bluuebunny/crossref_metadata_2025 huggingface dataset:
Systemic lupus erythematosus with C1q deficiency
Authors: Stone N.M. , Williams A. , Wilkinson J.D. , Bird G.
Published: March 2003

We report a case of systemic lupus erythematosus associated with C1q deficiency. Our patient presented at the age of 6 years with cutaneous lupus. She later developed Raynaud’s phenomenon, non-scarring alopecia, oral ulceration and grand mal seizures due to cerebral vasculitis. Complement C3 and C4 levels were consistently normal during flares of her lupus and haemolytic activity of her complement was absent, suggesting a deficiency of an early component of the complement cascade. No C1q could be detected.
Pseudoscleroderma associated with lung cancer: correlation of collagen type I and connective tissue growth factor gene expression
Authors: Querfeld C. , Sollberg S. , Huerkamp C. , Eckes B. , Krieg T.
Published: March 2003

Pseudoscleroderma as a paraneoplastic syndrome is a rare disease. We report here a patient with lung cancer (undifferentiated squamous cell carcinoma), who developed acrosclerosis. Using in situ hybridization, marked expression of α1(I)-collagen and connective tissue growth factor (CTGF) mRNA was found in fibroblasts scattered throughout the dermis. However, transforming growth factor (TGF)-β1 expression was not detected. The pattern of CTGF gene expression and collagen synthesis was similar to that in systemic scleroderma. The absence of TGF-β1 mRNA could indicate that tumour-derived factors induce the expression of CTGF.
Telomerase activity in acute myelogenous leukaemia: clinical and biological implications
Authors: Seol Jae Goo , Kim Eun Shil , Park Woo Hyun , Jung Chul Won , Kim Byoung Kook , Lee Young Yiul
Published: March 2003

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)2D3, and vitamin D3 analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D3‐sensitive HL‐60 cells, whereas vitamin D3 insensitive K562 cells did not change its activity. This down‐regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34− cells > leukaemic CD34+ cells > normal CD34− cells > normal CD34+ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S‐phase, cytotoxicity to cytosine arabinoside and percentage of CD34+ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.
Thrombotic thrombocytopenic purpura plasma enhances platelet–leucocyte interaction in vitro
Authors: Valant Peter A. , Jy Wenche , Horstman Lawrence L. , Mao Wei‐Wei , Ahn Yeon S.
Published: March 2003

In thrombotic thrombocytopenic purpura (TTP), intravascular platelet aggregation and formation of platelet‐rich thrombi impair the microcirculation. TTP plasma has been shown to induce aggregation of normal platelets in vitro. The present study investigates the formation of activated platelet aggregates (aPAg) induced by TTP plasma, with particular attention to their binding to leucocytes (LPAg). Results were compared with the effects of plasmas from normal controls (CTL) and from patients with immune thrombocytopenic purpura (ITP) or thrombosis (THR). Following addition of test plasma to normal whole blood (WB), aPAg and LPAg were assayed by flow cytometry using mAbs against CD41 (platelet marker), CD62p (platelet activation marker) and CD45 (pan‐leucocyte marker). Compared to control plasma, TTP plasma was more potent than ITP or THR plasma in increasing aPAg; only TTP plasma significantly promoted leucocyte binding to give increased LPAg. Prior removal of neutrophils (PMN) from WB by beads coated with anti‐CD15 mAb largely prevented formation of aPAg and LPAg. However, TTP plasma added to normal platelet‐rich plasma significantly increased aPAg, which suggested possible hindrance of aPAg formation by erythrocytes and other leucocytes in PMN‐depleted blood. We concluded that TTP plasma was most potent in the induction of aPAg and unique in promoting LPAg formation in WB. Neutrophils, and not other leucocytes, appear to be essential for LPAg formation. Enhanced PMN–platelet interaction in the microcirculation may facilitate platelet adhesion to vessel walls and promote the formation of platelet‐rich microthrombi in TTP.
Flow cytometric analysis of reticulated platelets: evidence for a large proportion of non‐specific labelling of dense granules by fluorescent dyes
Authors: Robinson  , Mackie  , Khair  , Liesner  , Goodall  , Savidge  , Machin  , Harrison
Published: March 2003

The labelling of platelets with thiazole orange (TO) has been utilized by various laboratories to determine the percentage of reticulated platelets within whole blood or platelet‐rich plasma (PRP). A proportion of TO labelling, however, is not entirely mRNA specific and remains to be fully defined. Almost half of the total TO‐positive signal within normal platelets (n = 5) was shown to be abrogated upon degranulation with 80 μm thrombin receptor activating peptide (TRAP) (P = 0.006), strongly suggesting that platelet granules are non‐specifically labelling with dye. We have confirmed this hypothesis by studying TO labelling of platelets within whole blood from dense granule deficient patients, e.g. Hermansky‐Pudlak syndrome (HPS) (n = 5) and storage pool disease (SPD) (n = 4). The levels of TO‐positive platelets were found to be significantly lower than normal (P = 0.0003 and P = 0.0002 respectively), but not significantly different from TRAP degranulated platelets. Upon degranulation of HPS and SPD platelets there was very little further reduction in the TO signal. Incubation of normals and SPD whole blood with different concentrations of either TO or coriphosphine‐O confirmed that dense granules were non‐specifically labelling even at high concentrations of both dyes. These findings suggest that although TO labelling is in part RNA specific, the dense granular pool of nucleotides appears to cause a substantial amount (∼50%) of non‐specific labelling observed under these conditions of assay. This can easily be controlled for by a degranulation step with a non‐enzymatic platelet agonist such as TRAP, and may have important consequences for the eventual standardization, clinical utilization and automation of reticulated platelet assays.
Renin in acute myeloid leukaemia blasts
Authors: Wulf Gerald G. , Jahns‐Streubel Gerlinde , Nobiling Rainer , Strutz Frank , Hemmerlein Bernhard , Hiddemann Wolfgang , Wörmann Bernhard
Published: March 2003

