bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of platelet glycoproteins and platelet/endothelial cell antibodies in patients with thrombotic thrombocytopenic purpura
Authors: Wright  , Wang  , Hornstein  , Hogarth  , Mody  , Garvey  , Blanchette  , Rock  , Freedman
Published: March 2003

Platelets and sera from 12 patients with thrombotic thrombocytopenic purpura (TTP) and 12 healthy normal control subjects were examined. As determined by quantitative flow cytometry, prior to plasma exchange therapy platelet surface glycoprotein (GP) Ib levels were similar in TTP patients and normal controls (mean 20 188 and 20 226 molecules/platelet, respectively). Platelets from patients with TTP did, however, have significantly reduced levels of GPIIb/IIIa prior to plasmapheresis (mean 36 348 v 52 505 molecules/platelet in controls; P = 0.0004) and of GPIV (mean 13 321 v 26 212 molecules/platelet in controls; P = 0.0002). An increase in activated platelets, as determined by CD62 expression, was observed in 82% of patients. Increased platelet‐associated immunoglobulins and/or complement was also seen in approximately 60% of the patients. In general, with return of platelet counts to normal levels following seven plasmaphereses, the above abnormalities were reversed, although often not to normal levels. Western blot analysis indicated the presence of antibodies reactive to platelet GPIV (88 kD) in 70% of pretreatment sera from patients with TTP; a similar band was observed in 80% of patient sera against microvascular endothelial cells. Immunofluorescence microscopic examination indicated the presence of antibody in pretreatment sera from patients with TTP to microvascular (73%) and large vessel (36%) endothelial cells. As measured by an indirect flow cytometric assay, pretreatment sera from 55% of patients with TTP were reactive with large vessel endothelial cells and 100% reacted with microvascular endothelial cells; reactivity was significantly greater against the microvascular endothelial cells (P = 0.0048) and was reduced following plasma exchange therapy. These results indicate abnormalities in platelet glycoprotein expression in TTP and suggest that anti‐platelet and anti‐endothelial cell antibodies play a role in the thrombocytopenia and vasculitis characteristic of this disorder.
Characterization of VLA‐4‐dependent myeloma cell adhesion to fibronectin and VCAM‐1
Authors: Sanz‐Rodríguez Francisco , Ruiz‐Velasco Natividad , Pascual‐Salcedo Dora , Teixidó Joaquin
Published: March 2003

The integrin VLA‐4 mediates attachment of myeloma cells to multiple myeloma (MM) bone marrow stroma. The alternatively‐spliced CS‐1 region of fibronectin (FN) and VCAM‐1 are main ligands for VLA‐4 and are both expressed on MM stroma. The H1 region is present in all FN isoforms and represents an additional binding site for VLA‐4. We employed FN fragments FN‐H89 and FN‐H0, that contain either the CS‐1 and H1, or only the H1 sites, respectively, as well as soluble VCAM‐1 (sVCAM‐1), to characterize VLA‐4‐mediated adhesion pathways used by myeloma cells to attach to MM stroma. CD38highCD45RA− cells from MM bone marrow, and the myeloma‐derived cell lines NCI‐H929, IM‐9 and RPMI 8226, specifically adhered, by different degrees, to FN‐H89, FN‐H0 and sVCAM‐1, and their VLA‐4‐dependent adhesion was substantially up‐regulated by the anti‐β1 antibody TS2/16, which increases the affinity of VLA‐β1 integrins. Furthermore, VLA‐4 function on NCI‐H929 cells was enhanced by TS2/16 during adhesion to MM stroma. The α4β7 integrin mediated a small portion of myeloma cell line adhesion to FN‐H89, mainly upon integrin activation with Mn2+. These results indicate that myeloma cells use VLA‐4 to interact with CS‐1/FN, H1/FN and VCAM‐1 on MM stroma, and that its function can be potentially up‐regulated, enabling higher degrees of cell adhesion to these VLA‐4 ligands, which might influence myeloma cell localization in the bone marrow.
Danish patients with untreated multiple myeloma do not harbour human herpesvirus 8
Authors: Rask Charlotte , Kelsen Jens , Olesen Gitte , Nielsen Johan Lanng , Obel Niels , Abildgaard Niels
Published: March 2003

The role of human herpesvirus 8 (HHV‐8) in multiple myeloma (MM) remains controversial. We examined 15 Danish MM patients before cytoreductive therapy. Mononuclear cells isolated from peripheral blood and bone marrow aspirates, as well as long‐term cultured bone marrow stromal cells, were assayed for the presence of HHV‐8 DNA. All material was tested by three simple unnested polymerase chain reaction (PCR) assays (amplifying regions of ORF26, ORFK1 and ORF75) and two nested PCR assays (amplifying regions of ORF26). HHV‐8 was not demonstrated in any of the samples. Our findings do not suggest an association between HHV‐8 and MM in the Danish population.
Neuropeptide control of bone marrow neutrophil production is mediated by both direct and indirect effects on CFU‐GM
Authors: Broome  , Whetton  , Miyan
Published: March 2003

