bluuebunny/crossref_metadata_2025 huggingface dataset:
Megakaryocytic differentiation is accompanied by a reduction in cell migratory potential
Authors: Mathur Anthony , Hong Ying , Martin John F. , Erusalimsky Jorge D.
Published: March 2003

Megakaryocytes (MKs) have been found in the peripheral circulation, suggesting that they can migrate out of the bone marrow. In order to evaluate if megakaryocytic differentiation confers a migratory phenotype, we investigated this property in the haematopoietic cell lines MO7e and UT‐7/mpl and in CD34+progenitor cells before and after induction of differentiation by thrombopoietin (TPO). Migration was studied using a bicompartmental culture system in the presence or absence of a bone marrow endothelial cell monolayer. Preincubation with TPO led to a significant reduction in stromal cell‐derived factor‐1 (SDF‐1)‐induced migration of MO7e cells (0·7% ± 0·08% for TPO‐treated vs. 2·6% ± 0·3% for controlsP < 0·05). A similar decreased migratory response was seen with UT‐7/mpl cells (7·4% ± 0·4% for TPO‐treated vs. 11·1% ± 0·01% for controls,P < 0·05), although these cells did not migrate in response to SDF‐1. CD34+cells partially differentiated with TPO showed decreased migration following further TPO‐induced maturation (13·9% ± 1·8% for TPO‐treated vs. 24·1% ± 1·8% for untreated,P < 0·05). This reduction was more pronounced in the large MK (≥ 4n) fraction. These results demonstrate that megakaryocytic differentiation is accompanied by a partial suppression of the haematopoietic cell migratory phenotype.
Monocytes, but not endothelial cells, downregulate the anticoagulant activity of activated protein C
Authors: Colucci Mario , Stramaglia Anna M. , Mascolo Elisa , Napoleone Emanuela , Lorenzet Roberto , Semeraro Nicola
Published: March 2003

Activated protein C (APC) is a natural anticoagulant and inhibits thrombin generation by degrading factors Va and VIIIa. We evaluated the ability of APC to inhibit blood coagulation triggered by lipopolysaccharide (LPS)‐stimulated [tissue factor (TF)‐expressing] human mononuclear cells (MNCs) or umbilical vein endothelial cells (HUVECs). Using a plasma recalcification assay, we found that APC (up to 53·3 nmol/l final concentration) had a poor anticoagulant effect in the presence of LPS‐stimulated MNCs, whereas it caused a marked prolongation of clotting time in the presence of LPS‐stimulated HUVECs. A poor response to APC was also observed when platelet‐free MNCs, monocyte‐enriched preparations or the monocytoid cell line U937 were tested. Using a TF‐independent (FXa‐induced) thrombin generation assay, we demonstrated that both LPS‐stimulated and unstimulated MNCs negated the inhibitory activity of APC. Direct determination of FVa activity indicated that MNCs were less efficient than HUVECs in promoting FVa inactivation by APC. Together, our results suggest that MNCs, at variance with HUVECs, protect factor Va from inactivation by APC, probably through the expression of a membrane component not present on endothelial cells. These strengthen the importance of monocytes in fibrin deposition associated with pathological conditions characterized by monocyte recruitment and activation.
Extracellular Tat activates c‐fos promoter in low serum‐starved CD4+ T cells
Authors: Gibellini Davide , Re Maria Carla , Ponti Cristina , Celeghini Claudio , Melloni Elisabetta , La Placa Michele , Zauli Giorgio
Published: March 2003

The regulatory human immunodeficiency virus‐1 (HIV‐1) Tat protein shows pleiotropic effects on the survival and growth of both HIV‐1‐infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c‐fos mRNA and protein in serum‐starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c‐fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c‐fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c‐fos is functional as demonstrated by induction of the AP‐1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat‐mediated induction of c‐fos and AP‐1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV‐1 replication, occurring predominantly in activated/proliferating CD4+ T cells.
Rapid protein‐based assays for the diagnosis of T–B+ severe combined immunodeficiency
Authors: Gilmour Kimberly C. , Cranston Treena , Loughlin Sam , Gwyther Jackie , Lester Tracy , Espanol Teresa , Hernandez Manuel , Savoldi Gianfranco , Davies E. Graham , Abinun Mario , Kinnon Christine , Jones Alison , Gaspar Hubert B.
Published: March 2003

The severe combined immunodeficiencies (SCID) are a heterogeneous group of conditions arising from a variety of molecular defects. The X‐linked form of SCID (X‐SCID) is caused by defects in the common gamma chain (γc), and is characterized by a T–B+NK− immunophenotype. This lymphocyte profile is seen in an autosomal recessive form of SCID caused by mutations in the JAK3 molecule. Thus, X‐SCID and JAK3‐deficient SCID are clinically and immunologically indistinguishable. Knowledge of the precise molecular defect is essential for antenatal diagnosis, carrier testing and for treatment using somatic gene therapy. To identify the molecular defect in children presenting with a T–B+NK− form of SCID, we have developed rapid assays based on flow cytometric analysis of γc, immunoblotting for JAK3 and γc, and detection of interleukin‐2 (IL‐2)‐induced tyrosine phosphorylation of JAK3. Sixteen T–B+NK− SCID patients from 15 families were examined. Nine had no detectable γc, four had abnormal γc expression and no IL‐2‐induced JAK3 tyrosine phosphorylation, and one had normal γc expression but no IL‐2‐induced JAK3 tyrosine phosphorylation, although JAK3 was present. All these patients had mutations identified in their γc gene. Two patients exhibited normal γc expression, but JAK3 was not detected by immunoblotting and these patients were confirmed as having JAK3 gene mutations. Thus, these protein‐based assays have led to rapid molecular diagnoses in T–B+ SCID that have subsequently been confirmed by genetic analysis.
Age as the major predictive factor of long‐term response to splenectomy in immune thrombocytopenic purpura
Authors: Fabris F. , Tassan T. , Ramon R. , Carraro G. , Randi M. L. , Luzzatto G. , Moschino P. , Girolami A.
Published: March 2003