Hypokalaemia is a clinical phenomenon in patients with acute myeloid leukaemia (AML) to which activation of the renin–angiotensin system (RAS) may contribute. Recently monocytes were found to express renin, the key initializing enzyme of the RAS. By RT/PCR, transcripts for renin were detected in four of 18 bone marrow samples from patients with AML. Three leukaemic cell lines, isolated monocytes, bone marrow stromal cells and 25 peripheral blood and 24 bone marrow samples of normal controls were negative for renin transcripts. In view of the importance of local RAS in other tissues, the expression of renin in the bone marrow of AML patients warrants further investigation.
Genes for thrombopoietin and c‐mpl are not responsible for familial thrombocythaemia: a case study
Authors: Kunishima Shinji , Mizuno Shinichi , Naoe Tomoki , Saito Hidehiko , Kamiya Tadashi
Published: March 2003

The underlying molecular basis for familial thrombocythaemia (FT), an extremely rare form of primary thrombocythaemia which occurs in an autosomal dominant manner, is currently unknown. We have investigated a family with FT and clarified whether we could detect alteration(s) in the genes coding for c‐mpl and its ligand, thrombopoietin (TPO). There was no difference in platelet c‐mpl mRNA expression levels between the affected and non‐affected individuals in the family. Nucleotide sequence analysis of the c‐mpl cDNA for the proband revealed no abnormality. We identified an intragenic tetranucleotide short tandem repeat system in the TPO gene and found non‐linkage between the TPO locus and the FT phenotype. We conclude that genes for c‐mpl and TPO are not responsible for thrombocythaemia in our FT family.
Complementary and antagonistic effects of IL‐3 in the early development of human megakaryocytes in culture
Authors: Dolzhanskiy Alexandr , Hirst John , Basch Ross S. , Karpatkin Simon
Published: March 2003

The effect of IL‐3 on the early steps in the growth and development of megakaryocytes (MK) in culture has been studied. Although thrombopoietin (TPO) by itself could support the development of mature CD41+ MK from pre‐MK, the number of cells produced was greatly augmented by the addition of IL‐3 and SCF. IL‐3 was also able to support the growth of MK colonies in semi‐solid media (CFU‐MK). The CD41+ cells that developed in suspension cultures containing IL‐3 differed phenotypically from those that developed without this agent. Cells grown in the presence of IL‐3 lost CD34 expression more rapidly, expressed lower levels of the platelet glycoproteins gpIIb‐IIIa and Ib and achieved lower degrees of polyploidy than in the absence of IL‐3. The inhibitory effects of IL‐3 were not a consequence of the dilution of the mature cells by increased numbers of immature cells since it was observed under conditions in which IL‐3 did not stimulate MK growth. The results obtained in these cultures suggest that IL‐3 plays an important role in early MK development, but inhibits further maturation after endoreduplication begins. Thus, prolonged contact with IL‐3 results in the appearance of cells that do not mature normally.
T‐helper phenotypes in the blood of myeloma patients on ECOG phase III trials E9486/E3A93
Authors: Kay Neil E. , Leong Traci , Bone Nancy , Kyle Robert A. , Greipp Phillip R. , Van Ness Brian , Oken Martin M.
Published: March 2003

T‐helper blood populations are frequently altered in multiple myeloma (MM). We measured the numbers of naive and activated cell subsets in the blood of a cohort of both previously untreated and treated MM patients. Two‐colour flow cytometry to detect total CD4+, CD4+, CD45RA+ (naive) and CD4+, CD45RO+ (activated) subsets was then used to quantify the T‐cell subsets in controls and MM patients. Previously treated MM patients either on or off treatment (n = 105) had significantly reduced (P < 0.0001) total CD4 and naive/activated cells than controls. Previously treated MM patients sampled for naive/activated cells while currently off therapy (n = 45) had no difference in the levels of CD4 and naive/activated cells compared to the currently treated patients (n = 60). However, newly diagnosed patients (n = 58) had a significantly reduced total CD4 (P = 0.023) and activated CD4 (P = 0.004), but not naive CD4 subsets, compared to controls. CD19+ cell levels above 125/μl were positively associated with higher T‐helper cell levels. There was a strong positive association for better overall survival for patients with > 395 CD4 cells/μl (P = 0.0001). These data indicate that MM patients at diagnosis have altered T‐helper subsets, with a selective reduction in activated but not naive cells. Subsequent chemotherapy or the disease process contributes to a further reduction in CD4 cells. Importantly, the association of higher CD19+ cell levels with higher T helper cells indicates that certain myeloma patients can be identified with a more quantitatively intact immune system.
A CD4+ proliferation of large granular lymphocytes expresses the protease activated receptor‐1
Authors: Macey  , Hou  , Milne  , Parameswaren  , Howe  , Cavenagh  , Howells  , Newland
Published: March 2003

The platelet‐type thrombin receptor was the first member to be identified in a family of protease activated receptors (PARs) and has been designated PAR‐1. We recently reported that the large granular lymphocytes (LGLs) in patients with proliferations of CD8+ cells co‐expressed PAR‐1 and the expression of PAR‐1 correlated with the expression of CD57. Here we show, by three‐colour immunofluorescence, that the LGLs from a patient with a rare CD4+ CD57+ monoclonal expansion also expressed PAR‐1. Northern blot analysis confirmed the presence of high levels of mRNA for PAR‐1 in these LGLs.
Expansion of dendritic cells derived from human CD34+ cells in static and continuous perfusion cultures
Authors: Soligo  , Lambertenghi Deliliers  , Quirici  , Servida  , Caneva  , Lamorte
Published: March 2003