Noradrenaline‐ and peptide‐containing nerve fibres project into the bone marrow and terminate in association with stromal cells and within the parenchyma. Peptidergic nerve terminals are also associated with antigen‐processing and ‐presenting cells throughout the body and have been shown to be important in leucocyte trafficking and wound healing, as well as haemopoiesis. Here, we tested the in vivo effects of deleting the peripheral neuropeptide network on haemopoiesis and also investigated whether the target cell population for these substances was myeloid progenitor cells (colony‐forming unit‐granulocyte/macrophage, CFU‐GM). Deletion of the neuropeptides, substance P (SP) and calcitonin gene‐related peptide (CGRP) by capsaicin abrogates normal blood cell production. These neuropeptides produced significant stimulation of colony formation from unfractionated bone marrow and elicited production of soluble factors capable of stimulating highly enriched CFU‐GM. CGRP also had a direct stimulatory effect on highly enriched CFU‐GM. Noradrenaline elicited factors that inhibited colony formation and had no direct effect on CFU‐GM. We conclude that the neuropeptides form the positive arm of a neural control system and that noradrenaline acts as a negative regulator.
Four novel non‐random chromosome rearrangements in B‐cell chronic lymphocytic leukaemia: 6p24–25 and 12p12–13 translocations, 4q21 anomalies and monosomy 21
Authors: Cuneo Antonio , Roberti Maria Grazia , Bigoni Renato , Minotto Claudia , Bardi Antonella , Milani Raffaella , Tieghi Alessia , Campioni Diana , Cavazzini Francesco , De Angeli Cristiano , Negrini Massimo , Castoldi Gianluigi
Published: March 2003

Nine patients with previously unreported chromosome changes were identified among 209 B‐cell chronic lymphocytic leukaemia (CLL) cases: three patients had a translocation involving 6p24–25; three had a 12p12–13 translocation; two had 4q21 involvement (one with coexisting 6p anomaly); and two had monosomy 21.

Interphase fluorescence in situ hybridization (FISH) detected some cryptic aberrations (+12, 6q–, 17p−, 11q−) in those patients with 6p translocations, whereas only a cytogenetically undetected 13q14 deletion was found in the remaining cases.

Atypical morphology was noted in six cases, including both cases with monosomy 21, two cases with 6p and 4q21 anomaly and one case with 12p involvement. Four of these cases also had more than one phenotype deviation with respect to the classical CLL phenotype. Disease progression after 21–51 months (median 41) was noted in two cases with 6p and 4q21 involvement and in one case with 12p anomaly and monosomy 21.

We arrived at the following conclusions: (i) 6p24–25 and, possibly, 4q21 lesions represent non‐random events in CLL, occurring in association with other well‐known unbalanced rearrangements; (ii) 12p rearrangements and monosomy 21 may possibly represent early chromosome defects that are not associated with the classical DNA gains and losses known to be present in the majority of CLL; and (iii) atypical morphology and immunophenotype as well as disease progression were frequently observed in these cases
Differential effect of interferon α on chronic myelogenous leukaemia and normal haematopoietic progenitors in a stromal cell co‐culture context: role of the flt3 ligand
Authors: Solanilla A. , El Andaloussi A. , Grosset C. , Duchez P. , Mossalayi M. D. , Mahon F. X. , Reiffers J. , Marit G. , Ripoche J.
Published: March 2003

Interferon alpha (IFN‐α) is used to treat chronic myelogenous leukaemia (CML) patients. However, its target(s) remain(s) unknown. One possibility is that there is a differing sensitivity of the leukaemic from the normal colony‐forming cell (CFC) compartments to IFN‐α. Co‐cultures of progenitors with stromal cells provide a valuable tool to dissect direct and indirect activities of IFN‐α. In this study, we have used endothelial cells (EC) as a source of stromal cells. In co‐cultures of normal progenitors with EC, IFN‐α increased the generation of clonogenic cells, mainly via an increased production of flt3 ligand (FL) by EC. In contrast, in co‐cultures of CML progenitors with EC, IFN‐α inhibited the generation of clonogenic cells, mainly by direct inhibition on the progenitors, the up‐regulation of FL production by stromal cells being unable to compensate for the direct inhibitory effects of IFN‐α. These data provide evidence for a differential effect of IFN‐α on the growth of CML and normal CFC cells in a stromal context and suggest that an alteration in the response of CML progenitor cells to FL is important in the explanation of this differential effect.
Interleukin 6, tumour necrosis factor α, interleukin 1β and interleukin 1 receptor antagonist promoter or coding gene polymorphisms in multiple myeloma
Authors: Zheng ChengYun , Huang DeRen , Bergenbrant Susanne , Sundblad Anne , Österborg Anders , Björkholm Magnus , Holm Göran , Yi Qing
Published: March 2003

Proinflammatory cytokines such as interleukin 6 (IL‐6), tumour necrosis factor α (TNF‐α) and IL‐1β are considered to be involved in the pathogenesis of multiple myeloma (MM). In the present study, we examined a G/C polymorphism at position −174 in the promoter region of IL‐6, a biallelic polymorphism at position −308 in the promoter region of TNF‐α, the TaqI restriction fragment length polymorphism in exon 5 of IL‐1β and a variable number of identical tandem repeat polymorphisms in intron 2 of IL‐1 receptor antagonist (IL‐1Ra) genes. The alleles of these loci are known to influence the level of production of the cytokines and the IL‐1Ra. Seventy‐three patients with MM, 27 with monoclonal gammopathy of undetermined significance (MGUS) and 129 healthy individuals were included. No difference was found between patients and healthy controls or between MM and MGUS patients in the distributions of genotypes and frequencies of alleles of the IL‐6 (−174), TNF‐α (−308), IL‐1βTaqI and IL‐1Ra gene polymorphisms. No associations between the polymorphisms at the loci under study and clinical factors such as age, sex, clinical stage at onset and M‐protein type were observed. Our results indicate that the cytokine (IL‐6, TNF‐α and IL‐1β) and IL‐Ra gene polymorphisms do not confer susceptibility to the development of MM.
IgG‐secreting lymphoplasmacytoid leukaemia: a B‐cell disorder with extensively mutated VH genes undergoing Ig isotype‐switching frequently associated with trisomy 12
Authors: Garand Richard , Sahota Surinder S. , Avet‐Loiseau Hervé , Talmant Pascaline , Robillard Nelly , Moreau Anne , Gaillard Fanny , Stevenson Freda K. , Bataille Régis
Published: March 2003

