bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of three potential genetic risk factors for thrombosis in 43 families carrying the factor V Arg 506 to Gln mutation
Authors: Le Cam‐Duchez Véronique , Gandrille Sophie , Trégouët David , Alhenc‐Gelas Martine , Emmerich Joseph , Fiessinger Jean‐Noël , Borg Jeanne‐Yvonne , Aiach Martine
Published: March 2003

The factor V (FV) Arg 506 to Gln mutation is the most common abnormality observed in familial thrombophilia. Many studies have shown that its clinical expression differs among families and among carriers. Some thrombotic patients carry an additional genetic risk factor such as protein C, protein S or antithrombin deficiency. We sought to identify other genetic risk factors potentially favouring expression of the thrombotic phenotype in 370 members of 43 families with the FV Arg 506 to Gln mutation. We analysed three candidate polymorphisms in genes involved in the PC anticoagulant pathway, consisting of two polymorphic sites in the 5′ non‐transcribed region of the PC gene, −1654 C/T and −1641 A/G, with three known combinations (TA, CA and CG) that influence the protein C plasma level; one polymorphic site (4070 A/G) in exon 13 of the FV gene, which influences the plasma factor V concentration, and one polymorphic site (677 C/T) in the methylenetetrahydrofolate reductase gene, which is often associated with moderate hyperhomocysteinaemia. The distribution of these different polymorphisms was similar in patients with a history of thrombosis and those who remained asymptomatic, ruling out the possibility that each of these polymorphisms alone can play a role in the onset of thrombosis in carriers of the FV Arg 506 to Gln mutation.
Factor V Leiden and the common haemochromatosis mutation HFE C282Y: is there an association in familial venous thromboembolic disease?
Authors: MacLean Rhona M. , Feeney Graham P. , Bowley Steven J. , Bowen Derrick J. , Worwood Mark , Collins Peter W.
Published: March 2003

The involvement in venous thrombosis of the two most common mutations of the hereditary haemochromatosis gene (HFE C282Y and HFE H63D) was investigated in 239 patients with objectively proven venous thrombosis. Neither mutation showed an increased prevalence in the cohort (HFE C282Y: 13.0% (95% CI 9.3–17.8) patients, 16.2% (95% CI 14.3–18.2) controls; HFE H63D: 28.3% (95% CI 22.9–34.3) patients, 28.1% (95% CI 25.8–30.6) controls. Neither mutation was increased in patients with factor V Leiden (FVL) compared to those without. However, HFE C282Y was increased among patients who had both FVL and a family history of thrombosis (7/20), compared with those with FVL and no family history (1/22) (relative risk 7.97, 95% CI 1.5–43.1, P = 0.016).
Selection and transplantation of autologous CD34+ B‐lineage negative cells in advanced‐phase multiple myeloma patients: a pilot study
Authors: Lemoli Roberto M. , Martinelli Giovanni , Olivieri Attilio , Motta Maria Rosa , Rizzi Simonetta , Terragna Carolina , Leopardi Giuliana , Benni MonicaA , Ronconi Sonia , Cantori Isabella , Rondelli Damiano , Mangianti Serena , Leoni Pietro , Montanari Mauro , Cavo Michele , Tura Sante
Published: March 2003

The feasibility of sequential positive and negative selection of mobilized CD34+ B‐lineage negative cells to achieve tumour‐free autografts in multiple myeloma (MM) patients was evaluated. Peripheral blood stem cells (PBSC) of 14 patients with advanced disease were mobilized. CD34+ cells were enriched in 12 of the patients by the avidin–biotin immunoabsorption technique. Subsequently, CD10+, CD19+, CD20+ and CD56+ cells (B‐lin cells) were removed by immunomagnetic depletion. Minimal residual disease (MRD) was detected by flow cytometry and PCR‐based molecular analysis of the patient specific IgH complementary‐determining region III (CDRIII). Positive selection of stem cells produced a median recovery of 54.7% of the initial content of CD34+ cells (median purity 71.9%). Negative depletion of B‐lineage cells reduced the number of CD34+ cells to 33.3% of the baseline value (median purity 72.7%). However, long‐term culture assays showed the recovery of >60% of primitive haemopoietic progenitor cells after depletion of the B‐lineage‐positive cells. All evaluable patients had detectable disease in PBSC collections. The first step of positive selection of CD34+ cells resulted in >2 logs of tumour cell purging. However, molecular assessment showed the persistence of the disease in 6/7 cases. Immunofluorescence analysis demonstrated 1 additional log of B‐cell purging by negative depletion. More importantly, molecular evaluation of IgH CDRIII region showed the disappearance of myeloma cells in 6/7 patients. 12 patients received a median of 3.9 × 106 CD34+ B‐lin− cells/kg after conditioning with high‐dose melphalan and showed a rapid reconstitution of haemopoiesis. These results were similar to two similar cohorts of patients who received either unmanipulated PBSC or positively selected CD34+ cells after the same conditioning regimen. Severe extrahaematological toxicity was limited to mucositis; no late infections were observed. We concluded that autotransplantation of purified CD34+ B‐lin− cells was associated with a rapid and sustained recovery of haemopoiesis and low peritransplant morbidity. Sequential positive and negative enrichment of stem cells reduced tumour cell contamination in B‐cell malignancies below the lower limit of detection of molecular analysis.
Intracellular cytokine production and cytokine receptor interaction of cord mononuclear cells: relevance to cord blood transplantation
Authors: Han P. , Hodge G.
Published: March 2003

