molecular defect in haemopoietic cells (in this case the c‐ABL translocation) persists and other aggressive clones with different molecular lesions eventually arise. Our observations and inferences are consistent with the hypothesis advanced by Fialkow et al (1991) to explain clonal remissions in acute non‐lymphocytic leukaemia.
The effects of gestation on circulating progenitor cells
Authors: Jones H. M. , Nathrath M. , Thomas R. M. , Edelman P. , Rodeck C. H. , Linch D. C.
Published: March 2008

The frequency of BFU‐E in second‐trimester fetal blood (484 ± 104/105) falls progressively during gestation to a value of 69 ± 41/105 in cord bloods of 36 weeks gestation and beyond, but this is still significantly greater than adult blood values of 14 ± 8 (P < 0.01). BFU‐E obtained from unfractionated peripheral blood mononuclear cells from fetuses/neonates less than 36 weeks gestation were more sensitive to erythropoietin than adult BFU‐E, but the sensitibvity of highly purified BFU‐E obtained from secondtrimester fetal liver was similar to that in adult cells. Almost maximal growth of BFU‐E from purified fetal progenitor cells could be achieved with erythropoietin alone, whereas adult cells required the presence of other factors with ‘burstpromoting activity’.
Detection of fetal RhD sequence from peripheral blood of sensitized RhD‐negative pregnant women
Authors: Lo Y‐M. D. , Noakes L. , Bowell P. J. , Fleming K. A. , Wainscoat J. S.
Published: March 2008

A sensitive PCR‐based assay was developed to amplify fetal‐derived rhesus D (RhD) sequence from peripheral blood of RhD‐negative pregnant women with circulating anti‐D. RhD‐PCR positivity was detected in 7/22 samples from women bearing RhD‐positive fetuses. despite the presence of varying levels of anti‐D. Evidence is presented with suggests that rising matermal anti‐D levels might reduced circulating fetal cell numbers. Further development of this assay may have implications in the clinical management of RhD‐sensitized pregnancies and aid the understanding of the physiology of feto‐maternal cell trafficking.
Development of acute myeloblastic leukaemia in a case of aplastic anaemia treated with granulocyte colonystimulating factor
Authors: Izumi Tohru , Muroi Kazuo , Takatoku Masa‐AKI , Imagawa Shigehiko , Hatake Kiyohiko , Miura Yasusanda
Published: March 2008

Summary. We report a case of aplastic anaemia (AA) treated with granulocyte colony‐stimulating factor (G‐CSF) terminating as acute myeloblastic leukaemia (AML). Because of severe pneumonia, 250 μg of G‐CSF was administered for 30d to promote neutrophil recovery. Following G‐CSF therapy, myeoblasts appeared, and the diagnosis of AML was then made. The myeloblasts proliferated in response to G‐CSF in vitro and in vivo. In AA. development of AML, after treatment with G‐CSF is rate. Therefore a careful observation for leukaemic transformation is necessary in long‐term administration of G‐CSF for AA.
Immunocytochemical and flow cytometric detection of proteinase 3 (myeloblastin) in normal and leukaemic myeloid cells
Authors: Dengler Robert , Muunstermann Ursula , Al‐Batran Salah , Hausner Irmela , Faderl Stefan , Nerl Christoph , Emmerich Bertold
Published: March 2008

Summary. Proteinase 3 (P3) is a serine proteinase present in the primary granules of neutrophils. We have investigated the expression of this protein in samples of bone marrow from healthy individuals and patients with different types of leukaemias by using immunocytochemical staining and flow cytometric quantitation. In normal bone marrow the enzyme was found in promyelocytes, myelocytes, metamye‐locytes, band forms and polymorphonuclear neutrophils, correlating with the synthesis of neutrophil serine proteinases during myeloid maturation. No staining was found within the lymphoid, erythroid and megakaryocytic lineage. In the leukaemic samples, only those of acute myeloid and chronic myeloid leukaemia patients were labelled with the antiproteinase 3 antibody. Cases of acute lymphoblastic and chronic lymphocytic leukaemia, as well as other malignant lymphomas, were consistently negative, indicating that P3 may be used as a specific marker for the discrimination between myeloid and lymphoid leukaemias. In addition. immunoreactivity of myeloperoxidase (MPO) was investigated and the expression of P3 and MPO correlated with the French‐American‐British (FAB) classification. P3 was not detected in minimally differentiated MO and Ml cases but was in predominantly labelled cells of M2 and M3 subtypes plus half of the M4 and one out of six M5 cases but not those of M6. These findings correspond to the differentiation stage in which P3 is expressed and stored in the primary granules. Therefore the enzyme may also be used as an adjunct to the classic morphological and cytochemical methods to elucidate further the stage at which the differentiation arrest of the leukaemic clone has occurred.
Induction of differentiation into monocyte/macrophage cell lineage of a human eosinophilic leukaemia cell line EoL‐1 by simultaneous stimulation with tumour necrosis factor‐α and interferon‐γ
Authors: Nakajima Toshiharu , Yamashita Naomi , Matsui Hideki , Suzuki Noboru , Kaneoka Hidetoshi , Mizushima Yutaka , Sakane Tsuyoshi
Published: March 2008