We investigated 16 patients with elevated serum monoclonal IgG and a leukaemic B‐cell lymphocytic disorder different from multiple myeloma. Their clinical history was that of a non‐aggressive disease with dominant splenomegaly and long survival. Whereas abnormal blood and bone marrow cells were predominantly small lymphocytes with a few lymphoplasmacytoid cells, histopathological features included a lymphoplasmacytic infiltrate in eight cases. Most frequently, abnormal blood cells displayed a CD19+CD5−CD23+/– immunophenotype different from that of chronic lymphocytic leukaemia, except in two cases with a CD19+CD5+CD23+ phenotype. Interestingly, a coexistent serum monoclonal IgM and/or surface IgMG+ with identical light chain was identified in 10 patients, whereas in the remaining six patients only IgG expression was determined. VH gene analysis was performed in eight patients to investigate the clonal origins of tumour cells. All cases utilized the VH3 family, with evidence of extensive somatic mutations and intraclonal homogeneity in all cases. VH gene analysis indicated a clonal relationship between cells expressing IgM and IgG, with one case being biclonal. Cytogenetic evaluation showed a high incidence of trisomy 12 (60%) and 13q14 deletion (40%). In conclusion, we have described an unusual subset of low‐grade lymphoma with high‐serum IgG and frequent lymphoplasmacytoid features in which tumour cells derive from post‐follicular memory B cells undergoing isotype switching with some cases arrested at both the IgM and IgG stage and others as IgG‐positive cells only.
Constitutive expression levels of CD95 and Bcl‐2 as well as CD95 function and spontaneous apoptosis in vitro do not predict the response to induction chemotherapy and relapse rate in childhood acute lymphoblastic leukaemia
Authors: Wuchter Christian , Karawajew Leonid , Ruppert Velia , Schrappe Martin , Harbott Jochen , Ratei Richard , Dörken Bernd , Ludwig Wolf‐Dieter
Published: March 2003

CD95 (Fas/APO‐1) expression and function and Bcl‐2 expression, as well as spontaneous apoptosis in vitro, have been shown to be predictive markers for the in vivo response to chemotherapy in acute myeloid leukaemia (AML). To determine the clinical significance of apoptosis‐regulating factors in acute lymphoblastic leukaemia (ALL), we investigated cell samples of children with ALL who had been included in the German ALL Berlin–Frankfurt–Münster (BFM) study using flow cytometry for constitutive expression levels of CD95 (n = 110) and Bcl‐2 (n = 110). Furthermore, we determined the extent of spontaneous apoptosis in vitro (n = 102) and susceptibility to anti‐CD95‐induced apoptosis (CD95‐sensitivity) (n = 97). We correlated these findings with the functional activity of the multidrug resistance (MDR)‐associated P‐glycoprotein (P‐gp), as detected by the rhodamine123 efflux test, immunophenotype, cytogenetics and clinical data of the patients examined. Good responders to initial prednisone therapy (‘prednisone response’) revealed significantly higher Bcl‐2 expression levels [5·4 ± 3·4 relative fluorescence intensity (RFI), n = 68] than poor responders (3·7 ± 2·6 RFI, n = 42; P = 0·002). There was no significant correlation between the other investigated parameters and prednisone response. Moreover, neither the CD95 and Bcl‐2 expression levels nor the extent of spontaneous apoptosis in vitro, CD95 sensitivity or P‐gp function were correlated with the response to induction chemotherapy or relapse rate, either for B‐cell precursor ALL or T‐cell ALL. No consistent pattern of change in CD95 (n = 10) and Bcl‐2 expression (n = 9) was noted in cases studied at both initial diagnosis and relapse. In conclusion, our findings underline the different cell biological features of primary AML and ALL cells.
Generation of the NUP98–HOXD13 fusion transcript by a rare translocation, t(2;11)(q31;p15), in a case of infant leukaemia
Authors: Shimada Hiroyuki , Arai Yasuhito , Sekiguchi Shinichiro , Ishii Toru , Tanitsu Shizue , Sasaki Michiko
Published: March 2003

We report a case of de novo acute myelomonocytic leukaemia with the t(2;11)(q31;p15) translocation in a Japanese female infant. The NUP98–HOXD13 fusion transcript generated by the translocation was detected in the patient's bone marrow cells by reverse transcription–polymerase chain reaction (RT‐PCR). Additionally, ectopic expression of the normal allele of the HOXD13 gene was observed in this patient, suggesting that it might be associated with leukaemogenic development. This case is the third report of t(2;11) leukaemia with NUP98–HOXD13 and the first report showing that NUP98 rearrangements are associated with infant leukaemia, as well as therapy‐related acute myelogenous leukaemia or myelodysplastic syndrome.
Surface expression of HLA‐DM on dendritic cells derived from CD34‐positive bone marrow haematopoietic stem cells
Authors: Min Yoo Hong , Lee Seung Tae , Choi Kyung Mi , Hahn Jee Sook , Ko Yun Woong
Published: March 2003