Sixty‐one consecutive patients undergoing splenectomy for chronic immune thrombocytopenia were retrospectively evaluated. Platelet response was considered as complete (CR) when platelet count rose to > 100 × 109/l, partial (PR) when 30–100 × 109/l or absent (NR) if otherwise. Follow‐up (mean time 7·6 years) was possible in 54 patients. Forty‐eight patients (88%) had an immediate response to splenectomy (39 CR, 9 PR) whereas six (12%) were NR. Thirty‐six responders (67%) had sustained remission (31 CR; 5 PR) without further treatment; thrombocytopenia recurred in 12 patients (33%). The probability curve of continued remission showed a constant relapse‐rate during the first 36 months; a further step of relapse was observed beginning 70 months after surgery. The only positive predictive factor for the long‐term response to splenectomy was age < 40 (P < 0·005). Neither duration of thrombocytopenia nor previous response to medical treatment (steroids and/or intravenous immunoglobulins) were related to splenectomy response.
The retinoblastoma family member, p107, is active in non‐dividing monocyte‐derived dendritic cells
Authors: Ardeshna K. M. , Ahmed F. , Devereux S. , Thomas N. S. B.
Published: March 2003

A member of the retinoblastoma family of cell cycle regulatory proteins, p107, is not normally expressed in non‐cycling cells. We demonstrate here that p107 is expressed in monocytes as they differentiate into dendritic cells under the influence of granulocyte‐macrophage colony‐stimulating factor and interleukin 4, a process shown not to involve cellular proliferation. We show that p107 is localized to the nucleus of these cells and is active, in that it binds an E2F‐DNA binding site, together with E2F transcription factors. These findings suggest a hitherto unknown role for p107 in non‐proliferating dendritic cells that warrants further investigation.
The biological significance of HLA‐DP gene variation in haematopoietic cell transplantation
Authors: Petersdorf Effie W. , Gooley Ted , Malkki Mari , Anasetti Claudio , Martin Paul , Woolfrey Ann , Smith Anajane , Mickelson Eric , Hansen John A.
Published: March 2003

Although it has been over 25 years since HLA‐DP was mapped to the major histocompatibility complex (MHC), its biological functions remain ill‐defined. We sought to test the hypothesis that HLA‐DP functions in a manner similar to that of other class II genes by measuring the risk of clinically severe grades III–IV acute graft‐vs.‐host disease (GVHD) associated with recipient HLA‐DP disparity after haematopoietic cell transplantation. HLA‐DPB1 exon 2 was sequenced in 205 patients who underwent transplantation from HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 allele‐matched unrelated donors. HLA‐DPB1 mismatched recipients experienced a significantly increased risk of acute GVHD compared with HLA‐DP‐identical transplants. Patients who were mismatched for a single HLA‐DPB1 allele had an odds ratio (OR) of 1·0 (0·5, 2·2; P = 0·99) and patients who were mismatched for two alleles had an OR of 2·2 (1·0, 4·9; P = 0·06) for developing acute GVHD. Compared with matched and single‐allele mismatched transplants, patients who were mismatched for two DPB1 alleles had an OR of 2·2 (1·2, 4·1; P = 0·01). HLA‐DP plays an important role in the alloimmune response. A threshold effect of multiple HLA‐DP disparities is evident in determining the risk of acute GVHD after haematopoietic cell transplantation from unrelated donors.
Influence of highly active anti‐retroviral therapy on response to treatment and survival in patients with acquired immunodeficiency syndrome‐related non‐Hodgkin's lymphoma treated with cyclophosphamide, hydroxydoxorubicin, vincristine and prednisone
Authors: Navarro José‐Tomás , Ribera Josep‐Maria , Oriol Albert , Vaquero Manuel , Romeu Joan , Batlle Montserrat , Flores Alonso , Millá Fuensanta , Feliu Evarist
Published: March 2003

Combined highly active anti‐retroviral therapy (HAART) with protease and reverse transcriptase inhibitors has modified the natural history of opportunistic infections and neoplasms in human immunodeficiency virus (HIV)‐infected patients. We analysed the influence of HAART on the response to treatment and survival in a series of 58 patients with acquired immune deficiency syndrome (AIDS)‐related non‐Hodgkin's lymphoma (NHL) treated with CHOP (cyclophosphamide, hydroxydoxorubicin, vincristine and prednisone). Two groups of patients were included: (i) forty‐one patients diagnosed with NHL between 1988 and 1996 who were not treated with HAART; (ii) seventeen patients diagnosed since 1996, who were receiving or commenced HAART when NHL was diagnosed. The response rate to CHOP was higher in group 2 (13 out of 17 cases; 75%) than in group 1 (14 out of 41 cases; 34%) (P = 0·003). The 2‐year probability of event‐free survival (EFS) [95% confidence interval (CI)] for group 1 was 0·5 (0·24–0·74), whereas for group 2 it was 0·85 (0·61–0·90) (P = 0·024). The lymphoma‐free survival (LFS) was also significantly different for both groups (2‐year LFS probability 0·53 vs. 1·0,P = 0·04). The median (95% CI) overall survival (OS) for group 1 was 7 months (range, 3–10·8 months), whereas it was not reached in group 2 (P = 0·0015). In the multivariate analysis for remission attainment, the only variables with a higher probability to achieve complete remission (CR) were HAART (P = 0·01) and International Prognostic Index score 1 (P = 0·02). The only statistically significant variable in the multivariate analysis for EFS was HAART (P = 0·049) and the variables with prognostic value for OS in the multivariate analysis were B symptoms (P = 0·01) and HAART (P = 0·003). Patients with AIDS‐related NHL treated with CHOP and HAART had a higher CR rate than those treated only with CHOP. In this study, HAART was an independent prognostic factor for CR, OS and EFS in patients with AIDS‐related NHL.
Deletion of the factor IX gene as a result of translocation t(X;1) in a girl affected by haemophilia B
Authors: Krepischi‐Santos A. C. V. , Carneiro J. D. A. , Svartman M. , Bendit I. , Odone‐Filho V. , Vianna‐Morgante A. M.
Published: March 2003