A ‘cytokine storm’ consisting of IL‐1, IL‐2, IL‐12, IFNγ and TNFα is considered important in the development of graft‐versus‐host disease (GvHD). These cytokines activate effector cells or damage host tissues. Cord blood transplantation has been associated with a low incidence of GvHD. We hypothesized that the low incidence of GvHD relates to the cord mononuclear cells being poor producers of pro‐inflammatory cytokines. The cytokine profile (IL‐1α/β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IFNγ and TNFα) of cord blood cells induced by immune stimuli was determined in heparinized whole blood. Compared to adult, cord blood CD3+ and NK cells produced less IFNγ, less cord blood CD3+ cells and monocytes produced TNFα and less monocytes produced IL‐1α/β. Although more cord T cells produced IL‐2 compared to adult T cells at 4 h, adult T cells produced more at 24 h. Cord blood had similar proportions of monocytes to adult producing IL‐6, IL‐10 and IL‐12. Both adult and cord mononuclear cells constitutively expressed receptors for IFNγ and TNFα but not IL‐12. In contrast to the adult cells, cord CD3+ and NK cells did not express IL‐12 receptor but did up‐regulate IL‐10 receptor after mitogenic stimulation. The findings of this study indicate that the cord blood cytokine‐receptor network is biased towards anti‐inflammatory activity compared to adult and helps to explain the decreased incidence of GVHD in cord blood transplantation.
Identification of patient‐specific peptides for detection of M‐proteins and myeloma cells
Authors: Szecsi Pal B. , Riise Erik , Roslund Lisbeth B. , Engberg Jan , Turesson Ingemar , Buhl Lone , Schafer‐Nielsen Claus
Published: March 2003

We have taken advantage of the selection power of phage display technology to define specific peptide mimotopes that recognize individual M‐proteins, isolated from patients with multiple myeloma. Preferred amino acid motifs of phages binding to M‐proteins were identified in 6/9 patients investigated. Chemically synthesized peptides, corresponding to the phage‐displayed peptide inserts, were used to verify the specificity of binding in competition assays. The peptides were able to bind to the M‐proteins, as well as the myeloma cells, with high sensitivity and specificity. Employing simple immunological techniques, < 0.01 g/l of M‐protein could be quantified, suggesting a novel way for monitoring minimal residual disease in the production of guidelines for adjusting or reintroducing conventional chemotherapy. The peptide mimotopes defined by this technology may be useful as tumour‐specific targeting agents and as a tool for purging cells in autologous bone marrow transplantation.
Monitoring chronic myeloid leukaemia therapy by real‐time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics
Authors: Branford  , Hughes  , Rudzki
Published: March 2003