HLA‐DM has been known to be largely absent from the cell surface of antigen‐presenting cells, accumulating instead in the intracellular compartment. In this study, we demonstrated that a population of HLA‐DM‐positive (HLA‐DM+) dendritic cells (DCs) can be identified in anin vitroculture of CD34+bone marrow haematopoietic stem cells. CD34+bone marrow cells of healthy donors were used to generate DCs with the recombinant human cytokines granulocyte–macrophage colony‐stimulating factor (GM‐CSF), tumour necrosis factor α (TNF‐α) and stem cell factor (SCF), both with and without interleukin 4 (IL‐4). Flow cytometric analysis demonstrated that HLA‐DM+cells comprised 2·5 ± 0·9% and 1·8 ± 0·4% of the CD34+cell‐derived progeny in the presence of GM‐CSF, TNF‐α and SCF after 7 d and 14 d of culture respectively. The number of HLA‐DM molecules expressed per HLA‐DM+cell on d 7 was significantly higher than that on d 14 (1410 ± 47 versus 370 ± 25,P < 0·05). The addition of IL‐4 to the cytokines from the commencement of culture increased the proportion of HLA‐DM+cells and increased the number of HLA‐DM molecules per HLA‐DM+cell significantly (P < 0·05). Although most of the HLA‐DM+cells expressed CD1a, CD80 or CD86 antigen, only a small proportion of CD1a+, CD80+or CD86+cells expressed HLA‐DM. About half the HLA‐DM+cells expressed CD83. The addition of IL‐4 resulted in a decrease in the expression of CD83 on the HLA‐DM+cells on d 7. Microscopic evaluations of sorted HLA‐DM+cells revealed the characteristic morphological features of DCs. Primary mixed lymphocyte cultures demonstrated that the HLA‐DM+cells elicited a vigorous proliferation of allogeneic T cells. The level of antigen‐specific T‐cell activation induced by antigen‐pulsed, chloroquine‐treated HLA‐DM+cells was substantially higher than that induced by HLA‐DM−cells (P < 0·05). These results show that HLA‐DM can be used as a useful DC lineage‐specific marker, as well as a tool for the characterization of DCs and human immunotherapy.
α‐Thalassaemia as a result of a novel splice donor site mutation of the α1‐globin gene
Authors: Harteveld C. L. , Beijer C. , Van Delft P. , Zanardini R. , Bernini L. F. , Giordano P. C.
Published: March 2003

We describe the characterization of an α+‐thalassaemia determinant as a result of a transition of G→A of the donor splice consensus site sequence of the first intron of the α1‐globin gene (α1IVS I‐1). The mutation was found in combination with the South‐East Asian α0‐thalassaemia deletion in an haemoglobin (Hb)H patient and her sister, both of Thai origin. Sequencing of the abnormally spliced mRNA product revealed the presence of a cryptic splice site in exon 1 of the α1‐globin gene. No normally spliced α1mRNA was detected. The abnormally spliced mRNA product from the α1‐gene carrying the mutation does not lead to functional protein and causes a mild HbH‐disease phenotype when in combination with the deletion type α0‐thalassaemia.
Variant translocations in sporadic Burkitt's lymphoma detected in fresh tumour material: analysis of three cases
Authors: Cario Gunnar , Stadt Udo zur , Reiter Alfred , Welte Karl , Sykora Karl‐Walter
Published: March 2003

Burkitt's lymphoma/Burkitt cell leukaemia (BL) is characterized by one of the reciprocal translocations involving the MYC oncogene on chromosome 8 and one of the immunoglobulin (Ig) loci on chromosomes 14, 2 or 22. In the few cell lines with the variant translocations t(2;8) and t(8;22) reported to date, the breakpoints on chromosome 8 were located downstream of MYC at a distance of up to 300 kb and more. Here, we describe three new cases with variant translocations. Fresh tumour material from paediatric patients, negative for the common translocation t(8;14), was analysed using a long‐distance (LD) polymerase chain reaction (PCR) approach. On chromosome 8, primers were derived from several different regions 3′ of MYC, and on chromosomes 2 and 22 from the constant regions of the Ig kappa (Igκ) and lambda (Igλ) genes. One translocation t(2;8) and two t(8;22) were detected. In the t(2;8) translocation, the chromosome 8 breakpoint was located 2 kb 3′ of the MYC exon 3 and the chromosome 2 breakpoint within an unrearranged Igκ locus. The breakpoints of the two translocations t(8;22) were detected 16 kb for one and 58 kb for the other downstream of MYC. Sequencing the t(8;22) translocation in one of the cases showed hypermutation of the translocated variable Vλ4b gene. The presence of hypermutated variable regions in the t(8;22) case suggests germinal centre B cells as the origin of this translocation. The t(2;8) translocation is the first description of a translocation t(2;8) involving an unrearranged Igκ gene. A mechanism different from V–J recombination and somatic hypermutation has to be proposed for this translocation.
Therapy‐related myelodysplastic syndrome of recipient origin after allogeneic bone marrow transplantation for acute lymphoblastic leukaemia
Authors: Au W. Y. , Lie A. K. W. , Ma S. K. , Leung Y. H. , Siu L. L. P. , Kwong Y. L.
Published: March 2003

Therapy‐related myelodysplastic syndrome (t‐MDS) is a very rare complication of allogeneic bone marrow transplantation (BMT). A woman with T acute lymphoblastic leukaemia (T‐ALL) received an allogeneic BMT from a donor with the β‐thalassaemic trait. Five years after BMT, the red cell indices returned to normal after an initial conversion to microcytosis, implying autologous haematopoietic regeneration. Seven years after BMT, thrombocytopenia developed and marrow examination confirmed t‐MDS, with a characteristic karyotype 46,XX,inv(3)(q21;q26), del(5)(q13),add(17)(p11). Retrospective molecular analysis of donor/recipient chimaerism showed gradual regeneration of recipient cells after BMT, culminating at the time of t‐MDS. Our findings illustrate the unusual occurrence of t‐MDS after allogeneic BMT. Re‐emergence of recipient haematopoesis may herald the development of a haematological malignancy different from the original neoplastic clone for which the BMT was performed.
Myeloma‐reactive allospecific cytotoxic T lymphocytes lyse target cells via the granule exocytosis pathway
Authors: Chiriva‐Internati Maurizio , Du Jianhui , Cannon Martin , Barlogie Bart , Yi Qing
Published: March 2003