Summary. Human myeloid leukaemia cell lines have been shown to differentiate into distinct cell lineages in vitro in response to several differentiation‐inducing agents. A human eosinophilic leukaemia cell line, EoL‐1, has been shown to differentiate into mature eosinophilic granulocytes by treatment with the culture supernatant of a human T‐cell line, HIL‐3. In this study we have studied whether the EoL‐1 cell line has potential to differentiate into cell lineage other than eosinophils. We found that EoL‐1 cells cultured in the presence of tumour necrosis factor (TNF)‐α (10u/ml) and interferon (IFN)‐γ (1000u/ml) for 2–4d differentiated into macrophage‐like cells in morphology, and expressed CD14 antigen on their cell surface. It is possible that the small subpopulation of EoL‐1 cells which contains non‐specific esterase (NSE) activity may be preferentially differentiated by TNF‐α and IFN‐γ. To clarify this issue, we have cloned the EoL‐1 cell line and obtained NSE negative and positive sublines. Both EoL‐1 sublines differentiated into monocyte/ macrophage‐like cells, because: (a) EoL‐1 sublines were induced to express CD14 antigen, and (b) they attached firmly to the plastic wells; (c) after differentiation they became strongly positive for NSE staining, and secreted TNF‐α in response to the stimulation with lipopolysaccharide; and (d) they exhibited potent phagocytic activity. Therefore, we found that the EoL‐1 cell line has the ability to differentiate not only into mature eosinophilic cells but also into monocyte/macrophage cell lineage, suggesting that EoL‐1 cells represent immature cells with ability to differentiate into multiple cell lineages.
Erythropoietin gene expression in fetal and adult sheep kidney
Authors: Darby I. A. , Evans B. A. , Fu P. , Lim G. B. , Moritz K. M. , Wintour E. M.
Published: March 2008

Summary. The exact location of the cells which express erythropoietin (Epo) in the kidney is still controversial, with conflicting reports suggesting that both peritubular interstitial cells or proximal tubular epithelial cells are possible sites of Epo expression. In the present study we have examined the location of Epo‐expressing cells in fetal and adult sheep kidneys after Epo expression was stimulated by anaemia. In situ hybridization histochemistry was performed using synthetic oligonucleotide probes complementary to part of the sheep Epo cDNA sequence or a riboprobe (cRNA) for ovine Epo of 520 bases. Epo expression was confined to peritubular cells of the kidney cortex, in general in the area close to the cortico‐medullary junction. In some severely anaemic adult sheep kidneys, Epo‐expressing cells were also found in the outer cortex. In addition we located Epo‐expressing cells in the kidneys not only of anaemic fetuses (89–140d of gestation, term = 150d) but also in kidneys from normal fetuses 60–110d of gestation. Again, Epo expression was seen only in peritubular cells of the kidney cortex. These findings confirm that the kidney is an important site of Epo production, in the sheep, from at least 0–4 gestation, but also show that there is no ontogenic change in the cellular site of production within the kidney.
Correlations between the intensity of fMLP‐dependent respiratory burst and cellular fatty acid composition in human neutrophils
Authors: Bellavite P. , Guarini P. , Biasi D. , Carletto A. , Trevisan M. T. , Caramaschi P. , Bambara L. M. , Corrocher R.
Published: March 2008

Summary. The peripheral blood neutrophils were isolated from a group of normal subjects and their fatty acid composition determined by capillary gas‐chromatography. The superoxide (O2) release by the same cell preparations in response to formyl‐methionyl‐leucyl‐phenylalanine was also determined following cytochrome c reduction in a microplate assay. A strong negative correlation was found between C18:2 (linoleic acid) (r = ‐0.703, P‐0.01) and C:16:0 (palmitic acid) (r= ‐0.569, P= 0.009) and fMLP‐stimulated O2 release, whereas C20:4 (arachidonic acid) correlated Positively (r= 0.448, P= 0.048). Other fatty acids, namely C12:0, C14:0, C16:1, C18:1, C18:3, C18:4, C20:0, C20:1, C20:2, C20:5, C22:0, C22:1, C22:6, C24:0 and C26:0, were not correlated with O2. No correlations were found between fatty acid composition and O2 release from resting cells and from cells stimulated by phorbol‐myristate acetate. These results suggest that the fatty acid composition of blood neutrophils may be a critical factor determining the capability of releasing free radicals in response to formylpeptides. Moreover, since a concomitant increase of arachidonic acid and decrease of its precursor linoleic acid has been found in high‐responsive neutrophils, the rate of the enzymes of the arachidonic acid biosynthetic pathway (elongases and desaturases) appear to play an important role.
Pharmacokinetics of the oral iron chelator deferiprone (L1) in patients with iron overload
Authors: AL‐Refaie F. N. , Sheppard L. N. , Nortey P. , Wonke B. , Hoffbrand A. V.
Published: March 2008