We have developed a rapid real‐time quantitative PCR method for measuring BCR‐ABL mRNA levels in peripheral blood in chronic myeloid leukaemia (CML). The technique was used to monitor minimal residual disease for the early detection of relapse and as an assessment of treatment response. Normal BCR mRNA was quantitated to control for RNA degradation and the results reported as a percentage of BCR‐ABL/BCR. Every patient measured at diagnosis (n = 21) had increased expression of BCR‐ABL of up to 5‐fold above the normal BCR levels. With effective treatment the BCR‐ABL levels decreased. The molecular data was correlated with Philadelphia chromosome levels in bone marrow and a good correlation was found when treatment induced a cytogenetic response (Spearman correlation = 0.94, P < 0.0001, n = 67 samples). In patients receiving interferon‐α therapy we found a significant difference in the BCR‐ABL levels between cytogenetic response groups. The method was sensitive, reproducible, and readily detected a change in BCR‐ABL transcript levels in serial blood samples. Sample throughput was high because post PCR processing was unnecessary. We conclude that real‐time quantitative PCR monitoring of peripheral blood can be used to reliably monitor disease response in CML.
Extracorporeal photopheresis induces apoptosis in the lymphocytes of cutaneous T‐cell lymphoma and graft‐versus‐host disease patients
Authors: Bladon J. , Taylor P. C.
Published: March 2003

Extracorporeal photopheresis (ECP) is used in the treatment of T‐cell‐mediated disorders. However, the mechanism by which ECP achieves its effect remains illusive. Over recent years the ability of ECP to induce apoptosis has been demonstrated by cell culture experiments and retrospective histological analysis. We investigated if apoptosis could be determined in samples tested ex vivo from the UVAR:ECP system. Lymphocytes from 11 patients (six with cutaneous T‐cell lymphoma, four with graft‐versus‐host disease, and one with scleredema) were isolated at three stages of the ECP process: immediately before ECP treatment, from the first buffy coat collected, and post UV irradiation, prior to re‐infusion. Using flow cytometry each stage was tested for the early apoptotic markers; Annexin V, ApoptestTM and Carboxy‐SNARF‐1‐AM. Comparisons of the pre‐ECP and pre‐infusion samples demonstrated a significant increase in apoptotic lymphocytes for all three flow cytometric techniques (P < 0.01). Increases between the pre‐ECP and first buffy coat, used as a measure of the extracorporeal manipulation, were much lower. These results demonstrate that ECP directly induces significant levels of apoptosis in lymphocytes of CTCL, GvHD and scleredema patients. The apoptosis of these lymphocytes may contribute to the ECP effect.
Familial clustering of Langerhans cell histiocytosis
Authors: Aricò Maurizio , Nichols Kim , Whitlock James A. , Arceci Robert , Haupt Riccardo , Mittler Uwe , Kühne Thomas , Lombardi Alessandra , Ishii Eiichi , Egeler R. Maarten , Danesino Cesare
Published: March 2003

Langerhans cell histiocytosis (LCH) is considered a non‐hereditary disorder. Evaluation of the few familial cases might provide insight into its aetiology and pathogenesis. We conducted a survey to identify familial LCH cases. Data on family history, zygosity assessment in twins, clinical and laboratory features, treatment outcome, and present status were collected. According to variable confidence for twins monozygosity assessment, we termed these pairs ‘presumed monozygotic’ (pMZ). Nine families had more than one affected relative: five with LCH‐concordant twin pairs, four with LCH in siblings or cousins. Three twin pairs not concordant for LCH were also studied. Overall, four of five pMZ twin pairs and one of three dizygotic (DZ) pairs were concordant for LCH. The pMZ twins had simultaneous and early disease onset (mean age 5.4 months); onset was at 21 months in the DZ pair. Clinical features were similar in the pMZ pairs. One pair of DZ twins had disseminated LCH. The three healthy twins (one pMZ, two DZ) remain asymptomatic 0.3, 5.9 and 4.7 years, respectively, after disease onset in their co‐twins. Of the two families with affected non‐twin siblings, one had known parental consanguinity and the other possible consanguinity. Potential consanguinity was also present in one of the two families with affected first cousins. Our data support high LCH concordance rates in pMZ twins and add the finding of LCH concordance in one of three dizygotic pairs studied. Taken together with our identification of LCH in siblings and first cousins from known or possibly consanguineous families, and with prior reports of three affected parent–child pairs, the data support a role for genetic factor(s) in LCH. The work‐up of newly diagnosed patients should include a careful, extensive family history and chromosome studies. When possible, constitutional and/or lesional DNA should be obtained for future study.
Reconstitution of the cellular immune response after autologous peripheral blood stem cell transplantation in patients with non‐Hodgkin's lymphoma
Authors: Nolte  , Buhmann  , Emmerich  , Schendel  , Hallek
Published: March 2003