Accumulating evidence indicates that a graft‐vs.‐myeloma effect (GVM) and its associated clinical remission of the disease can be induced by donor lymphocyte infusion in myeloma patients who have relapsed after allogeneic bone marrow transplantation. Although it is believed that GVM is induced by allospecific T cells, T‐cell subsets and the mechanisms involved in the killing of myeloma cells by donor T cells have not been studied. In this study, we generated allospecific cytotoxic T lymphocyte (CTL) lines against three different myeloma cell lines, ARK, ARP‐1 and U266, from unmatched healthy donors and examined their cytotoxicity against the target cells. Our results demonstrate that the allospecific CTLs efficiently lysed myeloma cells. The observed cytotoxicity was mediated mainly by CD8+ T cells and inhibited by MHC class I‐blocking antibody. Furthermore, the CTLs lysed the target cells via the perforin‐mediated pathway, as concanamycin A, but not brefeldin A (the selective inhibitors for perforin‐ or Fas‐mediated pathways respectively) or tumour necrosis factor‐α (TNF‐α)‐blocking antibody, abrogated the cytolytic activity of the cells. These CTLs expressed and produced predominantly TNF‐α and interferon‐γ (IFN‐γ), indicating that they belong to the type 1 T‐cell subsets. Taken together, these results indicate that CD8+ allospecific T cells may be responsible for mediating GVM and that the granule‐mediated lysis of target cells is the major pathway in the CD8+ T‐cell response against myeloma cells.
The fleet feet of haematopoietic stem cells: rapid motility, interaction and proteopodia
Authors: Frimberger Angela E. , McAuliffe Christina I. , Werme Kimberly A. , Tuft Richard A. , Fogarty Kevin E. , Benoit Brian O. , Dooner Mark S. , Quesenberry Peter J.
Published: March 2003

Haematopoietic stem cells (HSCs) have been extensively characterized regarding in vivo engraftment, surface epitopes and genetic regulation. However, little is known about the homing of these rare cells, and their intrinsic motility and membrane deformation capacity. We used high‐speed optical‐sectioning microscopy and inverted fluorescent videomicroscopy to study highly purified murine lineage‐negative, rhodamine‐low, Hoechst‐low HSCs over time under various in vitro conditions. We discovered extremely rapid motility, directed migration to stromal cells and marked membrane modulation. High resolution images with three‐dimensional reconstruction showed the general presence of microspikes. Further, pseudopodia (proteopodia) were observed that were induced by stromal‐derived factor‐1 and steel factor. Proteopodia were directed towards and were quenched by stromal cells, at times bridged HSCs, and could rapidly retract or detach from cells. Proteopodia were also observed in vivo with homed HSCs in frozen sections of murine spleen, lung and heart. This is the first demonstration that HSCs are both fast and highly malleable in phenotype.
Anti‐thrombotic and haemorrhagic effects of active site‐inhibited factor VIIa in rats
Authors: Ghrib F. , Léger P. , Ezban M. , Kristensen A. T. , Cambus J. P. , Boneu B.
Published: March 2003

Active site‐inhibited factor VIIa (FFR‐rFVIIa) competes with factor VIIa (FVIIa) for binding to tissue factor (TF) and exerts an anti‐thrombotic effect. We report an evaluation of the anti‐thrombotic properties of FFR‐rFVIIa in a model of thrombosis involving two thrombogenic surfaces. Uncoated glass capillaries or glass capillaries coated with TF were incorporated into an arterioarterial shunt in the rat and the occlusion time (OT) of the shunt was determined. An anti‐thrombotic activity of FFR‐rFVIIa was shown only on the TF‐coated surface: the OT of the shunt was significantly prolonged, from 167 ± 34 s in control animals to 312 ± 42 s after i.v. bolus administration of 4 mg/kg FFR‐rFVIIa. This OT was similar to those observed with the uncoated shunts in untreated animals (353 ± 84 s). In vitro preincubation of the TF‐coated shunt with FFR‐rFVIIa significantly prolonged the OT to 245 ± 45 s in the absence of detectable amounts of FFR‐rFVIIa in the plasma. rFFR‐rFVIIa weakly prolonged the tail template bleeding time by a factor of 1·5. This effect was more pronounced in animals pretreated with heparin. The anti‐thrombotic and prohaemorrhagic effects of FFR‐rFVIIa were totally reversed by administration of an equidose of rFVIIa. These results provide new information on the pharmacological properties of FFR‐rFVIIa that will be useful for its clinical development.
In vitro drug resistance and prognostic impact of p16INK4A/P15INK4B deletions in childhood T‐cell acute lymphoblastic leukaemia
Authors: Ramakers‐van Woerden N. L. , Pieters R. , Slater R. M. , Loonen A. H. , Beverloo H. B. , Van Drunen E. , Heyman M. , Calero Moreno T. , Rots M. G. , Van Wering E. R. , Kamps W. A. , Janka‐Schaub G. E. , Veerman A. J. P.
Published: March 2003

p16 gene deletions are present in about 70% of primary paediatric T‐cell acute lymphoblastic leukaemia (T‐ALL) and 20% of common/precursor B‐cell ALL cases. It is not clear what the impact of the frequent p16 deletions is within the subgroup of T‐lineage ALL. We studied the relationship between p16/p19ARF deletions, using fluorescence in situ hybridization, and in vitro drug resistance and prognosis in childhood T‐ALL at diagnosis. The cellular drug resistance was measured with the methyl thiazol tetrazoliumbromide assay using a panel of drugs and the thymidylate synthase inhibition assay for methotrexate. There was a complete overlap of individual LC50 values of p16 gene homozygously deleted and p16 germ‐line cases for most of the nine classes of drugs tested. The only difference was for dexamethasone: the p16‐deleted group was more sensitive than the germ‐line p16 group (P = 0·030). The homozygously deleted p16 T‐ALL patients (n = 34) treated with the modern multiagent chemotherapy schemes of the Dutch Childhood Leukaemia Study Group ALL‐VII/‐VIII or Co‐operative ALL‐92/‐97 protocols have a significantly lower 5‐year disease‐free survival (DFS) than germ‐line p16 T‐ALL (n = 25) (65·1 ± 9·1% vs. 95·5 ± 4·4%, Plog rank= 0·021). Hence, this study identifies a subpopulation of primary childhood T‐ALL that appears to have an extremely high DFS. However, the observed differences in outcome do not seem to be related to intrinsic resistance for the tested drugs.
Recognition of chronic myelogenous leukaemia cells by autologous T lymphocytes primed in vitro against the patient's dendritic cells
Authors: Müller Ludmila , Provenzani Carmelinda , Faul Christoph , Pawelec Graham
Published: March 2003