Summary. Single oral dose pharmacokinetics of the iron chelator deferiprone (L1) were studied in 24 patients with chronic iron overload and correlated with 24h urinary iron excretion (UIE) and creatinine clearance. Absorption of L1 was rapid with a t1/2 of 22·2 pL 17·7 (mean pL SD) min. The elimination half‐life (elt1/2) of the drug was 91·1 pL 33·1 min and of its metabolite, L1‐glucuronide (L1G) 147·7pL 52·0 min. Creatinine clearance of the patients correlated significantly with the elimination t1/2 of L1G (r= ‐0·79, P= 0·002). There was also a significant correlation between 24 h UIE in the 14 patients studied and L1 versus time area under the curve (AUC) (P= 0·007). The total amount of L1 recovered in urine in 24 h comprised 77·9 pL 13·3% of the L1 dose. L1 efficiency (the 24 h UIE divided by the amount of iron the oral dose of L1 is capable of binding) in the 14 patients was 3·8 pL 1·9%. These data show for the first time that the urinary elimination of L1G is influenced by the renal function of the patient. Although no significant accumulation of L1 and L1G will occur in most of the patients if L1 is given more than once daily, in some patients with impaired renal function, L1G may accumulate.
Clonal and non‐clonal karyotypically abnormal cells in haemophagocytic lymphohistiocytosis
Authors: KANEKO YASUHIKO , MASEKI NOBUO , SAKURAI MASAHARU , IDO MASARU , TSDNEMATSU YUKIKO , MIZUTANI SHUKI , HATTORI TATSUYA , SHIMIZU HIROYUKI , EGUCHI HARUHIKO , OKA TOSHIAKI , MIYAKE MUNENORI , HORIKOSHI YASUO , SUCHI TAIZAN
Published: March 2008

We studied chromosomes in bone marrow (BM) or peripheral blood cells of nine patients with haemophagocytic lymphohistiocytosis (HLH); three of them had a family history of HLH and four others underwent concurrent Epstein‐Barr virus (EBV) infection. In addition to a large population of normal mitotic cells, karyotypically abnormal clonal cells were found in two patients, abnormal clonal cells and a nonclonal (single) abnormal cell in one, and nonclonal abnormal cells in three. All the six patients with chromosome abnormalities died of progressive disease; one of them also had EBV infection and EBV‐associated clonal proliferation. Two of three patients with EBV infection and only normal mitotic cells in BM completely recovered from the disease.

Although HLH did not show histological and/or haema‐tological evidence of a neoplastic disease, clonal chromosome abnormalities and the fatal clinical outcome found in some of the patients suggest that the disease may be heterogenous and include malignancy. HLH patients with karyotypically abnormal clonal cells in BM should warrant more intensive chemotherapy than that presently being applied to them and should be considered as candidates for BM transplantation.
Enhanced Mg2+‐ATPase activity in ghosts from HS erythrocytes and in normal ghosts stripped of membrane skeletal proteins may reflect enhanced aminophospholipid translocase activity
Authors: VERMEULEN WILLEM P. , BRIEDE JACOB J. , BUNT GERTRUDE , DEN KAMP JOS A. F. OP , KRAAIJENHAGEN ROB J. , ROELOFSEN BEN
Published: March 2008

Hereditary spherocytosis (HS) is a congenital haemolytic anaemia which is characterized by a great variety of structural defects in the red cell's membrane skeleton and/or deficiencies in particular membrane (skeletal) proteins. Enhanced (Mg2+)‐dependent adenosine triphosphatase (Mg2+‐ATPase) activities, varying from 115% to 160%, were invariably found in erythrocyte ghosts derived from 13 HS patients. Similarly, an enhancement of Mg2+‐ATPase activity by 30% is observed in normal red cell ghosts that have been stripped of the greater part of their membrane skeletal proteins by treatment with a low ionic strength buffer. Reassociation of those stripped ghosts with spectrin reduces the enhanced Mg2+‐ATPase activity to its original level. Since in both cases, HS ghosts and stripped normal ghosts, the stabilizing effects that the membrane skeleton exerts on the maintenance of an endofacial localization of the aminophospholipids are impaired, the enhanced Mg2+‐ATPase activity is interpreted to reflect an increased activity of the aminophospholipid translocase. The present observations therefore support a role of the membrane skeleton in the stabilization of phospholipid asymmetry in the red cell membrane and consequently in reducing the energy consumption of the translocase.
Assessment of clonal evolution at Ig/TCR loci in acute lymphoblastic leukaemia by single‐strand conformation polymorphism studies and highly resolutive PCR derived methods: implication for a general strategy of minimal residual disease detection
Authors: BARUCHEL ANDRE , CAYUELA JEAN‐MICHEL , MACINTYRE ELISABETH , BERGER I ROLAND , SIGAUX FRANCOIS
Published: March 2008