Peripheral blood stem cell (PBSC) transplants may be depleted of lymphoid progenitors, thereby disabling the cellular immune response against viral pathogens after autologous PBSC transplantation (PBSCT). To monitor the cellular immune reconstitution after autologous PBSCT, we investigated the cytolytic activity (CLA) of peripheral blood T lymphocytes against Epstein–Barr virus (EBV) in 13 patients with non‐Hodgkin‘s lymphoma or multiple myeloma. The individual EBV‐directed CLA (EBV‐CLA) was determined by calculating the number of cytolytic effector cells in 106 T cells needed to lyse 25% of autologous EBV‐transformed B‐lymphoblastoid cells, expressed as lytic units (LU25). During the first 6 months after PBSCT, the EBV‐CLA was only 14.6% of the response of healthy controls (median 4.8 vs. 32.9 LU25). Thereafter, the EBV‐CLA increased to 28.15 LU25 (median) or 86% of healthy controls. Monthly follow‐up analyses in five selected patients showed that the EBV‐CLA was barely detectable at 4 weeks and recovered at 8–12 weeks after PBSCT in four out of five patients. Effector cells consisted mostly of CD8‐positive T lymphocytes, with small CD4‐ and CD3/CD56‐positive lymphocyte fractions. These results suggest that the reconstitution of the cellular immune response against EBV takes 8–12 weeks after autologous PBSCT.
Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow‐repopulating ability together with the expansion of committed progenitors
Authors: Ivanović  , Bartolozzi  , Bernabei  , Cipolleschi  , Rovida  , Milenković  , Praloran  , Sbarba
Published: March 2003

We developed previously a hypoxic culture system in which progenitors endowed with marrow‐repopulating ability (MRA), unlike committed progenitors, were selected and maintained better than in air. We report here an improvement to this system targeted at combining the maintenance of progenitors sustaining MRA with the numerical expansion of multipotent and committed progenitors. Murine bone marrow cells were incubated at 1% oxygen in liquid medium supplemented with stem cell factor, granulocyte colony‐stimulating factor, interleukin‐6 and interleukin‐3. In day 8 hypoxic cultures, the numbers of high proliferative potential and granulocyte/macrophage colony‐forming cells (HPP‐CFC and CFU‐GM) were increased with respect to time zero. Colonies generated by HPP‐CFC derived from hypoxic cultures exhibited a high replating ability, whereas colonies generated by HPP‐CFC derived from control cultures exhibited a low replating ability. MRA was fully maintained in hypoxia and markedly reduced in air. Thus, severe hypoxia is able to ensure a full maintenance of progenitors sustaining MRA, together with a significant expansion of in vitro‐detectable clonogenic progenitors, including those endowed with replating ability. This system could contribute to the improvement of current techniques for the in vitro treatment of human haematopoietic cell populations before transplantation.
Genetic studies of three Japanese patients with p22‐phox‐deficient chronic granulomatous disease: detection of a possible common mutant CYBA allele in Japan and a genotype–phenotype correlation in these patients
Authors: Yamada Masafumi , Ariga Tadashi , Kawamura Nobuaki , Ohtsu Makoto , Imajoh‐Ohmi Shinobu , Ohshika Eiki , Tatsuzawa Osamu , Kobayashi Kunihiko , Sakiyama Yukio
Published: March 2003

Chronic granulomatous disease (CGD) is a disorder caused by defects in the NADPH oxidase responsible for superoxide generation in phagocytes. Cytochrome b558, an essential component of this enzyme, is a heterodimer formed by a 91 kDa glycoprotein (gp91‐phox) and a 22 kDa polypeptide (p22‐phox). Mutations in the p22‐phox gene (CYBA) locus in 16q24 result in one of the rare autosomal recessive forms of CGD. We performed mutation analysis in three female CGD patients suspected of having this form of the disease and found two novel mutations in CYBA. Whereas patient 1 with severe phenotype had a homozygous nonsense mutation in exon 1 (C‐35 → T, Gln‐3 → stop), patients 2 and 3 with mild phenotype shared the same homozygous missense mutation in exon 2 (G‐98 → A, Gly‐24 → Arg). None of the parents of patients 2 and 3 is related. Therefore, this mutation could be a hot‐spot or a common mutation in the Japanese population. Patients 2 and 3, but not patient 1, were demonstrated to have detectable p22‐phox expression and significant granulocyte respiratory burst (ROB) activity. In this study, we were able to demonstrate an excellent correlation between genotype, p22‐phox expression, ROB activity and clinical phenotype in these patients.
Dyserythropoiesis associated with a Fas‐deficient condition in childhood
Authors: Bader‐Meunier  , Rieux‐Laucat  , Croisille  , Yvart  , Mielot  , Dommergues  , Ledeist  , Tchernia
Published: March 2003