A balanced de novo translocation t(X;1) is described in a girl with severe haemophilia B. The translocated X was shown cytologically to be preferentially active, and methylation analysis of the DXS255 locus confirmed the skewed X‐inactivation with the paternal allele being the active one. Cytogenetic and molecular analysis showed that this chromosomal rearrangement led to the deletion of at least part of the factor IX gene. Therefore, the girl was heterozygous for factor IX deficiency and expression of her clinical phenotype was the result of the inactivation of the normal maternal X chromosome. The localization of one of the X chromosome translocation breakpoints in YAC clone 957F9, that was demonstrated to map distally to the factor IX gene, revealed the complexity of this chromosomal rearrangement.
Anti‐beta 2 glycoprotein 1 and anti‐annexin V antibodies in women with recurrent miscarriage
Authors: Arnold J. , Holmes Z. , Pickering W. , Farmer C. , Regan L. , Cohen H.
Published: March 2003

While it has been established that anti‐phospholipid antibodies (aPL) are associated with recurrent miscarriage (RM), the importance of anti‐β2 glycoprotein 1 (GP1) IgG and anti‐annexin V IgG antibodies as risk factors for RM is undefined. We have investigated the prevalence of anti‐β2 GP1 IgG and anti‐annexin V IgG antibodies in 54 aPL‐positive and 48 aPL‐negative women with RM. The prevalence of IgG anti‐β2 GP1 antibodies was not significantly different in persistently aPL‐positive women with RM (7%), aPL‐negative women with RM (6%) and the normal parous control group (3%). Anti‐annexin V IgG antibody prevalence was significantly increased in aPL‐positive women with RM compared with aPL‐negative women with RM (P = 0·01). The elevations were found in 35%, 19% and 16% of aPL‐positive women with RM, aPL‐negative women with RM and the control group respectively. No women showed positivity for both anti‐β2 GP1 IgG and anti‐annexin V antibodies. Anti‐β2 GP1 IgG antibodies do not appear to be contributory to the investigation of women with RM. Anti‐annexin V antibody positivity, although associated with aPL positivity in women with RM, is not an independent risk marker.
Protein S levels are lower in women receiving desogestrel‐containing combined oral contraceptives (COCs) than in women receiving levonorgestrel‐containing COCs at steady state and on cross‐over
Authors: Mackie Ian J. , Piegsa Karin , Furs Sally‐ann , Johnson Juliet , Bounds Walli , Machin Samuel J. , Guillebaud John
Published: March 2003

This study aimed to identify specific haemostatic changes that might account for previous observations of higher venous thromboembolic risk among users of combined oral contraceptives (COCs) containing desogestrel (DSG) than levonorgestrel (LNG). Sixty‐three current users of monophasic 30 μg oestrogen COCs containing either LNG or DSG omitted one pill‐free interval (PFI), switching immediately either to the opposite formulation for one cycle or continuing with the same pill. Venesection followed the initial PFI after one cycle (21 tablets) and two cycles (42 tablets) of continuous pill taking, and after the following PFI. Protein S was lower in users of DSG than LNG formulations after the first PFI (mean ± SD, 0·67 ± 0·09 vs 0·76 ± 0·10, P < 0·001) and after one cycle (0·61 ± 0·09 vs 0·76 ± 0·09, P < 0·0001). Protein S decreased when switching from LNG to DSG pills (0·77 ± 0·07–0·65 ± 0·06, P < 0·0001), mirrored by an increase at switching from DSG to LNG formulations (0·61 ± 0·08–0·73 ± 0·10, P < 0·005). Mean protein S levels remained within the normal range. Three different markers of thrombin generation remained unaltered. Potential explanations for COC‐related thrombotic events are ‘acquired resistance to activated protein C’ or inhibition of fibrinolysis. A potential role has been described for protein S deficiency in both. A further triggering factor is a probable prerequisite for actual thrombosis, but pill‐takers whose levels of protein S were in the lowest percentiles may be at greatest risk.
Cost‐effectiveness of high‐dose chemotherapy in first‐line treatment of advanced multiple myeloma
Authors: Sampson F. C. , Beard S. M. , Scott F. , Vandenberghe E.
Published: March 2003

The cost‐effectiveness of high‐dose chemotherapy in multiple myeloma was considered as part of a UK National Health Service Executive regional evidence‐based appraisal process. The use of high‐dose chemotherapy supported by autologous stem cell transplantation in patients under 65 years of age was shown to provide a marginal benefit of 0·7 life‐years over conventional chemotherapy. This corresponded to an incremental cost ‘per life‐year gained’ figure of approximately £15 000, based upon initial treatment costs and trial‐period data only. The use of high‐dose chemotherapy in the first‐line treatment of advanced multiple myeloma improves event‐free and overall survival and appears to be a cost‐effective treatment option.
The role of thrombopoietin in post‐operative thrombocytosis
Authors: Folman Claudia C. , Ooms Marike , Kuenen B Bart , De Jong Shreyas M. , Vet Raymond J. W. M. , De Haas Masja , Von Dem Borne Albert E. G. Kr
Published: March 2003