Junctional sequences of immunoglobulin (Ig)/T‐cell receptor (TCR) gene rearrangements are used as patient‐specific PCR targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukaemias (ALLs). Clonal evolution of gene rearrangements is a major pitfall of this strategy. Using high‐resolution PCR‐based analyses (including denaturing gel electrophoresis and single‐stranded conformation polymorphism (SSCP)) we have compared Ig/TCR gene rearrangements at presentation and relapse in a series of ALLs. These methods allow an unambigous comparison of rearrangements taking into account junctional size and nucleotide sequence information and allow a precise assessment of the clonal evolution. V7‐J7 and V6j‐J6i rearrangements were analysed in 12 T‐ALLs. VH‐JH, V7‐J7, V62‐D63 and, in selected cases, DH‐JH rearrangements were studied in 14 B‐lineage ALLs. Clonal evolution, regarding major rearrangements, occurs for at least one of these loci in 2/12 T‐ALLs and in 5/14 B‐lineage ALLs. Clonal evolution is more marked for minor rearrangements than for major ones. As shown using SSCP analysis, rearrangements observed at relapse are sometimes found in minor clones at presentation which are therefore selected in vivo by a proliferative advantage. These data, as well as those from the available literature, suggest the use of at least two patient‐specific probes to detect MRD in ALLs. A general strategy including selected Ig/TCR rearrangements and chromosomal abnormalities as PCR targets is proposed.
Clinical significance of serum thymidine kinase in adult T‐cell leukaemia and acute myeloid leukaemia
Authors: SADAMORI NAOKI , ICHIBA MOTOKO , MINE MARIKO , HAKARIYA SATOMI , HAYASHIBARA TOSHIHISA , ITOYAMA TAKAHIRO , NAKAMURA HIDEO , TOMONAGA MASAO , HAYASHI KUNIAKI
Published: March 2008

To clarify the clinical and biological significance of serum thymidine kinase (TK) in adult T‐cell leukaemia (ATL) associated with human lymphotropic virus type‐I (HTLV‐I) and in acute myeloid leukaemia (AML), TK was measured in 52 patients with ATL (acute ATL, 35 patients; lymphoma ATL, two patients; chronic ATL, 12 patients; smouldering ATL, three patients), and in 27 patients with AML (one FAB MO, one Ml, 10 M2, seven M3, five M4, one M5, one M6, one MU). In ATL patients, statistical analysis disclosed a close correlation between TK level and the leucocyte count (P<0–01), and absolute number of abnormal lymphocytes (P<0–01). However, no correlation was observed between serum lactic dehydrogenase (LDH) level and these items. Concerning the therapeutic response, a statistical difference was present in TK between complete remission and no response (P<005), but not in LDH. We also investigated a significant inverse correlation between TK level as well as LDH level and the length of survival after the initial diagnosis (P<001). In AML patients a close correlation of TK level with the count of leucocytes (P<001), percentage of blasts in the blood (P<005), therapeutic response (P<0–01) and the length of survival after the initial diagnosis (P< 005) was present.
Clonal analysis of haemopoietic cells in essential thrombocythaemia
Authors: KASSAR NAHED EL , HETET GILLES , LI YAN , BRIERE JEAN , GRANDCHAMP BERNARD
Published: March 2008