Defective lymphocyte apoptosis caused by mutations of the Fas gene can result in an autoimmune lymphoproliferative syndrome (ALPS) in humans. We report two cases of dyserythropoiesis associated with a Fas‐deficient condition in childhood. In both cases, dyserythropoiesis predominated on the more mature erythroblasts, and was associated with a lymphoproliferative syndrome as well as with haemolytic anaemia, hypergammaglobulinaemia and the expansion of an unusual population of CD4− CD8− T cells that express the α/β T‐cell receptor. The regression of dyserythropoiesis under steroid therapy suggested that it resulted from an autoimmune mechanism, itself secondary to the lymphocyte Fas apoptosis deficiency. Fas‐defective apoptosis may be a new aetiology for childhood dyserythropoiesis.
Rapid detection of α‐thalassaemia deletions and α‐globin gene triplication by multiplex polymerase chain reactions
Authors: Liu  , Old  , Miles  , Fisher  , Weatherall  , Clegg
Published: March 2003

We describe a sensitive, reliable and reproducible method, based on three multiplex PCR assays, for the rapid detection of seven common α‐thalassaemia deletions and one α‐globin gene triplication. The new assay detects the α0 deletions – –SEA, – (α)20.5, – –MED, – –FIL and – –THAI in the first multiplex PCR, the second multiplex detects the –α3.7 deletion and αααanti3.7 variant, the third multiplex detects the –α4.2 deletion. This simple multiplex method should greatly facilitate the genetic screening and molecular diagnosis of these determinants in populations where α‐thalassaemias are prevalent.
A study of the iron and HFE status of blood donors, including a group who failed the initial screen for anaemia
Authors: Boulton  , Collis  , Inskip  , Paes  , Garlick
Published: March 2003

A complete data set (age, weight, diet and recent donation history; venous blood cell count, serum ferritin and soluble transferrin receptor concentrations and transferrin saturation; HFE genotype) was obtained from 113 male and 122 female blood donors. Progressive iron depletion and deficiency — most apparent from serum concentrations of soluble transferrin receptor divided by the logarithm of ferritin concentrations (the TfR‐F index) — developed in men donating up to six times in 2 years, although the serum ferritin alone was also informative; however, no prediction could be made for those iron‐depleted individuals who will develop iron deficiency after donation. Iron stores in the groups of donors with ‘low‐normal’ haemoglobin (Hb) concentrations were indistinguishable from those in donors with higher Hb values, whereas donors failing the anaemia screen had reduced stores. This supports the UK policy of accepting donations from people whose Hb concentration is up to 0.5 g/dl below the recommended European threshold. Women eating red meat once a week sustained higher ferritin concentrations, and the iron status of first‐time women donors resembled that of men donating twice each year. Homozygosity for either HFE variant allowed greater iron retention in the face of regular donation, but among heterozygotes the findings were inconclusive.
Effects of intravenous immunoglobulin in a patient with intermittent thrombotic thrombocytopenic purpura
Authors: Kondo Haruki , Imamura Takaaki
Published: March 2003

We describe a patient with a 9‐year history of thrombotic thrombocytopenic purpura (TTP) who exhibited four relapses. Intravenous immunoglobulin (IVIg) was effective for these four episodes. The patient was well and the laboratory findings were within normal ranges between each episode, although unusually large von Willebrand factor multimers were observed during remission. Our results suggest the usefulness of IVIg at the time of relapse in the treatment of patients with TTP who have multiple relapses over a long period.
Clinicopathological features of aggressive large granular lymphocyte leukaemia resemble Fas ligand transgenic mice
Authors: Lamy Thierry , Bauer Frank A. , Liu Jin Hong , Li Yong Xiang , Pillemer Eric , Shahidi Homayoon , Gregory Stephanie A. , Zambello Renato , Marcolongo Renzo , Semenzato Gianpietro , Loughran Jr Thomas P.
Published: March 2003