We examined the effects of different cytokine combinations and culture conditions on the expansion and modulation of cell surface antigens of CD34+ derived dendritic cells (DCs), the most efficient antigen‐presenting cells capable of stimulating resting T cells in the primary immune response. Cells with a dendritic morphology and expressing HLA‐DR, CD1a, S100 and CD83 were maximally expanded under serum‐free conditions with the addition of SCF, GM‐CSF, TNF‐α, TGF‐β and Flt‐3 ligand (fold increase of CD1a+ cells = 102 ± 32 after 2 weeks of culture). CD34+ cells were also grown under continuous flow conditions in an artificial capillary system; after 14 d of culture, the expansion in the total cell number was lower than that of the static cultures (3.3 ± 2 v 18.9 ± 4) but the percentage of CD1a+/CD83+/CD80+ cells was considerably higher, whereas the CD14+ cells were significantly reduced (8.9 ± 2 v 26 ± 13). In continuous perfusion cultures, low levels of DC precursors and of LTC‐IC were still present up to day 14. The DCs generated under flow conditions stimulated the mixed leucocyte reaction (MLR) more than the cells grown in static cultures. By electron microscopy, cells grown in the continuous flow system showed an increased number of large cells with numerous dendritic processes and abundant multilamellar complexes. The cells expanded under these conditions were sorted on the basis of their light‐scatter properties into two fractions: one containing a predominance of CD1a+/S100+/CD83+/CD80+/CD14−‘large cells’ with great internal complexity (mature DCs); the second including ‘small cells’ either CD33+/CD14+, CD33+/CD15+ or CD33+/CD13±/CD14−. The DCs generated and selected with this method are therefore particularly well suited for immunotherapeutic protocols.
Arg260‐Cys mutation in severe factor XIII deficiency: conformational change of the A subunit is predicted by molecular modelling and mechanics
Authors: Ichinose Akitada , Tsukamoto Hiroaki , Izumi Tomonori , Yamazaki Tomio , Togashi Masaki , Takamatsu Junki , Saito Hidehiko , Umeyama Hideaki
Published: March 2003

To explore the implications of the structure/function relationships in factor XIII, a patient with severe A subunit deficiency was examined at the DNA and RNA levels. Nucleotide sequence analysis of the patient's DNA amplified by PCR revealed that the patient had a replacement of C by T in the codon for Arg260. RT‐PCR analysis demonstrated that only one kind of mRNA coding for the Arg260‐Cys mutation was expressed in the patient at a normal level. Another possible defective allele of the A subunit gene with a G‐A polymorphism was not expressed (null allele). The substitution of Arg260 by Cys located on the interface of two A subunits would preclude the reciprocal ionic interaction (salt bridge) between Arg260 and Asp404. Molecular modelling and, for the first time, molecular mechanics calculated that Cys260 changed the local conformation of the A subunit and reduced the electrostatic interaction between two monomers, suggesting destabilization of the molecule's dimer.
A common polymorphism in the tumour necrosis factor‐alpha gene associated with high TNF levels is not a risk factor for venous thromboembolism
Authors: Brown Karen , Luddington Roger , Baglin Trevor
Published: March 2003

The odds ratio of homozygosity or heterozygosity for the TNF2 polymorphism in 575 patients with venous thromboembolism compared to controls was found to be 1.0 (95% CI 0.4–2.1) and 1.1 (95% CI 0.8–1.4) respectively. Comparing subgroups of patients and controls with the factor V Leiden mutation the odds ratio for the TNF2 polymorphism was 0.7 (95% CI 0.2–2.2). Despite evidence of a link between high tumour necrosis factor (TNF) levels and hypercoagulability, our results do not indicate a link between the genetic regulation of TNF production and venous thromboembolic disease.
The effect of HFE mutations on serum ferritin and transferrin saturation in the Jersey population
Authors: Merryweather‐Clarke Alison T. , Worwood Mark , Parkinson Lisa , Mattock Chris , Pointon Jennifer J. , Shearman Jeremy D. , Robson Kathryn J. H.
Published: March 2003

High frequencies of the haemochromatosis‐related HFE C282Y mutation have been reported in North European populations, in which a high proportion of patients with the disease are homozygotes. However, the degree of penetrance of this genotype is unknown. We determined the HFE C282Y and H63D genotypes of 411 consenting volunteer blood donors on Jersey, and the serum ferritin and transferrin saturation levels of 204 of these volunteers. The C282Y allele frequency was found to be 8.3% in 822 chromosomes, indicating a homozygote frequency of 1/145. Consistent with this, four C282Y homozygotes were detected in 411 volunteers. As there are only 18 patients presently receiving treatment for haemochromatosis on Jersey, out of a total population of about 85 000, there is a large discrepancy between the number of haemochromatosis patients and the number of C282Y homozygotes in this population. In a preliminary study of 204 consenting volunteers we found a correlation between transferrin saturation and HFE H63D/C282Y genotype (P = 0.017) and between serum ferritin and genotype (P = 0.056). We also observed elevated values of transferrin saturation in the two C282Y homozygotes assayed. These results suggest that a large proportion of the many undetected C282Y homozygotes on Jersey and in similar populations could be in the preclinical stages of haemochromatosis, and warrant investigation. However, there may be a wide variation in the expression of the condition, and a more extensive study of the level of disease penetrance encompassing a large number of hitherto undetected C282Y homozygotes is therefore imperative.
Identification of a novel point mutation (Leu72Pro) in the NADH‐cytochrome b5 reductase gene of a patient with hereditary methaemoglobinaemia type I
Authors: WU YU‐Shui , Huang Chang‐Hui , Wan Yao , Huang Qiao‐Jia , Zhu Zhong‐Yong
Published: March 2003