Essential thrombocythaemia (ET) is a myeloproliferative disease (MPD) predominantly involving the platelet lineage, with a diagnosis often difficult to establish in practice. The demonstration of a clonal haemopoiesis can contribute to diagnosis. The clonal origin of blood cells can be assessed in female patients using X‐chromosome‐linked polymorphisms, assuming a random inactivation of the X chromosome. The human androgen receptor gene HUMARA has been used for this purpose, taking advantage of a highly polymorphic trinucleotide repeat in the first exon. The close proximity of the polymorphic trinucleotide repeat to four methylation sites permits a clonal analysis based on the polymerase chain reaction. We have used this technique to study the clonality of haemopoietic cells in 32 patients with ET and 23 age‐matched control heterozygotes for the HUMARA polymorphism. A monoclonal pattern of haemo‐poiesis was detected in unfractionated nucleated blood cells from 22 patients, suggesting that haemopoiesis is essentially monoclonal in a majority of cases in this disease. In some patients a monoclonality of granulocytes was documented, whereas the mononuclear fraction had a polyclonal pattern of X‐inactivation. Finally, in two patients for whom a polyclonality had been found in unfractionated blood, analysis of G6PD transcripts in platelets revealed a clonal megakaryocytopoiesis. These findings confirm the heterogeneity of haematological abnormalities in ET patients and the potential utility of a multifaceted laboratory approach to investigate these patients.
A monoclonal antibody (B724) to von Willebrand factor recognizing an epitope within the Al disulphide loop (Cys509‐Cys695) discriminates between type 2A and type 2B von Willebrand disease
Authors: CHRISTOPHE O. , ROUAULT C. , OBERT B. , PIETU G. , MEYER D. , GIRMA J. P.
Published: March 2008

Monoclonal antibody (MoAb) B724 to von Willebrand factor (vWF) completely inhibits its interaction with heparin, sulphatides and botrocetin and consequently botrocetin‐induced binding of vWF to platelets. MoAb B724 has no effect on the binding of vWF to collagen or to ristocetin‐treated platelets nor on vWF‐dependent platelet aggregation induced with ristocetin and asialo‐vWF‐mediated platelet aggregation. MoAb B724 preferentially recognizes a conformation of native vWF, in solution, or immobilized through a coated antibody. It exhibits a markedly lower affinity for vWF immobilized onto collagen or plastic surfaces. Using proteolytic fragments of vWF, B724 epitope was localized within the 512–673 sequence of the Al disulphide loop of vWF, MoAb B724 was used as second antibody in a two‐site ELISA to test a series of patients with type 1, 2 A, 2B and 2N vWD or haemophilia A and recombinant wild type or mutated vWFs. Results were compared with those obtained by control ELISAs performed using polyclonal antibodies. Using MoAb B724, strikingly lower levels of vWFAg were observed in plasma from most patients with type 2B vWD, and in seven out of the eight rvWF mutated close to or within the Al disulphide loop. Therefore MoAb B724, which interferes with this loop involved in the function of vWF, appears to be a useful tool for rapid screening of conformational changes in this region.
Resistance to activated protein C activity of an anti‐/2 ‐glycoprotein I antibody in the presence of β‐glycoprotein I
Authors: MATSUDA Juzo , GOTOH MORITAKA , GOHCHI KENGO , KAWASUGI KAZUO , TSUKAMOTO MIYO , SAITOH NORIKO
Published: March 2008

Some researchers claim that lupus anticoagulant‐positive plasma may cause a false‐positive reaction in the test for activated protein C (APC) resistance, a hereditary thrombophilic state characterized by abnormal factor V, which frequently causes venous thrombosis, We investigated whether anti‐/32 ‐glycoprotein I antibody (aGPI), which has recently come to be regarded as an anti‐cardiolipin antibody (aCL) itself, might have an effect on the APC resistance test.
Expression of an erythropoietin‐like gene in the trout
Authors: SHIELS A. , WICKRAMASINGHE S. N.
Published: March 2008

Cross‐species Northern blot hybridization and radioimmunoassay have provided evidence for the expression of an erythropoietin (EPO)‐like gene in the rainbow trout. The principal site of EPO‐like mRNA and antigen expression in the adult trout appears to be the kidney which also acts as the major erythropoietic organ. The data suggest that a locally produced EPO‐like factor may regulate renal erythropoiesis in bony fish and, furthermore, they identify a possible evolutionary origin for renal EPO production in mammals.
The fusion of TEL and ABL in human acute lymphoblastic leukaemia is a rare event
Authors: JANSSEN JOHANNES W. G. , RIDGE SUSAN A. , PAPADOPOULOS PANOS , COTTER FINBARR , LUDWIG WOLF‐DIETER , FONATSCH CHRISTA , RIEDER HARALD , OSTERTAG WOLFRAM , BARTRAM CLAUS R. , WIEDEMANN LEANNE M.
Published: March 2008

We have recently identified a common ALL patient which harboured a chromosomal fusion between the TEL gene on chromosome 12 and the ABL gene on chromosome 9. We designed an RT‐PCR assay to screen 186 adult ALL and 30 childhood ALL patients for this novel translocation. We were unable to identify any additional cases with a TEL/ABL fusion product.
Characterization of insulin‐like growth factor binding proteins (IGFBP) and regulation of IGFBP‐4 in bone marrow stromal cells
Authors: GRELLIER PASCALE , YEE DOUGLAS , GONZALEZ MYRNA , ABBOUD SHERRY L.
Published: March 2008