Fas ligand triggers cell death after interaction with its receptor Fas. Altered expression of Fas has been associated with lymphoproliferation and autoimmune disorders in both mice and man. Apoptosis of lung and liver tissue is seen in Fas ligand transgenic mice. It is not known whether constitutive expression of Fas ligand can cause a similar human disease. Four patients with aggressive large granular lymphocyte (LGL) leukaemia involving lung and liver were studied. All four patients were severely ill with pulmonary involvement. Two patients presented with hypoxia and were oxygen dependent; the other two patients had severe pulmonary hypertension. Lung biopsies showed interstitial infiltration by leukaemic LGL. The infiltrating lymphocytes expressed both Fas and Fas ligand, whereas normal pneumocytes expressed only Fas. Similar findings were observed in liver biopsies from these patients. Features mimicking the pathological changes of graft‐versus‐host disease were observed, including pneumocyte apoptosis. All four patients had high levels of circulating Fas ligand. Successful treatment with oral methotrexate or 2‐chlorodeoxyadenosine was associated with disappearance or marked reduction of circulating Fas ligand. These results suggest that dysregulated expression of Fas ligand can lead to human disease with pathological features resembling graft‐versus‐host disease.
Signal antonymy unique to myelodysplastic marrows correlates with altered expression of E2F1
Authors: Mundle Suneel D. , Mativi B. Yifwayimare , Cartlidge Jonathan D. , Dangerfield Bruce , Broady‐Robinson LaTanya , Li Biarou , Shetty Vilasini , Venugopal Parameswaran , Gregory Stephanie A. , Preisler Harvey D. , Raza Azra
Published: March 2003

Myelodysplastic syndromes (MDS) have previously been reported to show competitively high rates of apoptosis and proliferation in the bone marrow (BM). Using a double‐labelling technique in the present study, we demonstrated that a significantly high number of S‐phase cells were simultaneously apoptotic (signal antonymy; SA) in MDS (mean ± s.e.m. 53·5 ± 6·7%, n = 24, P < 0·001). In contrast, SA was negligible in all other specimens studied, including normal control BM (n = 13) from non‐Hodgkin's lymphoma (NHL) patients, BM from patients with de novo acute myelogenous leukaemia (1′AML; n = 5), or secondary AML that had transformed from MDS (2′AML; n = 10), or the solid tumours from patients with NHL (n = 9) or head and neck squamous cell carcinoma (HNSCC; n = 10). Subsequently, the expression of a transcription factor, E2F1, was studied in density‐separated BM aspirate mononuclear cells from MDS patients (n = 9) and a normal control. Two separate sets of primers were used that recognized the regulatory retinoblastoma (Rb) protein‐binding region and the functional DNA‐binding region of E2F1. Interestingly, although the latter manifested the expected band (280 bp) in all samples, the Rb‐specific primers showed the expected band (380 bp) in the normal and in 4/9 MDS specimens. Two other MDS specimens also showed a smaller band (∼ 325 bp), whereas 3/9 MDS patients showed exclusively the smaller band. The levels of SA were significantly higher in those MDS cases that showed the smaller Rb‐specific band either alone or in addition to the expected band (median 19·5%, n = 4, P = 0·037) than in those showing exclusively the expected band (median 0·4%, n = 3). Our present studies show SA as a characteristic feature of MDS and, importantly, demonstrate its link with an altered expression of E2F1 in some MDS patients.
Granulocyte colony‐stimulating factor (G‐CSF) induces the production of cytokines in vivo
Authors: Xu Shengyuan , Höglund Martin , Håkansson Lena , Venge Per
Published: March 2003

Granulocyte colony‐stimulating factor (G‐CSF) is a haematopoietic growth factor required for the proliferation and differentiation of haematopoietic precursors of neutrophil granulocytes and is now used to overcome congenital and acquired neutropenia. In addition to increasing the numbers of neutrophils in vivo and modulating neutrophil functions, G‐CSF may induce the production of cytokines such as tumour necrosis factor α (TNF‐α). In the present study, the plasma levels of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) in six healthy volunteers given G‐CSF at 10 μg/kg once daily for 6 d were measured and found to be elevated. The elevated levels (P < 0·05) were detected on day 2, peaked on days 6–7 and returned to baseline on day 12. In vitro, G‐CSF did not enhance the secretion of TNF‐α and GM‐CSF from mononuclear cells, whole blood or endothelial cells. However, in the co‐presence of whole blood and endothelial cells, the secretion of TNF‐α was significantly enhanced by G‐CSF at low concentrations. The GM‐CSF secretion, however, was unaltered. G‐CSF pretreatment of whole blood suppressed lipopolysaccharide (LPS)‐induced secretion of TNF‐α and GM‐CSF in a dose‐dependent manner. These results together with our previous findings suggest that G‐CSF induces the production of TNF‐α and GM‐CSF in vivo, and that this production may be due to the co‐effects of endothelial cells and whole blood under the influence of G‐CSF through an as yet unknown network of cells and cytokines. Treatment of whole blood with G‐CSF suppresses LPS‐induced secretion of TNF‐α and GM‐CSF.
Mutation analysis in the HFE gene in patients with hereditary haemochromatosis in Saguenay–Lac‐Saint‐Jean (Quebec, Canada)
Authors: Rivard Sylvain R. , Mura Catherine , Simard Hervé , Simard Raynald , Grimard Doria , Le Gac Gérald , Raguenes Odile , Férec Claude , De Braekeleer Marc
Published: March 2003