Thrombopoietin (Tpo), the main regulator of thrombocytopoiesis, is a probable candidate to play a role in the increase in platelet counts that is frequently seen after surgery. In the current study, serial blood samples of patients that underwent major surgery were analysed with respect to Tpo kinetics, platelet turnover and inflammatory cytokines. Platelet Tpo content and plasma Tpo levels rose before platelet counts increased, suggesting that Tpo was indeed responsible for the elevation in platelet counts. In addition, an increase in interleukin 6 (IL‐6) levels, but not in IL‐11 and tumour necrosis factor alpha levels, was seen before the rise in Tpo concentration. In vitro, IL‐6 was shown to enhance Tpo production by the HepG2 liver cell line. Thus, increased Tpo levels after surgery, possibly resulting from enhanced Tpo production under the influence of IL‐6 or other inflammatory cytokines, are involved in an enhanced thrombocytopoiesis.
Variables to predict engraftment of umbilical cord blood into immunodeficient mice: usefulness of the non‐obese diabetic–severe combined immunodeficient assay
Authors: Ballen Karen K. , Valinski Helen , Greiner Dale , Shultz Leonard D. , Becker Pamela S. , Hsieh Chung Cheng , Stewart F. Marc , Quesenberry Peter J.
Published: March 2003

Umbilical cord blood is an alternative stem cell source for patients without matched family donors. In this study, we examined several parameters that have not been studied in detail – radiation dose, cell dose, age of mice, and maternal and neonatal characteristics of the cord blood donor – that affect engraftment of cord blood in non‐obese diabetic–severe combined immunodeficient (NOD–scid) mice. Engraftment, measured using flow cytometry analyses of human CD45+ cells, was highest in 400 cGy‐treated mice. Successful engraftment was demonstrated up to 6 months, with a mean engraftment of 31% (range 0–67%) of human cells in recipient bone marrow. Engraftment was skewed to B lymphocytes. The radiation dose of 350 cGy resulted in superior survival of the murine recipients compared with 400 cGy (P = 0.03). The sex of the NOD–scid recipients had a significant effect on survival (female superior to male, P = 0·01), but not on engraftment. There were high levels of variability among different cord units and among animals injected with the same cord unit. This variability may limit the clinical usefulness of the NOD–scid mice as hosts for the quantification of human stem cells.
A prospective analysis of the pattern of immune reconstitution in a paediatric cohort following transplantation of positively selected human leucocyte antigen‐disparate haematopoietic stem cells from parental donors
Authors: Eyrich Matthias , Lang Peter , Lal Shangara , Bader Peter , Handgretinger Rupert , Klingebiel Thomas , Niethammer Dietrich , Schlegel Paul G.
Published: March 2003

Transplantation of haematopoietic stem cells from human leucocyte antigen (HLA)‐disparate parental donors presents a promising new approach for the treatment of patients lacking a HLA‐matched donor. Success against major obstacles such as graft‐versus‐host disease (GvHD) and graft rejection has recently been demonstrated, so that immune reconstitution is one of the prime factors that determines the long‐term prognosis following transplantation. Twenty children transplanted with megadoses of highly purified CD34+ haematopoietic stem cells after rigorous T‐cell depletion were prospectively monitored for their immune reconstitution during the first post‐transplant year. Natural killer (NK) cells showed a marked increase on d +30. T and B cells began to reconstitute on d +72 and +68 respectively. During extended follow‐up, their numbers and proliferative capacity upon mitogen stimulation continually increased. Early reconstituting T cells were predominantly of a primed, activated phenotype with severely skewed T‐cell receptor (TCR)‐repertoire complexity. Naive T cells emerged 6 months post transplantation, paralleled by an increase in TCR‐repertoire diversity. All patients self‐maintained sufficient immunoglobulin levels after d +200. This study demonstrates that paediatric recipients of highly purified, haploidentical stem cells are able to reconstitute functioning T‐, B‐ and NK‐cell compartments within the first post‐transplant year. This, together with the absence of significant GvHD, provides a strong indication for this approach to be considered in children who lack a HLA‐matched donor.
Rapid genotyping of human platelet antigen 5 with fluorophore‐labelled hybridization probes on the LightCycler
Authors: Kretzschmar Evelyne , Von Ahsen Nicolas , Trobisch Heiner
Published: March 2003

Genotyping of human platelet antigens (HPAs) can be useful for the diagnosis and therapy of alloimmune thrombocytopenic syndromes such as neonatal alloimmune thrombocytopenic purpura, post‐transfusion purpura and refractoriness to platelet transfusion therapy. We developed a single‐tube method for HPA‐5 genotyping on the LightCycler that combines rapid‐cycle polymerase chain reaction with allele‐specific fluorescent probe melting for mutation detection. Our method is fast, robust and suitable for routine HPA‐5 typing. This work extends recent studies on HPA‐1 typing using the LightCycler.
Evidence of a role for a non‐matrix‐type metalloproteinase activity in the shedding of syndecan‐1 from human myeloma cells
Authors: Holen Ingunn , Drury Noel L. , Hargreaves Philip G. , Croucher Peter I.
Published: March 2003