Summary. Bone marrow stromal cells synthesize and secrete insulin‐like growth factor (IGF)‐I and IGF‐binding proteins (IGFBP). IGFBPs may modulate the action of IGF‐I or IGF‐II on haemopoiesis. However, the specific IGFBPs produced by various stromal cell types have not been identified. We examined six different stromal phenotypes for IGFBP protein and IGFBP‐1 to ‐6 mRNA expression. [125I]IGF‐I ligand blot analysis of conditioned medium demonstrate different patterns of IGFBP secretion by each cell type. The most prominent IGFBPs were 24 and 29 kD species, consistent with IGFBP4 and IGFBP5, respectively. RNase protection assays demonstrate that, overall, stromal cells express IGFBP‐2 to ‐6 mRNAs, with IGFBP4, IGFBP5 and IGFBP6 mRNAs predominating. Since agents that modulate cAMP levels may influence haemopoiesis via the release of stromal‐derived cytokines, we determined the effect of forskolin, a cAMP agonist, on IGFBP4 expression in TC‐1 cells. Forskolin (10 5 M) up‐regulated IGFBP4 mRNA and protein secretion in a time‐dependent manner. These findings suggest that IGFBP‐4, ‐5 and ‐6 released by stromal cells may be key modulators of the haemopoietic response to IGFs. Release of IGFBP4 by agents that increase cAMP may be an important mechanism involved in regulating IGF bioavailability in the marrow microenvironment.
All‐trans retinoic acid shows multiple effects on the survival, proliferation and differentiation of human fetal CD34+ haemopoietic progenitor cells
Authors: ZAULI GroRGio , VISANI GIUSEPPE , VITALE MARCO , GIBELLINI DAVIDE , BERTOLASO LUCIA , CAPITANI SILVANO
Published: March 2008

Summary. To evaluate the effect of all‐trans retinoic acid (RA) on fetal haemopoiesis, we performed serum‐free liquid and semisolid cultures using CD34+ cells purified from mid‐trimester human fetal blood samples. RA, at both physiological (10‐n and 10‐12M) and pharmacological (10‐6 and l(r7M) concentrations, significantly (P<0.01) promoted the survival of fetal CD34+ cells in liquid cultures from day 3 onwards, by suppressing apoptosis induced by serum and growth factor deprivation. On the other hand, RA alone had no significant effect on the proliferation and differentiation of fetal haemopoietic progenitors.

In the presence of optimal concentrations of recombinant interleukin‐3 (IL‐3), stem cell factor (SCF), granulocyte/ macrophage‐colony stimulating factor (GM‐CSF), and erythropoietin (Epo), low and high doses of RA induced striking differential effects on CD34+ cell proliferation in liquid cultures and colony formation in semisolid assays. In fact, 1CTU M and 1CT12M RA were able to: (i) significantly (P<0.05) increase 3H‐thymidine uptake by fetal CD34+ cells in liquid cultures, and (ii) variably promote the growth of pluripotent (CFU‐GEMM, P<0.05), early (BFU‐meg) and late (CFU‐meg, P<0.01) megakaryocyte, granulocyte/macrophage (CFU‐GM. P<001) and erythroid (BFU‐E) progenitors in semisolid cultures. On the contrary, 10‐6 and 10‐7 M RA induced: (i) an overall inhibition (P<0.01) of CD34+ cell growth in liquid cultures; (ii) a marked suppression of BFU‐E colony formation (P<0.01) at all Epo concentrations examined (0‐002‐4IU/ml); and (iii) a significant (P<0.()1) stimulation of CFU‐GM with a shift from mixed granulocyte/ macrophage to pure granulocyte colonies, whereas it had little effect on the growth of CFU‐GEMM, BFU‐meg and CFU‐meg.

Our data, as a whole, demonstrate that RA has direct complex effects on the survival, growth and clonal expansion of fetal haemopoietic progenitor cells, mainly depending on the presence of recombinant cytokines, the type of progenitor and the concentrations of RA.
A phase I/II trial of recombinant human interleukin‐6 in patients with aplastic anaemia
Authors: SCHREZENMEIER HUBERT , MARSH JUDITH C. W. , STROMEYER PATRICIA , MULLER HILKB , HEIMPEL HERMANN , GORDON‐SMITH EDWARD C. , RAGHAVACHAR ARUNA
Published: March 2008

Summary. In a phase I/II study, 11 patients with marrow failure (10 with acquired aplastic anaemia and one with pancytopenic Fanconi anaemia) were treated with recombinant human interleukin‐6 (rhIL‐6) to assess the safety and tolerability of rhIL‐6 and its effects on peripheral blood counts, bleeding complications and transfusion requirements. All patients with acquired aplastic anaemia were refractory to immunosuppressive treatment or had relapsed after immunosuppressive therapy and were not bone marrow transplantation candidates. Recombinant hIL‐6 was to be given as a once‐daily subcutaneous injection for 28 d at doses ranging from 0‐5 to 5‐0βg/kg. After an observation period of 2 weeks, five patients received a second treatment course of 28 d.