Utilizing polymerase chain reaction (PCR) related technology, we investigated the b5R gene of a Chinese patient with hereditary methaemoglobinaemia type I and found a novel missense mutation (CTC‐CCC) at codon 72 in exon 3 of the gene. As the mutation generates an Apa I recognition site, homozygosity for the mutation was confirmed by restriction analysis of PCR‐amplified fragments from the patient's genomic DNA. We predicted that the residue replacement of Leu with Pro of the mutant enzyme would account for the b5R deficiency in the patient. The results further confirm the genetic polymorphism of b5R gene mutations found in the RCM type I.
Linkage of four polymorphisms on the αIIb gene
Authors: Ruan  , Peyruchaud  , Nurden  , Bourre
Published: March 2003

The subunits of the platelet integrin αIIbβ3 are encoded by two genes located on chromosome 17. Two pathologies are associated with structural modifications of this complex: Glanzmann's thrombasthenia and alloimmune thrombocytopenia. The former is a hereditary bleeding disorder, the latter is due to an immune response linked to the presence of specific epitopes defined by single amino acid substitutions called human platelet alloantigen (HPA) systems. Analysing the αIIb gene from 112 independent chromosomes, we have defined two new silent polymorphisms in complete linkage disequilibrium. They are reciprocally linked to HPA‐3 and a previously reported 9 pb deletion in intron 21. Linkage of these four DNA markers spanning a 5 kb fragment of genomic DNA provides a new tool for analysing αIIb gene pathology and evolution.
The outcome of children requiring admission to an intensive care unit following bone marrow transplantation
Authors: Hayes Corinne , Lush Richard J. , Cornish Jacqueline M. , Foot Annabel M. , Henderson John , Jenkins Ian , Murphy Peter , Oakhill Anthony , Pamphilon Derwood H. , Steward Colin G. , Weir Patricia , Wolf Andrew , Marks David I.
Published: March 2003

We report the results of a retrospective study of the role of intensive care unit (ICU) admission in the management of 367 children who underwent bone marrow transplantation (BMT) at a tertiary referral institution. 39 patients (11%) required 44 ICU admissions for a median of 6 d. 70% received marrow from unrelated donors, half of which were mismatched; 80% had leukaemia and two‐thirds were considered high‐risk transplants. Respiratory failure was the major reason for admission to ICU. 75% of admissions required mechanical ventilation (for a median of 5 d) and 20 patients had lung injury as defined by the criteria of the Seattle group. None of 11 patients with proven viral pneumonitis survived (P = 0.06) and only one of 20 patients with lung injury survived (P < 0.01). Six of seven patients with a primary neurological problem survived (P < 0.001); these appear to represent a good outcome group. Age, the presence of graft‐versus‐host disease, the use of inotropes, isolated renal or hepatic impairment, and paediatric risk of mortality (PRISM) score were not predictive of outcome. In total, 12 patients (27% of admissions) survived and were discharged from hospital 30 d or more after admission and eight (18%) survived >6 months. ICU admission can be beneficial to selected children post‐BMT but it may be less useful in proven viral pneumonitis. Where mechanical ventilation is required, the duration of this support should be limited unless there is rapid improvement.
Critical study of prognostic factors in childhood acute lymphoblastic leukaemia: differences in outcome are poorly explained by the most significant prognostic variables
Authors: Donadieu Jean , Auclerc Marie‐FranÇoise , Baruchel AndrÉ , Leblanc Thierry , Landman‐Parker Judith , Perel Yves , Michel Gérard , Cornu Gérard , Bordigoni Pierre , Sommelet Danielle , Leverger Guy , Hill Catherine , Schaison for the Fralle group Gérard
Published: March 2003

We determined the proportion of survival variability explained by the usual prognostic factors in childhood acute lymphoblastic leukaemia (ALL) during a prognostic study of 1552 patients enrolled in three consecutive Fralle group protocols (Fralle 83, Fralle 87 and Fralle 89). The event‐free survival rates at 5 years were 54.8% (SD 1.9), 43.1% (SD 2.7) and 55.6% (SD 2.2), respectively. In the univariate analysis the following variables were predictive of poor outcome: male gender, elevated leucocytosis (> 50 × 109/l), circulating blastosis, haemoglobin >12 g/dl, platelet count <100 × 109/l, age under 1 year or over 9 years, enlarged mediastinum, nodes, spleen and liver, T phenotype, absence of CD10+ cells; testicular and meningeal involvement, poor response to induction therapy (CCSG M3), and LDH >400 U/l. Among the cytogenetic features, hyperdiploidy had a protective effect, whereas hypodiploidy, translocation and other structural abnormalities had a negative influence, particularly in cases of t(9;22) or t(4;11). Multivariate analysis summarized the prognostic information in terms of four variables: age, gender, leucocytosis and cytogenetic features. Missing data had little influence on the results. However, despite their significance in the multivariate analysis, these four variables each had very low predictive power (1.1% for gender, 2.0% for age, 3.5% for leucocytosis, and 1.6% for cytogenetic features). Thus, the most significant prognostic factors in childhood ALL each explain no more than 4% of the variability in prognosis. This may explain the disappointing practical value of these factors and underlines the need for prognostic tools in childhood ALL.
Desferioxamine increases iron depletion and apoptosis induced by ara‐C of human myeloid leukaemic cells
Authors: Leardi Annalisa , Caraglia Michele , Selleri Carmine , Pepe Stefano , Pizzi Claudia , Notaro Rosario , Fabbrocini Antonietta , De Lorenzo Sonya , MusicÒ Manuela , Abbruzzese Alberto , Bianco Angelo Raffaele , Tagliaferri Pierosandro
Published: March 2003