Defects in immune responses are common in patients with chronic myelogenous leukaemia (CML). However, using dendritic cells (DCs) to promote T‐cell immunity in vitro may nonetheless elicit potent specific anti‐tumour responses for use in immunotherapy. Here, we show that DCs generated from CML patients had a typical dendritic phenotype and were able to stimulate autologous T cells. Three primed T‐cell lines were studied in more detail in one patient. They were stimulated by autologous CML cells, but not by normal non‐leukaemic cells from the patient's HLA‐identical sibling. This was blocked by HLA‐DR‐specific, but not HLA‐DQ‐ or HLA‐DP‐specific antibodies. CML‐stimulated cytokine secretion, including interferon‐γ and granulocyte macrophage‐colony stimulating factor, suggested a Th1‐type phenotype for these sensitized anti‐leukaemic T cells. This study therefore shows that cells with a functional dendritic phenotype can be generated from the blood of CML patients and are potent inducers of T‐cell responses to tumour cells. This approach allows sensitization of patients' T cells by their own particular tumour without the need to identify the exact leukaemia antigens involved, and may find application in immunotherapy of CML.
Short‐term phytohaemagglutinin‐activated mononuclear cells induce endothelial progenitor cells from cord blood CD34+ cells
Authors: Kang Hyoung Jin , Kim Sung Chun , Kim Young Ju , Kim Chul Woo , Kim Joong Gon , Ahn Hyo Seop , Park Sang Ick , Jung Myeong Ho , Choi Bum Chae , Kimm Kuchan
Published: March 2003

Endothelial progenitor cells (EPCs) were recently demonstrated to exist in human cord blood. Phytohaemagglutinin (PHA), a potent mitogen for mononuclear cells was used to induce EPCs from unsorted cord blood mononuclear cells (CBMCs). Adherent cells in clusters appeared approximately 24 h after CBMCs were cultured in plain Roswell Park Memorial Institute media containing 10% fetal bovine serum (culture media) and PHA. Adherent cells were further propagated for 1 week in plain culture media. Flow cytometry and Di‐I staining analyses showed that CD45−, CD34+, Flk‐1+, CD31+ or VE‐cadherin+ EPCs were induced and that they were mainly from the CD34+ cell compartment. When enriched CD34+ cells alone were stimulated with culture supernatant of the PHA‐activated CBMCs, they neither proliferated readily nor induced EPCs. Because EPCs first appeared within the clustering cells that expressed high levels of fibronectin and vascular endothelial growth factor (VEGF), our data suggest that both cell–cell/cell–matrix interaction and the local VEGF action are important in the induction of EPCs. Thus, we demonstrate for the first time that EPCs are induced from human cord blood stem cell populations that interact with neighbouring PHA‐activated CBMCs. This finding may have a significant implication in inflammatory cell‐mediated vasculogenesis and angiogenesis in vivo.
Greater inhibition of platelet procoagulant activity by antibody‐derived glycoprotein IIb–IIIa inhibitors than by peptide and peptidomimetic inhibitors
Authors: Lages Bruce , Weiss Harvey J.
Published: March 2003

Antibody‐derived GPIIb–IIIa antagonists, such as the c7E3 Fab fragment abciximab, have been shown to inhibit platelet procoagulant activity as well as platelet aggregation. Whether low‐molecular‐weight peptide‐derived and peptidomimetic antagonists also inhibit platelet procoagulant activity in a similar manner has not been fully investigated. We compared the effects of the antibody‐derived antagonists c7E3 Fab and m10E5 IgG, the peptide‐derived antagonists eptifibatide, MK‐852 and RGDS, and the peptidomimetic RO44–9883 on platelet procoagulant activity and on the stimulated cytosolic calcium increases that promote procoagulant activity. Procoagulant activity was measured as prothrombinase activity in gel‐filtered platelets, activated by collagen plus thrombin or collagen alone, with and without stirring. The stimulated increases in cytosolic calcium were measured in parallel samples of platelets loaded with fura‐2AM. Both c7E3 and m10E5 inhibited prothrombinase activity by 40–50% under all conditions of activation tested and inhibited cytosolic calcium increases to a similar extent in stirred, but not unstirred, platelets. The low‐molecular‐weight antagonists caused significantly less inhibition of prothrombinase activity in collagen plus thrombin‐stimulated platelets, and produced no inhibition but rather a slight enhancement of activity in platelets stimulated by collagen alone. These antagonists also had little or no effect on the cytosolic calcium increases in stirred platelets. These differential effects of antibody‐derived versus non‐antibody GPIIb–IIIa antagonists on procoagulant activity may be a factor contributing to the differing anti‐thrombotic effects of these antagonists seen in clinical trials.
Donor interleukin 1 receptor antagonist genotype associated with acute graft‐versus‐host disease in human leucocyte antigen‐matched sibling allogeneic transplants
Authors: Cullup H. , Dickinson A. M. , Jackson G. H. , Taylor P. R. , Cavet J. , Middleton P. G.
Published: March 2003