Only one patient had a sustained increase in platelet count from 18 000 to 72 000/fA. Bleeding occurred in four patients and caused premature discontinuation of rhIL‐6 therapy in three patients. A deterioration of pre‐existing anaemia was observed in nine patients. No significant changes of leucocyte counts were observed during the first cycle. During the second cycle the peripheral blood monocyte counts decreased significantly. No significant changes in bone marrow cellularity were observed. Recombinant hIL‐6 induced a dose‐dependent increase in acute‐phase reactants in all patients. Other adverse events included fever, headache, arthralgia, tachycardia and hypertension. In conclusion, rhIL‐6 given alone at low doses does not increase platelet counts in the majority of patients with aplastic anaemia and can precipitate a sudden worsening of pre‐existing anaemia and thrombocytopenia. This study was discontinued prematurely on account of the toxicity of rhIL‐6 seen in patients with aplastic anaemia.
Bacterial infection‐associated improvement of platelet counts in two patients with chronic and unresponsive idiopathic thrombocytopenic purpura with normal platelet survival studies
Authors: BORDIN JOSE O. , SMITH JAMES W. , HAYWARD CATHERINE P. M. , WARKENTIN THEODORE E. , WASI PARVEEN , KELTON JOHN G.
Published: March 2008

Summary. Approximately 20% of adult patients with idiopathic thrombocytopenic purpura (FTP) do not respond to splenectomy and require alternative therapies to achieve a clinically safe platelet count. A small percentage of these patients have very refractory disease and are either unresponsive or poorly responsive to almost any therapy. In this report we describe two patients with chronic and unresponsive ITP with normal platelet survivals. Neither patient had responded to a large variety of treatments including corticosteroids, splenectomy, IVIgG, anti‐D, chemotherapy, and ascorbic acid. However, both had a rapid, but short‐lived, rise in their platelet count following a bacterial infection. One patient had a rise in platelet count for 6 months following the acute episode of bacteraemia. The second patient had a shorter response of 1 week. It is possible that these two patients represent a subset of patients with ITP who may benefit from cytokine therapy.
Biallelic DNA rearrangements and deletions within the BCL‐6 gene in B‐cell non‐Hodgkin's lymphoma
Authors: NAKAMURA YUICHI , MIKI TOHRU , KAWAMATA NORIHIKO , HIROSAWA SHINSAKU , SAITO KENJI , ENOKIHARA HIDEO , FURIJSAWA SHINPEI , SHISHIDO HIDEO
Published: March 2008

Summary. Chromosomal translocations involving band 3q27 are recently described common specific cytogenetic abnormalities in B‐cell neoplasms, and the BCL‐6 gene, identified on 3q27, was shown to be disrupted and over‐expressed in lymphoma cells having these chromosomal translocations. In the present study we found rearrangements within the BCL‐6 gene in seven out of 3 5 cases with B‐cell non‐Hodgkin's lymphoma (NHL). Further analysis revealed that three of these patients with BCL‐6 abnormality had multiple rearranged bands hybridized with probes from a single restriction fragment within the major translocation cluster (MTC). suggesting that independent DNA rearrangements would occur on both alleles. Additionally, Southern blot analysis indicated that three patients carry deletions encompassing the area containing the first exon of the BCL‐6 gene. Our results suggest that biallelic DNA rearrangements and deletions would occasionally occur in NHL patients with BCL‐6 abnormality.
Establishment and characterization of IRTA17 and IRTA21, two novel acute non‐lymphocytic leukaemia cell lines with t(16;21) translocation
Authors: HIYOSHI MOTUFUMI , YAMANE TAKAHISA , HIRAI MANABU , TAGAWA SHINICHI , HATTORI HIDEKI , NAKAO YOSHITAKE , YASUI YOSHINORI , KOH KEY‐RYANG , HINO MASAYUKI , TATSUMI NORIYUKI
Published: March 2008

Summary. The t(16;21)(pll;q22) translocation is an infrequent chromosomal abnormality, but seems specific to acute non‐lymphocytic leukaemia (ANIL). We established two cell lines with t(16;21)(pll;q22) from the bone marrow of a patient with ANLL in relapse. Their morphological, karyotypic, immunohistochemical and genetic features are examined. Although both cell lines show monocytoid features morphologically, they express only CD13 (My 7) and CD34, and neither expressed monocytoid or lymphoid markers. Reverse transcription‐polymerase chain reaction showed that both cell lines expressed a similar TLS‐ERG chimaeric mRNA as a result of the t(16;21)(pll;q22) translocation. As far as we know, there is no report of a leukaemia cell line with t(16;21). These cell lines represent a useful tool for leukaemia research.
Persistence of multilineage host haemopoiesis following allogeneic bone marrow transplantation
Authors: PRICE CATHY M. , COLMAN SUSAN M. , KANFER EDWARD J.
Published: March 2008