We investigated whether changes in iron metabolism and the transferrin receptor (TRF‐R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara‐C). Treatment with 100 n M ara‐C for 48 h reduced thymidine uptake and increased the surface expression of the TRF‐R on leukaemic blasts derived from 13/16 (81%) patients and on the HL‐60 and U‐937 cell lines. Whereas intracellular non‐haem iron was strongly depleted 24 h after ara‐C addition, TRF‐R up‐regulation and recovery of intracellular non‐haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara‐C and the iron chelator desferioxamine (DSF) on the growth of HL‐60 and U‐937 cells. We found that desferioxamine strongly potentiated the effects of ara‐C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara‐C and an iron chelator in terms of antileukaemic effects.
Inhibited apoptosis and drug resistance in acute myeloid leukaemia
Authors: Smith B. Douglas , Bambach Barbara J. , Vala Milada S. , Barber James P. , Enger Cheryl , Brodsky Robert A. , Burke Philip J. , Gore Steven D. , Jones Richard J.
Published: March 2003

Despite extensive investigation into mechanisms of drug resistance in acute myeloid leukaemia (AML), the aetiology of therapeutic resistance is unclear. We found that five leukaemia cell lines (K562, HL‐60, CEM, CEM induced to overexpress bcl‐2, and REH) displayed parallel sensitivity to four antileukaemia drugs with different mechanisms of action, with K562 generally being the least sensitive and REH being the most sensitive. The amount of spontaneous apoptosis in the cell lines after serum‐free culture paralleled their drug sensitivity: K562 cells displayed the least apoptosis at 24 h (2.50 ± 0.24%) and REH the most (24.47 ± 8.22%). The extent of spontaneous apoptosis of leukaemic blasts from 39 patients with newly diagnosed de novo AML also correlated with the success of the intensive, infusional cytarabine‐based induction therapy. There was a median of 19.5% (range 3.6–64%) apoptotic AML cells after 24 h of serum‐free culture in patients who entered a complete remission compared with 4.2% (1.8–7.0%) apoptotic AML cells in patients who did not achieve a complete remission (P = 0.0007). Thus, inhibited apoptosis was associated with both in vitro and in vivo pan‐resistance to antileukaemic chemotherapy. The cause of inhibited apoptosis in AML is probably a function of interactions among multiple signals that influence apoptosis. Assessment of spontaneous apoptosis may serve as an important prognostic factor for AML.
Functional granulocyte/macrophage colony stimulating factor receptor is constitutively expressed on neoplastic plasma cells and mediates tumour cell longevity
Authors: Villunger Andreas , Egle Alexander , Kos Marion , Egle Daniel , Tinhofer Inge , Henn Traudl , Überall Florian , Maly Karl , Greil Richard
Published: March 2003

It has been shown that granulocyte/macrophage colony stimulating factor (GM‐CSF) is able to support myeloma cell propagation in cooperation with interleukin (IL)‐6, the major growth factor for malignant plasma cells, although the biological mechanisms involved remain unknown. Therefore we investigated (i) the expression levels of the GM‐CSF receptor (GM‐CSFR) constituents in three malignant plasma cell lines and in native malignant plasma cells, (ii) the ability of the receptor to mediate common signalling pathways regulating proliferation and cell survival in malignant plasma cell lines, and (iii) the effects of GM‐CSF on tumour cell biology.

The GM‐CSFRα subunit was detected in the malignant plasma cell lines RPMI‐8226, MC/CAR, IM‐9 as well as 6/6 native myeloma cell samples derived from the bone marrow of patients with overt disease. Furthermore, GM‐CSFR expression was also detected in the CD19+ fraction from 2/3 bone marrow samples and 5/8 peripheral blood samples derived from patients with malignant plasma cell disorders, but not in the CD19+ fraction of peripheral blood from healthy donors. The expressed cytokine receptor α‐subunit was able to constitute a functional signalling complex with the ubiquitously expressed GM‐CSFRβ subunit, as demonstrated by the fact that GM‐CSF induced the p21‐ras/mitogen‐activated protein kinase (MAPK) signalling cascade in malignant plasma cell lines. Since this signalling cascade plays an essential role in the mediation of both proliferation and cell survival, we investigated the impact of GM‐CSF on these two events. Application of GM‐CSF led to an increase of DNA‐synthesis in MC/CAR, IM‐9 and RPMI‐8226 cells. Furthermore, it increased longevity of these malignant plasma cell lines by reducing the rates of spontaneous apoptosis. We conclude that (i) the functional GM‐CSFR is commonly expressed on malignant plasma cells and that (ii) GM‐CSF promotes the clonal expansion of myeloma cells by inhibiting spontaneous apoptosis and promoting DNA synthesis.
Low‐dose aspirin does not lower in vivo platelet activation in healthy smokers
Authors: Pernerstorfer  , Stohlawetz  , Stummvoll  , Kapiotis  , Szekeres  , Eichler  , Jilma
Published: March 2003

Smoking causes atherosclerosis, and smokers have increased thromboxane (TXA2) formation. As aspirin inhibits TXA2 production we speculated that smokers would preferentially profit from inhibition of the TXA2 pathway by aspirin. Increased expression of P‐selectin, a constituent of the alpha‐granules of platelets, and increased levels of circulating (c)P‐selectin in plasma are markers for platelet activation.