Interleukin 1 (IL‐1) is involved in various autoimmune and inflammatory diseases. IL‐1 receptor antagonist (IL‐1Ra) is the naturally occurring antagonist to IL‐1α and ‐1β. Polymorphisms of IL‐1β have been associated with variations in IL‐1β production (nucleotides +3953 and −511). A variable number tandem repeat (VNTR) polymorphism in the IL‐1Ra gene has been associated (allele 2) with increased IL‐1Ra production. We examined these polymorphisms in human leucocyte antigen (HLA)‐matched allogeneic bone marrow transplant patients and donors. IL‐1Ra VNTR (allele 2) in the donor genotype was more frequent with milder acute graft‐versus‐host disease (aGvHD) grades 0–II (29 out of 59 transplants) than severe GvHD grades III–IV (2 out of 18 transplants) (P = 0·0032). This association was confirmed in a subgroup with cyclosporine monotherapy prophylaxis: donor possession of allele 2 was again associated with milder aGvHD, grades 0–II (19 out of 38 transplants), than grades III–IV (1 out of 14) (P = 0·0042) transplants. No association was found between the IL‐1β−511 or IL‐1β+3953 polymorphism and severity of GvHD. Recipient IL‐1Ra VNTR genotype (allele 2) showed a strong trend towards association with aGvHD severity (P = 0·0697). Thus, the donor genotype for the IL‐1Ra polymorphism has an apparent protective role against acute GvHD following transplantation and may be an additional factor for individual risk assessment for complications, including GvHD, post transplant.
A cautionary tale: how to delete mouse haemopoietic stem cells with busulphan
Authors: Jopling Chris , Rosendaal Martin
Published: March 2003

In this study treating mice with the ‘correct’ dose of busulphan did not necessarily destroy all haematopoietic stem cells. In certain circumstances host stem cells survived undetected and subsequently resumed haemopoiesis. This may apply to the use of busulphan clinically. We found that the following conditions determined the deletion of mouse stem cells using busulphan: (1) graft size – grafting more than 106 marrow cells (∼0·3% of the animal's stem cells) concealed the survival of stem cells; (2) dose of busulphan – insufficient busulphan did not kill all host stem cells; (3) old or improperly stored busulphan failed to delete all host stem cells; furthermore (4) the survival of host stem cells should be assessed by typing many kinds of circulating cells; and (5) tests should be carried out to determine if busulphan has killed all host stem cells by typing circulating blood cells at appropriate intervals.
The storage defects in grey platelet syndrome and αδ‐storage pool deficiency affect α‐granule factor V and multimerin storage without altering their proteolytic processing
Authors: Hayward Catherine P. M. , Weiss Harvey J. , Lages Bruce , Finlay Marisa , Hegstad Anna‐Catharina , Zheng Shilun , Cowie Alison , Massé Jean‐Marc , Harrison Paul , Cramer Elisabeth M.
Published: March 2003

Among proteins stored in α‐granules, multimerin and factor V share unusual features: they bind to each other, are proteolysed to unique forms and are stored eccentrically in α‐granules. These unique features of their processing led us to study these proteins in alpha delta storage pool deficiency (αδ‐SPD) and grey platelet syndrome (GPS, α‐SPD), two conditions known to impair α‐granule protein storage. Platelet factor V and multimerin were severely reduced in GPS, whereas they ranged from reduced to normal in αδ‐SPD. The platelet levels of factor V and multimerin in these disorders indicated multimerin deficiency was not predictive of platelet factor V deficiency, although it reduced the amount of multimerin associated with platelet factor V. In GPS only, the defect in storing proteins was associated with increased multimerin and multimerin‐factor V complexes in plasma. Like normal platelets, GPS and αδ‐SPD platelets contained factor V mainly in granules. Platelet factor V and multimerin were proteolysed to normal platelet forms in GPS and αδ‐SPD platelets, indicating that these conditions preserve some aspects of normal α‐granule protein processing. Although we found factor V can be stored in platelets deficient in multimerin, our data indicate that multimerin storage influences the point at which multimerin binds factor V.
Detection and clinical significance of human acute myeloid leukaemia progenitors capable of long‐term proliferation in vitro
Authors: Sutherland Heather , Blair Allison , Vercauteren Suzanne , Zapf Richard
Published: March 2003

Acute myeloid leukaemia (AML) blasts within individual patients are heterogeneous in their surface antigen expression and proliferative ability suggesting that, subsequent to transformation, differentiation occurs creating a hierarchy of progenitors in AML. Cells that can produce AML colonies (colony forming units, CFU) in growth factor containing suspension cultures (SC) over 4–8 weeks have a phenotype similar to AML progenitors that engraft non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mice, but different from bulk AML blasts, suggesting that these AML SC‐initiating cells (SC‐IC) may be early progenitors. In this study, we evaluated culture conditions that provide for optimal growth of AML progenitors capable of long‐term proliferation. The frequency of CFU, both initially and after 2–4 weeks of SC, varied over four logs between individual patients and was not related to French–American–British subtype. Using limiting dilution, the proliferative potential of individual SC‐IC varied from 1 to 480 CFU. The frequency of CFU from SC after > 4 weeks was prognostic for patient survival, and correlated with NOD/SCID engrafting ability. These results suggest the use of long‐term culture as an assay for AML cells with leukaemia sustaining potential.
Acute myeloblastic leukaemias of FAB types M6 and M4, with cryptic PML/RARα fusion gene formation, relapsing as acute promyelocytic leukaemia M3
Authors: Virchis Andres , Massey Edwin , Butler Tina , Devaraj Prema , Wright Faith , Secker‐Walker Lorna , Prentice H. Grant , Mehta Atul
Published: March 2003