Syndecan‐1 is a cell surface proteoglycan that is expressed on human myeloma cells and is thought to act as a co‐receptor for certain extracellular matrix proteins and growth factors. The ectodomain of syndecan‐1 is thought to be shed from the surface of myeloma cells, although the exact mechanism of release remains unclear. In this study, we used a panel of inhibitors to identify the class of proteinase responsible for shedding the soluble syndecan‐1 ectodomain from human myeloma cells. Using enzyme‐linked immunosorbent assay, flow cytometry and immunocytochemistry, we demonstrated that myeloma cell lines expressed syndecan‐1 on their surface and that this was shed constitutively, but to a varying extent. In addition, phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C, stimulated a marked loss of cell surface syndecan‐1 from each of the cell lines and this was associated with a corresponding increase in soluble syndecan‐1. Inhibitors of serine and cysteine proteinases, and matrix‐type metalloproteinases, did not inhibit constitutive or PMA‐stimulated syndecan‐1 shedding from JJN3 and RPMI 8226 cells. However, BB‐94, a hydroxamate‐based, broad‐spectrum, metalloproteinase inhibitor, substantially suppressed constitutive and PMA‐stimulated syndecan‐1 loss from myeloma cells. These data indicate that a non‐matrix‐type metalloproteinase is responsible for syndecan‐1 shedding from the surface of myeloma cells.
Enhanced activation of B cells in a granulocyte colony‐stimulating factor‐mobilized peripheral blood stem cell graft
Authors: Tayebi Hakim , Lapierre Valérie , Saas Philippe , Lienard Agnes , Sutton Laurent , Milpied Noel , Attal Michel , Cahn Jean‐Yves , Kuentz Mathieu , Blaise Didier , Hervé Patrick , Tiberghien Pierre , Robinet Eric
Published: March 2003

In a randomized study that compared human leucocyte antigen‐identical allogeneic granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood stem cell (PBSC) versus bone marrow (BM) transplantation, the expression of activation markers, CD23, CD25 and CD45RO by B cells, was compared in blood before and after G‐CSF mobilization and in PBSC versus BM grafts. The fractions of CD23+ and CD25+ B cells were higher in PBSC than in BM grafts. Moreover, we observed a G‐CSF‐induced increase in B‐cell fractions in blood as well as in PBSC grafts when compared with BM grafts. Such an enhanced B‐cell activation could contribute to the accelerated kinetics of immuno‐haematological reconstitution, the occurrence of acute haemolysis in the ABO minor incompatibility setting, as well as the increased incidence of chronic graft‐versus‐host disease observed after PBSC transplantation.
Donor–recipient incompatibility at CD31‐codon 563 is a major risk factor for acute graft‐versus‐host disease after allogeneic bone marrow transplantation from a human leucocyte antigen‐matched donor
Authors: Balduini Carlo L. , Frassoni Francesco , Noris Patrizia , Klersy Catherine , Iannone Angela M. , Bacigalupo Andrea , Giorgiani Giovanna , Di Pumpo Michele , Locatelli Franco
Published: March 2003

Disparities at minor histocompatibility antigens (mHA) are thought to be responsible for acute graft‐versus‐host disease (aGVHD) in patients receiving bone marrow transplantation (BMT) from a human leucocyte antigen (HLA)‐matched donor. Although some mHA have been identified in humans, their role in aGVHD has not. Patients (n = 150) receiving a BMT from an HLA‐matched donor were investigated for a correlation between aGVHD and donor/recipient incompatibility for seven polymorphisms previously proposed for mHA (HA‐1, H‐Y, CD31‐codon 125, CD31‐codon 563, HPA‐1, HPA‐3 and HPA‐5). Only mismatch at CD31‐codon 563 predicted grade II–IV aGVHD. The risk derived from CD31‐codon 563 mismatch was the same as that derived from the use of bone marrow from an unrelated donor. We suggest that donor/recipient compatibility at CD31‐codon 563 should be added to HLA‐typing for donor selection and/or adjustment of aGVHD prophylaxis.
A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand
Authors: Fullard John , Murphy Ronan , O'Neill Sarah , Moran Niamh , Ottridge Brian , Fitzgerald Desmond J.
Published: March 2003

We have identified a patient designated as (GTa) with Glanzmann's Thrombasthenia (GT) diagnosed on the basis of a prolonged bleeding time and failure of the patient's platelets to aggregate. The number of glycoprotein (GP)IIb/IIIa receptors on the platelet surface was 37% of normal and those receptors displayed a defect in soluble fibrinogen binding. Nevertheless, GTa platelets showed increased adhesion to solid‐phase fibrinogen and binding affinity for the RGD‐mimetic 3H‐SC52012, a non‐peptide GPIIb/IIIa antagonist. Dithiothreitol (DTT) and ADP enhanced the affinity for [3H]‐SC52012 in normal platelets, but had little effect in GTa platelets. These findings suggested that GTa platelets were locked in an altered affinity state. Genetic analysis showed that GTa was a compound heterozygote for the GPIIIa gene. One allele showed a deletion at the 3′ end of exon 3 resulting in a premature stop codon. The second GPIIIa allele had a G to A transition at nucleotide 577, resulting in a Val193Met substitution. HEK 293T cells transfected with mutant GPIIb/IIIaV193M bound [3H]‐SC52012 with a higher affinity than wild‐type GPIIb/IIIa, and this was not increased by DTT. The mutant receptor distinguishes between platelet adhesion and aggregation, and demonstrates the phenotype that may be expected when platelet aggregation alone is inhibited.
Increased apoptosis of bone marrow CD34+ cells and impaired function of bone marrow stromal cells in patients with systemic lupus erythematosus
Authors: Papadaki Helen A. , Boumpas Dimitrios T. , Gibson Frances M. , Jayne David R. , Axford John S. , Gordon‐Smith E. C. , Marsh Judith C. W. , Eliopoulos George D.
Published: March 2003