A mutation analysis of the HFE gene followed, when applicable, by sequencing was performed on 47 patients with hereditary haemochromatosis (HH) living in Saguenay–Lac‐Saint‐Jean. The C282Y and H63D mutations were present on 50% and 20·3% of the HH chromosomes respectively. These frequencies were very different from those found in other populations and could be, at least partially, the result of a founder effect. No new mutation was identified among the remaining 28·1% of the HH chromosomes. Five of the eight probands with no mutation in the HFE gene had a severe and early onset suggestive of juvenile haemochromatosis.
Control of pH in human long‐term bone marrow cultures with low‐glucose medium containing zwitterion buffer lengthens the period of haemopoietic activity
Authors: Tennant G. B. , Truran L. N. , Bailey‐Wood R. , Burnett A. K.
Published: March 2003

Primary long‐term bone marrow cultures grown in 40 mm HEPES‐buffered McCoy's 5A medium produced granulocyte–macrophage colony‐forming units (CFU‐GM) for a median of 9 weeks compared with 7 weeks with CO2/bicarbonate‐buffered cultures. Reducing the medium glucose concentration (from 12·5 to 2·75 mm) extended the culture longevity to 17 weeks. The median period of erythroid colony detection increased from 6 to 8 weeks. Secondary cultures (5 × 106 cord blood mononuclear cells seeded on irradiated stroma) showed statistically similar myeloid and erythroid longevity to primary cultures. Improved control of medium pH significantly improved the capacity of long‐term stromal layers to maintain stem cells in vitro.
Hydroxyurea therapy decreases the in vitro adhesion of sickle erythrocytes to thrombospondin and laminin
Authors: Hillery Cheryl A. , Du Ming C. , Wang Winfred C. , Scott J. Paul
Published: March 2003

The adhesion of sickle erythrocytes to the vascular endothelium and subendothelial matrix probably contributes to the pathogenesis of vaso‐occlusive disease. The chemotherapeutic agent hydroxyurea (HU) decreases the frequency of vaso‐occlusive crises in patients with sickle cell disease. However, the exact mechanism(s) of HU's effect on vaso‐occlusive crises is not fully understood. The goal of this study was to determine the effect of HU therapy on the adhesion of sickle erythrocytes to the subendothelial matrix proteins thrombospondin (TSP) and laminin under conditions of flow in vitro. Erythrocytes from patients with severe sickle cell disease on HU therapy (n = 14) had significantly less adhesion to TSP (687 ± 92 erythrocytes/mm2, mean ± SE) than untreated patients with severe disease (n = 18, 1176 ± 117 erythrocytes/mm2, P = 0·003). In addition, there was significantly less adhesion of erythrocytes to immobilized laminin in patients treated with HU (1695 ± 293 erythrocytes/mm2) than in the untreated patients (2590 ± 296 erythrocytes/mm2, P = 0·02). Erythrocytes from an additional nine patients with severe sickle cell disease were studied both before and after initiation of HU therapy. Erythrocytes from these patients became less adhesive to both TSP (P = 0·001) and laminin (P = 0·01), a change that was sustained in most patients throughout the duration of the study (2 months to > 12 months). This study suggests that HU modulates the adhesive phenotype of sickle erythrocytes, an effect that may be in addition to, or independent of, other known effects of HU, such as an increase in fetal haemoglobin level.
Efficient depletion of alloreactive donor T lymphocytes based on expression of two activation‐induced antigens (CD25 and CD69)
Authors: Fehse Boris , Frerk Oliver , Goldmann Michal , Bulduk Maria , Zander Axel R.
Published: March 2003