Summary. The survival of host cells following high‐dose cytotoxic therapy and allogeneic marrow transplantation has been established previously, but the identity of these cells has not been elucidated in detail. Four patients who received sex‐mismatched marrow have been studied for up to 12 months post‐transplant using a simultaneous immuno‐phenotyping/fluorescence in situ hybridization technique. The results demonstrate residual host T cells (CD3+), B cells (CD22+) and myeloid cells (CDllc+ and CD13+), and additionally cells of progenitor cell phenotype (CD34+). The long‐term persistence of host haemopoiesis may have major relevance to the post‐transplant complications of marrow rejection, graft‐versus‐host disease, and malignant relapse.
Deletion of chromosome 13 (band ql4) but not trisomy 12 is a clonal event in B‐chronic lymphocytic leukaemia (CLL)
Authors: JABBAR SHIREEN A.B. , GANESHAGURU KANAGASABAI , WICKREMASINGHE RANMOHAN G. , HOFFBRANU A. VICTOR , FORONI LETIZIA
Published: March 2008

Summary. Chromosomal abnormalities are detected by conventional cytogenetic or FISH analysis in 50% of chronic lymphocytic leukaemias (CLL). Trisomy 12 and del 13ql4 account for 70% of these abnormalities. The incidence of these two abnormalities was studied in CLL patients by Southern blot analysis using a highly purified B‐cell malignant population (CD5>95%, CD3 < 5%). Probes for the Dl 3S25 marker on chromosome 13 band ql4 and for the RBTN3 gene on chromosome 12 band pi2‐13, were used. Deletion of the Dl 3S2 5 was detected in 17/42 patients (43%) in a homozygous (9‐5%) or heterozygous (30%) configuration. Deletion of the D13S25 marker appears to be a clonal and early event in CLL development since it is detected in >95% of the malignant clonal population. Conversely, trisomy 12 is rarely a clonal event (5/33 patients, 15%) and a varying proportion of cells carrying this abnormality can be demonstrated in 30% of CLL patients (10/33 patients).
Coronary artery bypass surgery in patients with inherited antithrombin deficiency
Authors: CLARK P. , WALKER ISOBEL D. , NEILSON R. F.
Published: March 2008

Summary. Antithrombin (AT) replacement in coronary artery bypass grafting procedures in three individuals with inherited antithrombin deficiency is described. All three had a significant personal or family history of thrombotic disease. All patients achieved satisfactory AT levels throughout bypass and in the postoperative period. All received heparin prophylaxis in the postoperative period. None suffered thrombotic or bleeding complications.
Impaired cytokine production by bone marrow stromal cells of immunodeficient mice
Authors: HAMBURGER ANNE W.
Published: March 2008

Summary. The aetiology of the bone marrow suppression in HIV‐infected patients is unknown. We have demonstrated previously that the ability of bone marrow cells, derived from mice made immunodeficient by infection with the retrovirus LP‐BM5, to establish long‐term stromal cultures is impaired. In this study we determined the ability of bone marrow stromal cells from these immunodeficient mice to produce cytokines important in haemopoiesis. Neither SCF, IL‐3, GM‐CSF nor TNFa were found in conditioned media of long‐term bone marrow cultures (LTBMC) of normal or MAIDS mice. We failed to detect mRNA for TNF or IL‐lα, by reverse transcription polymerase chain reaction (RT‐PCR), in cultures derived from either normal or immunodeficient mice. Steady‐state levels of transcripts for IL‐6 were equal in cells from normal and MAIDS mice. Steady‐state levels of mRNA for TGF β1, a known inhibitor of haemopoiesis, were decreased in cultures derived from MAIDS mice at late stages of infection. The mRNA level of the multipotent haemopoie‐tic regulator stem cell factor was also decreased in MAIDS cultures as compared with normals. Transcripts encoding the transmembrane form of the growth factor were almost absent. Addition of soluble GM‐CSF and SCF only transiently increased the production of CFUs (BFUE and CFU‐G/M) in MAIDS LTBMC. These findings suggest that derangements in cytokine production in stromal cells of immunodeficient mice may contribute to the suppression of haemopoiesis observed in this disease. One mechanism of HIV‐induced depression of haemopoiesis may be via alterations of the haemopoietic microenvironrnent.