Demonstration of either the translocation t(15;17)(q22;q21) or the fusion of PML and RARα genes is regarded as diagnostic for acute myeloid leukaemia (AML) of FAB type M3, but has occasionally been seen in other FAB types. We present two such cases. Case 1 presented with FAB type M6 and a complex karyotype involving chromosomes 1, 2, 11 and 17. Bone marrow relapse of FAB type M3 followed autologous bone marrow transplantation. Subsequent marrow dysplasia and an M6 relapse were accompanied by a new cytogenetic clone involving chromosomes X, 2, 4, 6, 7 and 16. Fluorescence in situ hybridization (FISH) of metaphase chromosomes at diagnosis showed insertion of material from chromosome 17 into a ‘normal’ 15 with juxtaposition of PML and RARα. Case 2 presented as AML M4 and relapsed as M3. Cytogenetic analysis at diagnosis and in relapse showed 46,XY,t(15;17)(q22;q11),del(16)(q22). FISH analysis showed this to be a three‐way translocation involving chromosomes 15, 16 and 17 again with juxtaposition of PML and RARα. Reverse transcription‐polymerase chain reaction (RT‐PCR) revealed PML/RARα fusion at diagnosis, in remission and in first relapse. These examples strengthen the case for RT‐PCR screening of all AML patients for these fusion genes.
Coexistence of a novel homozygous nonsense mutation in exon 13 of the factor V gene with the homozygous Leiden mutation in two unrelated patients with severe factor V deficiency
Authors: Van Wijk Richard , Montefusco Maria Claudia , Duga Stefano , Asselta Rosanna , Van Solinge Wouter , Malcovati Massimo , Tenchini Maria Luisa , Mannucci Pier Mannuccio
Published: March 2003

A novel homozygous 3571C→T nonsense mutation predicting the synthesis of a truncated factor V (FV) molecule was identified in exon 13 of the human coagulation factor V gene in two unrelated Italian probands with undetectable plasma levels of FV antigen and activity. Both patients were also homozygous for the FV Leiden mutation. Reverse transcription polymerase chain reaction studies showed strongly reduced mRNA levels of the mutant FV allele and FV heavy and light chains were not measurable in the plasma of the probands and reverse transcriptase. Haplotype analysis indicated that the nonsense mutation in both families had a common founder a long time ago.
Dramatic improvement of POEMS syndrome following autologous haematopoietic cell transplantation
Authors: Rovira M. , Carreras E. , Bladé J. , Graus F. , Valls J. , Fernández‐Avilés F. , Montserrat E.
Published: March 2003

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, serum monoclonal protein and skin changes) is a rare plasma cell disorder of unknown pathogenesis and is diagnosed by the demonstration of a plasma cell proliferation at the biopsy of an osteoesclerotic lesion. When the lesions are in a limited area, radiation therapy is usually highly effective. Patients with disseminated disease require systemic chemotherapy, which is not effective in most cases. A patient with severe widespread POEMS syndrome resistant to melphalan who experienced a dramatic improvement after high‐dose melphalan followed by autologous haematopoietic cell transplantation (AHCT) is reported. We believe that this is the first reported case of POEMS syndrome treated with AHCT, a procedure that could be considered in similarly affected patients.
Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells
Authors: Rodella Luigi , Zamai Loris , Rezzani Rita , Artico Marco , Peri Giovanni , Falconi Mirella , Facchini Andrea , Pelusi Giuseppe , Vitale Marco
Published: March 2003

Human natural killer (NK) cells constitutively express the β‐ and γ‐chains of the interleukin 2 (IL‐2)/IL‐15 receptor, and both IL‐2 and IL‐15 are able to activate NK cell proliferation and cytotoxicity. When IL‐2‐primed human NK cells are exposed to sensitive targets (i.e. K562) they undergo apoptosis mediated by the β2‐integrin CD18. Here, we demonstrate that: (i) endothelial cells, similar to K562 tumour target cells, induce apoptosis of IL‐2‐primed NK cells; (ii) endothelial‐ and K562 cell‐induced apoptosis is significantly lower in IL‐15 than in IL‐2‐stimulated NK cells; (iii) a critical role in the apoptosis of IL‐2‐primed NK cells is played by the α‐chain of the IL‐2 receptor. Our data show for the first time that IL‐2‐activated NK cells can die by apoptosis upon contact with the vascular endothelium, which is a necessary step for their extravasation, with a direct pathophysiological relevance on the strategy of adoptive immunotherapy of cancer. On the other hand, IL‐15, although generating a similar level of activation of NK cells, largely prevents their apoptotic fate. Therefore, IL‐15 produced early in the immune response, when T cells are not yet activated, generates lymphokine‐activated killer cells that are efficient killers relatively protected from apoptosis. Once activated, T cells produce IL‐2 that overcomes the effect of IL‐15 on NK cells, paving the way for their death by apoptosis.
Early response to chemotherapy as a prognostic factor in childhood acute lymphoblastic leukaemia: a methodological review
Authors: Donadieu J. , Hill C.
Published: March 2003

Published studies of the prognostic value of the early response to induction treatment in childhood acute lymphoblastic leukaemia (ALL) were analysed. Three criteria were used to judge the early treatment response: persistence of peripheral blasts (PPB) or of bone marrow blasts (PBMB) during induction therapy and minimal residual disease (MRD) after completion of induction therapy. Studies with more than 50 patients, published between 1980 and 2000, were reviewed. Among 13 659 distinct articles published on ALL, we identified only 43 applicable studies. Within‐ and between‐laboratory variations were evaluated in only one study. Treatment modalities differed among, and sometimes within, studies. The cut‐off points used in the statistical analyses were never discussed, and in many studies appeared to be selected after multiple tests. The proportion of missing data was > 30% in almost all studies of MRD, as a result of technical difficulties and not missing samples. PPB and PBMB were associated with shorter survival in, respectively, 13 out of 14 and 15 out of 16 studies. Detection of MRD was associated with poor outcome in 12 of the 13 studies. Because none of the parameters used to measure the early response to induction therapy for childhood ALL have been properly assessed as prognostic factors, we conclude that they should be considered only as candidate prognostic indicators pending more thorough studies.