The aim of this study was to compare P‐selectin expression on platelets between smokers and nonsmokers, and to compare with placebo the effect of 2 weeks administration of 100 mg/d aspirin on platelet activation in smokers. Smokers exhibited higher P‐selectin expression on platelets than non‐smokers (2.7 ± 1.8% v 1.6 ± 0.6%, P = 0.018), thus confirming increased platelet activation. Aspirin did not reduce platelet activation as demonstrated by unchanged P‐selectin expression on platelets and cP‐selectin plasma levels.
Protein Z in healthy human individuals and in patients with a bleeding tendency
Authors: Ravi Subramaniasivam , Mauron Thomas , LÄmmle Bernhard , Wuillemin Walter A.
Published: March 2003

Protein Z is a vitamin‐K‐dependent plasma glycoprotein; its physiological function is not clear. Low protein Z levels were found in patients with otherwise unexplained bleeding disorders. It was therefore suggested that low protein Z levels might be associated with a bleeding diathesis.

In the present study we measured protein Z levels in plasma samples of 48 patients with a suspected bleeding disorder and in plasma samples of 200 healthy adult individuals. We found protein Z to have a wide normal range in healthy men and women. Significantly lower protein Z levels were observed in women compared to men, whereas no correlation was found with age or other vitamin‐K‐dependent coagulation proteins. None of the 48 patients with a bleeding disorder had a protein Z level below the normal range. However, protein Z was significantly lower in the group of male patients with a bleeding history as compared to healthy men.

In conclusion, our data indicate that low‐normal protein Z levels are not associated with a bleeding tendency. However, it remains to be determined whether a low protein Z level is a weak cofactor associated with an increased bleeding tendency and whether decreased or absent protein Z (conditions not detected in our patients) might constitute a haemorrhagic diathesis.
Low CD4 counts rather than superantigenic‐like effects account for differences in expressed T‐cell receptor (TCR) repertoires between HIV‐1 seropositive long‐term non‐progressors and individuals with progressive disease
Authors: Westby Michael , Vaughan Andrew N , Balotta Claudia , Galli Massimo , Clerici Mario , Dalgleish Angus G
Published: March 2003

Analysis of HIV‐infected individuals who have stable CD4 counts many years after seroconversion may provide an insight as to how the host's immune system can successfully control HIV infection. In this study we analysed the T‐cell receptor (TCR) Vβ repertoire in 13 HIV+ individuals, seven of whom were classed as long‐term non‐progressors (LTNP), using a technique which couples anchor PCR (AnPCR) amplification of β‐chain cDNA to differential probe hybridization with non‐radioactively labelled Vβ family specific oligonucleotide probes. There were no significant differences in the expressed TCR repertoires between the LTNP group and the other HIV‐infected individuals. However, there was a statistically significant inverse correlation between CD4 count and the number of Vβ family‐specific perturbations in the recent seroconverters (SC) and those with progressive infection (PI), consistent with other shared features of clinical disease progression (Th1/Th2 switch and high frequency of viral isolation). We conclude that long‐term clinical non‐progression in HIV‐1 infection is not associated with the loss or expansion of a particular Vβ family; instead, low CD4 count in the PI and SC individuals was correlated with increased number of Vβ family‐specific perturbations relative to the LTNP group and that it is hence unlikely that HIV encodes a superantigen.
Aetiology of severe iron overload in a family with hereditary haemolytic anaemia
Authors: Dykes Anne , GÜrtler Volker , Firkin Frank
Published: March 2003

Severe iron overload is a reported complication of certain erythroid disorders which are characterized by increased erythropoietic activity. Proposed mechanisms include enhancement of iron absorption secondary to increased erythroid activity and coexistent heterozygosity or homozygosity for haemochromatosis. We performed PCR‐based analysis for the haemochromatosis‐related HFE C282Y mutation in an extended family with inherited haemolytic anaemia in which several members exhibited iron overload. The results demonstrated iron overload was associated with homozygosity but not heterozygosity for this mutation. Such an association may also exist in other erythroid disorders in which iron overload has been reported.
A role for tumour necrosis factor‐α, Fas and Fas‐Ligand in marrow failure associated with myelodysplastic syndrome
Authors: Gersuk Geoffrey M. , Beckham Cassandra , Loken Michael R. , Kiener Peter , Anderson Jeanne E. , Farrand Alan , Troutt Anthony B. , Ledbetter Jeffrey A. , Joachim Deeg
Published: March 2003