The changes in bone marrow (BM) stem cell reserve and function and stromal cell function in patients with active systemic lupus erythematosus (SLE) were investigated. The study was carried out on seven SLE patients and 28 healthy controls using flow cytometry and in vitro cell culture assays. We found that patients had low CD34+ cells, compared with the control group, reflecting the decrease of both CD34+/CD38− and CD34+/CD38+ cells. Patient CD34+/Fas+ but not CD34−/Fas+ cells were significantly increased. Apoptotic (7AADdim) cells were higher among CD34+/Fas+ than among CD34+/Fas− cells, and individual values of apoptotic CD34+ cells strongly correlated with the number of CD34+/Fas+ cells. These findings are suggestive of a Fas‐mediated apoptosis accounting for the low CD34+ cells in SLE patients. Moreover, we found that patients had low numbers of granulocyte‐macrophage colony‐forming units (CFU‐GM) and erythroid burst‐forming units (BFU‐E), compared with the control group, and that the generation of colony‐forming cells in long‐term BM cultures was significantly reduced. Patient BM stroma failed to support allogeneic progenitor cell growth. In one patient, CD34+ cells were increased, apoptotic CD34+/Fas+ cells were normalized and defective stromal cell function was restored after autologous stem cell transplantation. We concluded that defective haemopoiesis in SLE patients is probably caused, at least in part, to the presence of autoreactive lymphocytes in BM.
Burkitt's acute lymphoblastic leukaemia transformatation after myelodysplastic syndrome
Authors: Ikeda Takashi , Sato Ken , Yamashita Takuya , Kanai Yoshiaki , Kuwada Naruo , Matsumura Takuya , Nakamura Yukitsugu , Kimura Fumihiko , Motoyoshi Kazuo
Published: March 2003

We describe a patient with myelodysplastic syndrome (MDS) that transformed to Burkitt's acute lymphoblastic leukaemia (ALL). The leukaemic blasts were negative for peroxidase staining, and expressed CD10, CD19, CD22, CD38, human leucocyte antigen (HLA)‐DR and surface immunoglobulin (sIg) M, but neither sIgD nor sIgG were expressed. Chromosomal study during the ALL phase showed t(8;22)(q24;q11) in addition to the karyotypes determined during the MDS phase. Furthermore, overexpression of c‐myc mRNA was confirmed in ALL blasts. These findings indicate that MDS transformed to Burkitt's ALL through multiple cytogenetic evolutions, the final event of which seems to be overexpression of the c‐myc gene.
Correction of stromal cell defect after bone marrow transplantation in aplastic anaemia
Authors: Scopes John , Ismail Medhat , Marks Karen J. , Rutherford Timothy R. , Draycott Gwen S. , Pocock Christopher , Gordon‐Smith Edward C. , Gibson Frances M.
Published: March 2003

Defects in stromal cell function have been demonstrated in a number of aplastic anaemia (AA) patients. Here we have studied a patient with severe AA and abnormal stromal cell function who underwent bone marrow transplantation (BMT). The objective of this study was to investigate the timing and the mechanism of correction of the stromal defect after transplantation. The patient, a 25‐year‐old woman with severe AA, underwent BMT from her brother. BM was obtained from the patient on five occasions: 2 weeks pre BMT, and 3, 8, 16 and 21 months post BMT. Stromal cells were grown to confluence and recharged with purified CD34+ cells from normal donors. The support of such cells, as assessed by weekly colony‐forming assay (CFU) of non‐adherent cells, was compared with that of stromal layers grown from normal BM. A novel technique of combined fluorescence in situ hybridization (FISH) and immunocytochemistry was used to determine the origin of specific stromal cell types on cytospins of stroma post BMT. Stromal function was defective at 2 weeks pre BMT and at 3 months post BMT, but returned to normal at 8 and 16 months post BMT. At 21 months post BMT, stromal fibroblasts and endothelial cells were shown to be of recipient origin, and macrophages and T cells were of donor origin. We present here evidence in a case of severe AA for defective stromal function before BMT and delayed normalization of function after BMT. This correlated with engraftment of donor macrophages and T cells, but not fibroblasts and endothelial cells.
Analysis of clonal B‐cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B‐chronic lymphocytic leukaemia
Authors: Jelinek Diane F. , Tschumper Renee C. , Geyer Susan M. , Bone Nancy D. , Dewald Gordon W. , Hanson Curtis A. , Stenson Mary J. , Witzig Thomas E. , Tefferi Ayalew , Kay Neil E.
Published: March 2003

Recent reports suggest that the expression of germline (GL) Ig variable region heavy‐chain genes (VH) is a negative prognostic factor for B‐cell chronic lymphocytic leukaemia (B‐CLL) patients and that CLL B‐cell CD38 expression may be a surrogate marker of Ig VH gene status. Currently, however, the usefulness of this surrogate marker is controversial. Therefore, our goal was to study the ability of CD38 to act as a surrogate marker for Ig VH somatic mutation (SM), and to identify differences in overall survival (OS), progression‐free survival (PFS) and response in B‐CLL patients based on these two markers. We first assessed the relationship between CD38 expression and Ig VH status on 131 B‐CLL patients, including 66 patients enrolled in three North Central Cancer Treatment Group Trials. Although the mean percentages of CD38+ clonal B cells were significantly higher for patients classified as GL versus SM, CD38 was not a reliable marker for clonal B‐cell SM. Overall, GL patients exhibited significantly shorter OS and PFS times than SM patients. Despite the inability of clonal B‐cell CD38 expression to predict Ig VH mutation status, patients with ≥ 30% CD38+ cells did have shorter PFS and OS times than did CLL patients with < 30% CD38+ cells. Thus, the relationship between CD38 expression and Ig VH mutation status in B‐CLL is not straightforward. Nevertheless, analysis in a co‐operative group clinical trial setting suggests that both B‐cell markers alone or in combination may have clinical usefulness. These data strongly encourage the study of these biological markers as they relate to disease heterogeneity in B‐CLL.
Matrix metalloproteinase and tissue inhibitors of metalloproteinase secretion by haematopoietic and stromal precursors and their production in normal and leukaemic long‐term marrow cultures
Authors: Marquez‐Curtis L. A. , Dobrowsky A. , Montaño J. , Turner A. R. , Ratajczak J. , Ratajczak M. Z. , Janowska‐Wieczorek A.
Published: March 2003