Apoptosis of haemopoietic cells in the marrow of patients with myelodysplastic syndrome (MDS) has been suggested as a mechanism for peripheral cytopenias. We determined the expression of Fas (CD95), Fas‐Ligand (Fas‐L) and TNF‐α factors known to be involved in apoptosis, in the marrow of 44 patients with MDS and characterized their functional relevance in in vitro assays of haemopoiesis. Multidimensional flow cytometry revealed phenotypically aberrant blasts as defined by orthogonal light scatter and CD45 expression in the marrow of 24/44 patients. Among those blasts Fas expression was increased on CD34‐positive cells and on cells co‐expressing HLA‐DR. In addition, Fas‐L was expressed on some CD34+ cells of MDS patients but was never detected on CD34+ cells in normal marrow. Fas and Fas‐L mRNAs as well as mRNA for TNF‐α, known to increase Fas expression in normal marrow, were up‐regulated in patients with MDS. TNF‐α protein and sTNF‐R1 levels in marrow plasma were higher in MDS patients than in controls (P < 0.002 and <0.003, respectively). However, results were dependent upon disease category: TNF‐α levels were significantly higher in patients with refractory anaemia (RA) than in patients with RA with excess blasts (RAEB) or RAEB in transformation (RAEB‐T) (P = 0.043). Conversely, the proportion of Fas‐L‐positive cells was lowest in patients with RA (P = 0.037). In marrow cultures, Fas‐Ig, rhuTNFR:Fc or anti‐TNF‐α antibody, by blocking Fas or TNF mediated signals, respectively, significantly increased the numbers of haemopoietic colonies compared to untreated cells (P < 0.001, P < 0.003, P < 0.001, respectively). These results show significant dysregulation in the expression of TNF‐α, Fas and Fas‐L in the marrow from MDS patients. Altered expression of these molecules appears to be of functional relevance in the dysregulation of haemopoiesis in MDS and may be amenable to therapeutic interventions.
Serological evidence of an association between chlamydial infections and malignant lymphomas
Authors: Anttila  , Lehtinen  , Leinonen  , Bloigu  , Koskela  , Lehtinen  , Saikku
Published: March 2003

Chronic infections may predispose to malignant growth. Recently, serological markers of chronic Chlamydia pneumoniae infection have been associated with lung cancer. Our aim was to study the possible association between chronic chlamydial infections and malignant lymphomas.

The present case–control study involved 72 patients with lymphoma (31 females and 41 males) and matched controls. 53 patients had non‐Hodgkin's lymphoma (NHL) and 19 had Hodgkin's disease. The sera, collected at the time of diagnosis, were tested for IgG antibodies and immune complexes to C. pneumoniae and C. trachomatis by a micro‐immunofluorescence method and ELISA and for IgG antibodies and immune complexes to Helicobacter pylori by ELISA.

The serological markers suggesting chronic chlamydial infection were associated with malignant lymphoma. The association was most evident for the presence of C. pneumoniae‐specific immune complexes in NHL (OR = 7.3, 95% CI 2.2–25) and appeared to be limited to men. No association between H. pylori antibodies or immune complexes and malignant lymphomas could be demonstrated.

Our study provides seroepidemiological evidence of an association between chronic chlamydial infections and lymphomas.
Unbalanced expression of Fas and CD40 in mantle cell lymphoma
Authors: Clodi Katharina , Snell Virginia , Zhao Shourong , Cabanillas Fernando , Andreeff Michael , Younes Anas
Published: March 2003

B cells are characterized by the dual expression of CD40 and Fas receptors, which can mediate their survival and death, respectively. The balance between the dynamically opposing functions of these two receptors is important for B‐cell selection, maturation and homeostasis. We found that mantle cell lymphoma (MCL) cells had a high level of CD40 and low or absent level of Fas, therefore favouring the CD40 cell survival pathway. Exogenous Fas ligand had no effect on MCL cells, whereas exogenous CD40 ligand enhanced their survival and rescued them from fludarabine‐induced apoptosis. Our data raise the possibility that the prolonged survival of MCL cells in vivo may be explained by the unbalanced expression of Fas and CD40.
Factors associated with successful mobilization of peripheral blood progenitor cells in 200 patients with lymphoid malignancies
Authors: Ketterer Nicolas , Salles Gilles , Moullet Isabelle , Dumontet Charles , Eljaafari‐Corbin Assia , Tremisi Pierre , Thieblemont Catherine , Durand Brigitte , Neidhardt‐Berard Eve‐Marie , Samaha Hanadi , Rigal Dominique , Coiffier Bertrand
Published: March 2003

Peripheral blood progenitor cells (PBPC) were mobilized and harvested in 200 patients treated for non‐Hodgkin's lymphoma (n = 148), Hodgkin's disease (n = 22) and multiple myeloma (n = 30). The variables predicting the collection of a minimal (>2.5 × 106/kg) or a high (>10 × 106/kg) CD34+ cell count were analysed. Patients were mobilized with haemopoietic growth factors following either standard chemotherapy (n = 49) or high‐dose cyclophosphamide, given alone (n = 55) or combined with high‐dose VP16 (n = 86). 10 patients received haemopoietic growth factors only. The first mobilization resulted in a PBPC harvest with enough CD34+ cells in 179/200 patients (90%). High‐dose cyclophosphamide, with or without VP16, did not mobilize a higher progenitor cell yield than standard chemotherapy. When performing multiple regression analysis in the 190 patients who received chemotherapy‐containing mobilization, only the number of previous chemotherapy regimens and the exposure to fludarabine predicted for a failure to collect a minimal PBPC count (P = 0.06 and 0.0008 respectively). The target to collect a high CD34+ cell count was negatively associated with the number of previous chemotherapy regimens (P = 0.002). When only non‐Hodgkin's lymphoma patients were considered for multivariate analysis, low‐grade histology with fludarabine appeared to be associated with poor PBPC cell yield (P = 0.08 and 0.005 respectively). This data confirms that PBPC harvest should be planned early in the disease course in transplant candidates, and can be obtained after a standard course of chemotherapy.
PRAME, a gene encoding an antigen recognized on a human melanoma by cytolytic T cells, is expressed in acute leukaemia cells
Authors: Van Baren  , Chambost  , Ferrant  , Michaux  , Ikeda  , Millard  , Olive  , Boon  , Coulie
Published: March 2003