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate the turnover of the extracellular matrix and may modulate the biology of haematopoietic cells. We investigated whether MMPs and TIMPs are produced in long‐term marrow cultures (LTMCs) established from normal donors and acute myelogenous leukaemia (AML) patients, and by fibroblast‐ (F), granulocyte macrophage‐ (GM) and megakaryocyte‐ (Meg) colony‐forming unit (CFU) and erythroid burst‐forming unit (BFU‐E)‐derived precursor cells. ProMMP‐9 levels were highest (> 400 ng/ml) at week 1 of normal LTMC, whereas proMMP‐2, TIMP‐1, TIMP‐2 and TIMP‐3 levels peaked (up to 1000 ng/ml) after the establishment of the adherent layer. In LTMC from AML patients, these patterns of secretion were reversed. Moreover, we found that after a 24 h incubation in serum‐free media, normal CFU‐GM‐, BFU‐E‐ and CFU‐Meg‐derived cells secreted proMMP‐9 and CFU‐F‐derived cells proMMP‐2, in contrast to cells from LTMC adherent layer which secreted both active and latent forms of MMP‐2 and MMP‐9 under serum‐free conditions. However, when these adherent cells were incubated in 12·5% fetal calf or horse serum or complete LTMC growth media, active forms of MMP‐2 and MMP‐9 were no longer detectable, and TIMP levels increased. Hence, we concluded that (i) MMPs/TIMPs are secreted by normal human bone marrow haematopoietic and stromal cells and may play an important role in intercellular cross‐talk in haematopoiesis; and (ii) only latent forms of MMPs are present under LTMC conditions, indicating that the specific media used for weekly re‐feeding of LTMC can block activation of MMP‐2 and MMP‐9, maintaining the integrity of the stromal layer and supporting haematopoiesisin vitro.
Outcome for children after failed transplant for primary haemophagocytic lymphohistiocytosis
Authors: Ardeshna K. M. , Hollifield J. , Chessells J. M. , Veys P. , Webb D. K. H.
Published: March 2003

Primary haemophagocytic lymphohistiocytosis is a rare disorder of childhood, which is usually fatal without allogeneic stem cell transplantation (SCT). For children who lack a matched family or closely matched unrelated donor, SCT using haploidentical parental stem cells has been used, but is associated with an increased risk of graft failure. The most appropriate subsequent management for those children who survive after graft rejection is currently unclear. We report the outcome for three such children. After a period of disease quiescence lasting 4 months to 8 years, disease recurrence and subsequent death occurred in each case. Accordingly, a second SCT is recommended.
Transformation of T‐cell large granular lymphocyte leukaemia into a high‐grade large T‐cell lymphoma
Authors: Matutes Estella , Wotherspoon Andrew C. , Parker Norman E. , Osuji Nnenna , Isaacson Peter G. , Catovsky Daniel
Published: March 2003

We describe a case of T‐cell large granular lymphocyte (LGL) leukaemia that transformed into a large‐cell T‐cell lymphoma 11 years from diagnosis. A 29‐year‐old asymptomatic female presented in 1989 with lymphocytosis, neutropenia and mild bone marrow infiltration. The circulating cells were LGL with a CD2+, CD3+, CD8+, CD4–, CD16+, CD56+, CD57– phenotype. In August 2000, she developed fever, a large submandibular mass and hepatosplenomegaly. Biochemistry showed abnormal liver function tests and raised lactate dehydrogenase (LDH) levels. A serological screen for Epstein–Barr virus, cytomegalovirus, human T‐lymphotropic virus‐I, human herpes virus (HHV)‐6 and HHV‐7 was negative. Histology of the mass was consistent with the diagnosis of peripheral T‐cell lymphoma composed of large cells, and immunohistochemistry showed that the lymphoma cells had a phenotype identical to the mature LGL. Molecular analysis with the polymerase chain reaction (PCR) demonstrated rearrangement of the T‐cell receptor (TCR) γ‐chain gene with a band of identical size in both bone marrow mature LGL and lymph node cells. The patient was treated with CHOP (cyclophosphamide, vincristine, doxorubicin and prednisolone), resulting in the disappearance of the mass and improvement of the hepatosplenomegaly, LDH and liver abnormalities. She underwent splenectomy, and spleen histology showed involvement by T‐cell LGL leukaemia with no evidence of transformation. This case illustrates that transformation or Richter syndrome may occur in a minority of patients with T‐cell LGL leukaemia, a disease that has a benign clinical course in most cases. This is the first case documented by molecular methods of the transformation of the pre‐existing clone.
Myeloma interacts with the bone marrow microenvironment  to induce osteoclastogenesis and is dependent on osteoclast activity
Authors: Yaccoby Shmuel , Pearse Roger N. , Johnson Cherie L. , Barlogie Bart , Choi Yongwon , Epstein Joshua
Published: March 2003

Summary. Myeloma tumour growth, except in the most advanced stages of the disease, is restricted to the bone marrow. We used the severe combined immunodeficient‐human (SCID‐hu) host system, in which primary human myeloma cells grow in, disseminate to and interact with a human microenvironment, to study the interactions between myeloma cells and cells in the bone marrow microenvironment. We used inhibitors of osteoclast activity to determine the role of osteoclasts and their products in supporting myeloma cell growth. Treatment of myelomatous SCID‐hu hosts with an inhibitor of osteoclast activity (pamidronate or zoledronate) or with a specific inhibitor of the receptor activator of NF‐κB ligand (RANKL) halted myeloma‐induced bone resorption, when present, and resulted in inhibition of myeloma cell growth and survival. In contrast, myeloma cells from patients with extramedullary disease had a different growth pattern in the SCID‐hu hosts and were not inhibited by these interventions, indicating that, while still dependent on a human microenvironment, these cells no longer required the bone marrow microenvironment for survival. This study demonstrates the dependence of myeloma cells on osteoclast activity and their products, and highlights the importance of the myeloma–osteoclast–myeloma loop for sustaining the disease process. Breaking this loop may help control myeloma.
Syngeneic stem cell transplant for spent‐phase polycythaemia vera: eradication of myelofibrosis and restoration of normal haematopoiesis
Authors: Richard Shambavi , Isola Luis , Scigliano Eileen , Singh Harvinder , Najfeld Vesna , Gilbert Harriet , Weinberg Rona S. , Fruchtman Steven
Published: March 2003

Summary. We report a patient with spent‐phase polycythaemia vera (S‐PV) and massive splenomegaly who failed to engraft after a syngeneic granulocyte colony‐stimulating factor‐primed peripheral blood stem cell transplant (SCT), but later engrafted after splenectomy. Bone marrow (BM) showed resolution of myelofibrosis (MF) and absent endogenous erythroid colonies. This case demonstrated that (1) normal haematopoiesis can be restored after syngeneic SCT despite extensive MF, and (2) fibrosis can regress following a total body irradiation‐containing regimen and syngeneic SCT. As a graft‐versus‐BM stroma effect is non‐existent in syngeneic transplants, there may be a role for autologous SCT to obliterate MF in S‐PV.
Low frequency of clonotypic Ig and T‐cell receptor gene rearrangements in t(4;11) infant acute lymphoblastic leukaemia and its implication for the detection of minimal residual disease
Authors: Peham Martina , Panzer Simon , Fasching Karin , Haas Oskar A. , Fischer Susanna , Marschalek Rolf , Gadner Helmut , Panzer‐Grümayer E. Renate
Published: March 2003

Summary. Infant t(4;11) acute lymphoblastic leukaemia (ALL) is a rare but cytogenetically well defined subgroup of immature B‐cell precursor (BCP) ALL. To date, the configuration of their antigen receptor genes has not been studied in a large group of patients so far. In this study on 27 t(4;11) infant ALL, we have used standardized primer sets for the detection of all incomplete and complete immunoglobulin (Ig) heavy chain (IGH) rearrangements, as well as for the Ig light chain kappa (IGK), T‐cell receptor delta (TCRD) and gamma (TCRG) rearrangements that are most common in childhood BCP ALL. Only 52% of cases displayed clonotypic antigen receptor gene rearrangements (IGH in 48%, IGK, TCRD and TCRG in 12%, 41% and 6% respectively). This low frequency suggests, together with the findings of predominantly incomplete DJh joins and monoallelic IGH rearrangements, that they are derived from an immature progenitor cell. As 48% of the t(4;11) infant ALL cases had no detectable antigen receptor gene rearrangements that could be used for minimal residual disease (MRD) analysis, we established an expression‐independent, leukaemia‐specific polymerase chain reaction (PCR) using the genomic sequence of the MLL–AF4 fusion genes. This method had high sensitivity and specificity and resulted in identical estimations of tumour loads when compared with IGH targets. Thus, genomic MLL–AF4 fusion genes are a good alternative target for the analysis of MRD in patients with t(4;11) leukaemias.
Deletion of sequences flanking the t(9;22) breakpoint: a secondary genetic event associated with loss of cytogenetic response to interferon in a Philadelphia‐positive chronic myeloid leukaemia patient
Authors: Arranz Eva , Gil‐Fernández Juan José , Ramiro Soraya , Blas Carlos , Renedo Mónica , Alegre Adrián , Escudero Antonio , Fernández‐Rañada Jose María
Published: March 2003

Summary. We present a Ph‐positive chronic myeloid leukaemia patient who lost a complete cytogenetic response (CCR) of 23 months duration at the time of detection of a deletion, not previously observed, of chromosomes 9 and 22 sequences flanking the translocation breakpoint on the derivate 9 chromosome. To our knowledge, this is the first case in which a deletion at the t(9;22) breakpoint has arisen as a secondary genetic alteration produced after formation of the t(9;22) translocation. It remains to be determined whether this genetic abnormality has the same prognostic importance as when observed at diagnosis.
p53 expression in K562 cells is associated with caspase‐mediated cleavage of c‐ABL and BCR–ABL protein kinases
Authors: Di Bacco Alessandra M. A. , Cotter Thomas G.
Published: March 2003

Summary.  The chimaeric BCR‐ABL oncoprotein is the molecular hallmark of chronic myeloid leukaemia (CML). Expression of Bcr‐Abl has been associated with arrested differentiation as well as resistance to apoptosis. The downstream pathway involved in apoptosis resistance has been extensively studied, whereas the role of Bcr‐Abl in cell differentiation is largely unclear. A recent report has shown that Bcr‐Abl expression alone is sufficient to increase the number of multipotent and myeloid lineage‐committed progenitors in a dose‐dependent manner while suppressing the development of committed erythroid progenitors. In accordance with this model, downregulation of c‐Abl and Bcr‐Abl has been observed during differentiation in different systems, although the mechanism is still largely unknown. To investigate the relationship between erythroid differentiation and c‐Abl and Bcr‐Abl levels, we induced differentiation in K562 cells using a temperature‐inducible p53 mutant (p53Val1335). It was found that p53‐induced erythroid differentiation in K562 cells required caspase activity. During this process, caspase‐dependent cleavage of c‐Abl and Bcr‐Abl tyrosine kinases was observed, suggesting a new mechanism for the downregulation of the kinases during erythroid differentiation.