Gene PRAME was found to encode an antigen recognized on a human melanoma cell line by an autologous cytolytic T‐lymphocyte clone. This gene is expressed at a high level in a very large fraction of tumours, such as melanomas, non‐small‐cell lung carcinomas, sarcomas, head and neck tumours and renal carcinomas. It is therefore a candidate for tumour immunotherapy even though some low expression is found in certain normal tissues. We tested by RT‐PCR the expression of PRAME on more than 250 bone marrow or blood samples from patients with a haematological malignancy. Approximately 25% of the acute leukaemia samples were positive. Remarkably, all acute myeloblastic leukaemias that carried the chromosomal translocation t(8;21), which fuses the genes AML1 and ETO, expressed PRAME at a high level.
The ATM gene and susceptibility to childhood T‐cell acute lymphoblastic leukaemia
Authors: Takeuchi Seisho , Koike Michiaki , Park Susan , Seriu Taku , Bartram Claus R. , Taub Harry E. , Williamson Ian K. , Grewal Jeffrey , Taguchi Hirokuni , Koeffler H. Phillip
Published: March 2003

Ataxia‐telangiectasia (A‐T) is a multisystem recessive disease characterized by cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency and increased risk of cancer. The ATM gene, responsible for A‐T, was recently cloned at human chromosome band 11q22‐23, a region of frequent alterations in childhood acute lymphoblastic leukaemia (ALL). Children with A‐T frequently develop T‐ALL. We investigated 18 T‐ALL samples for ATM mutations and loss of heterozygosity (LOH) at the ATM locus. No mutations of ATM were found within the coding region in the 18 T‐ALL samples, and LOH at the ATM locus was detected in three. The ATM gene appears to be an infrequently altered tumour suppressor gene in childhood T‐ALL.
VAD or VMBCP in multiple myeloma refractory to or relapsing after cyclophosphamide–prednisone therapy (protocol MY 85)
Authors: Mineur  , MÉnard  , LE LOËT  , Bernard  , Grosbois  , Pollet  , Azais  , Laporte  , Doyen  , DE Gramont  , Wetterwald  , Euller‐Ziegler  , Peny  , Monconduit  , Michaux
Published: March 2003

263 patients (median age 65±10 years) with multiple myeloma were treated with cyclophosphamide– prednisone. Out of this cohort, 103 patients had progressive disease and were randomly assigned to either VAD (vincristine, doxorubicin, dexamethasone; 50 cases) or VMBCP (vincristine, BCNU, cyclophosphamide, melphalan and prednisone; 53 cases). There were no statistical differences between the two groups with the respect to clinical, biological and radiological parameters.

There was no difference in survival between the VAD and VMBCP groups. The 4 months response rate was similar in the two groups (50% VAD, 56% VMBCP). With multivariate analysis for survival (Cox model), two factors had a statistically significant impact: Karnofsky index (>60) and albuminaemia (<34 g/l). With both Karnofsky index > 60 and albuminaemia  34 g/l, the median survival was 29 months v 2 months with a Karnofsky index   60 and albuminaemia < 34 g/l (P < 0.05).

In conclusion, VAD or VMBCP had similar activity for salvage treatment in MM refractory or relapsing to first‐line treatment with cyclophosphamide–prednisone.
Relationships between severe neonatal thrombocytopenia and maternal characteristics in pregnancies associated with autoimmune thrombocytopenia
Authors: VAlat Anne SYlvie , CAUlier Marie THÉRÈse , Devos Patrick , Rugeri Lucia , Wibaut BÉNÉdicte , Vaast PASCAl , Puech Francis , Bauters Francis , Jude Brigitte
Published: March 2003

In pregnant women with antecedents of autoimmune thrombocytopenia (AITP), no predictive factor for severe fetal thrombocytopenia has been identified. We evaluated the relationships between the course of the maternal disease before and during pregnancy and the risk of severe fetal thrombocytopenia, in 64 pregnant women with known chronic AITP antecedents, over a 12‐year period. 28 pregnant women had undergone splenectomy before pregnancy and 17 experienced severe thrombocytopenia (< 50 × 109/l) during pregnancy (monthly determination). Eight infants presented with severe thrombocytopenia at birth (12.5%), and four in the following days (6.25%). No severe haemorrhage was observed. Severe thrombocytopenia at birth was present in 57% (CI 95% 18–90%) of the infants born to mothers with severe pregnancy‐associated thrombocytopenia and splenectomy antecedents, and in 0% (CI 95% 0–15%) of the infants born to mothers who presented none of these antecedents (P = 0.001). In thrombocytopenic mothers the infant platelet counts at birth were positively correlated to the nadir maternal platelet count during the index pregnancy (r = 0.42, P = 0.0075).

These results suggest that severe autoimmune disease is a risk factor for severe fetal thrombocytopenia, and that pregnant women with no antecedent of splenectomy nor severe thrombocytopenia during pregnancy have a very low risk of severe fetal thrombocytopenia.
Phase I/II trial of PIXY321 (granulocyte‐macrophage colony stimulating factor/interleukin‐3 fusion protein) for treatment of inherited and acquired marrow failure syndromes
Authors: Taylor Douglas S. , Lee Yisheng , Sieff Colin A. , Homans Alan , Garrison Leslie , Guinan Eva C.
Published: March 2003

Fourteen paediatric patients with advanced amegakaryocytic thrombocytopenia (AMT) or other bone marrow (BM) failure syndromes were enrolled on one of two phase I/II dose escalation studies of PIXY321. PIXY321 was administered subcutaneously in doses ranging from 250 to 750 mg/m2/d. No dose‐limiting toxicity was observed. Peak absolute neutrophil count (ANC) was higher than baseline in all patients. Most transfusion‐independent patients demonstrated elevation in haematocrit and/or platelet count. Trilineage haemopoietic responsiveness was evident in the three transfusion‐independent patients. In these paediatric populations PIXY321 is well tolerated and merits consideration as a potential therapy.