T lymphocytes play an important role in allogeneic bone marrow/stem cell transplantation by supporting engraftment and immune recovery. Moreover, donor T cells have been shown to mediate the so‐called graft‐versus‐leukaemia effect and are, therefore, increasingly used for adoptive immunotherapy. However, T‐cell infusions are associated with the risk of a graft‐versus‐host reaction, which may lead to a life‐threatening disease. To overcome this problem, we followed a new strategy for the exclusive depletion of alloreactive cells. We activated allogeneic T cells by cultivation on an adherent cell layer derived from peripheral blood. We then depleted activated cells based on the expression of CD25, CD69 or both activation‐induced antigens using magnetic cell sorting. Mixed lymphocyte culture (MLC) reactions and helper T‐lymphocyte precursor cell frequency (HTLP‐f) assays demonstrated that this technique led to a significant decrease in alloreactivity of ‘donor’ cells, which at the same time preserved reactivity against third‐party cells. The lowest level of alloreactivity was found when CD25 and CD69 antibodies were used together for depletion. This corresponds with our observation that expression of CD25 or CD69 may partially represent different activation pathways. We conclude that ex vivo depletion of CD25‐ and CD69‐expressing alloreactive cells may help to overcome limitations of adoptive immunotherapy.
Autologous stem cell transplantation for anaplastic large‐cell lymphomas: results of a prospective trial
Authors: Deconinck E. , Lamy T. , Foussard C. , Gaillard F. , Delwail V. , Colombat P. , Casassus P. , Lemevel A. , Brion A. , Milpied N. ,   for the Groupe Ouest‐est d'Etude des Leucémies et Autres Maladies du Sang (GOELAMS)
Published: March 2003

Autologous stem cell transplantation (ASCT) in the front line treatment of non‐Hodgkin's lymphoma (NHL) remains controversial. Anaplastic large‐cell lymphoma (ALCL) is known to have its own clinical and biological features. The outcome of ALCL patients treated with high‐dose chemotherapy and ASCT as part of their first‐line therapy was analysed in 202 intermediate or high‐grade NHL patients in a prospective randomized trial. First‐line chemotherapy comprised two alternating anthracycline‐containing regimens. Responding patients were autografted after a BEAM (BCNU, cytarabine, etoposide and melphalan) regimen. Patients with bulky or residual masses were irradiated. Fifteen patients with ALCL were identified by morphological and immunological features (CD30 was expressed in 14 out of 15 patients, three patients expressed B‐cell markers, five patients expressed T‐cell markers and seven patients did not express cell markers). Anaplastic lymphoma kinase (ALK) expression was confirmed in seven cases. The median age was 39 years with a predominant male sex ratio (2·75). Thirteen patients were stage ≥ III and six presented with two or more adverse prognostic factors. According to the international age‐adjusted prognostic index, the expected complete remission (CR), event‐free survival (EFS) and overall survival (OS) rates were 69%, 71% and 69%. Two deaths were observed (one due to interstitial pneumonitis, one due to pulmonary carcinoma). All patients entered CR, no relapse occurred and EFS and survival reached 87% with a follow‐up of more than 5 years. These results differ significantly from those observed in the other 176 lymphoma patients: event‐free survival was only 53 ± 5% and OS reached 60 ± 4% with a median follow‐up of 56 months (P = 0·006). Intensified chemotherapy with autologous stem cell support appeared effective in the treatment of ALCL, offering patients the real chance of a cure.
Interleukin 6 receptor expression by human cord blood‐ or peripheral blood‐derived primitive haematopoietic progenitors implies acquisition of different functional properties
Authors: Minamiguchi Hitoshi , Yahata Naoyuki , Kimura Takafumi , Fujiki Hiroshi , Harada Sachio , Wang Jianfeng , Okuda Keiko , Kaneko Hiroto , Hodohara Keiko , Banba Tadao , Yasukawa Kiyoshi , Ohyashiki Junko H. , Ohyashiki Kazuma , Abe Tatsuo , Sonoda Yoshiaki
Published: March 2003

The significance of interleukin 6 receptor (IL‐6R) expression by cord blood (CB)‐ and peripheral blood (PB)‐derived primitive haematopoietic progenitors was investigated. IL‐6R was preferentially expressed by PB‐derived myeloid progenitors. Most PB‐derived erythroid bursts (BFU‐E) and mixed colony‐forming cells (CFU‐Mix) did not express this receptor. However, CB‐derived primitive progenitor cells possessed multipotentiality, irrespective of IL‐6R expression. Interestingly, the long‐term culture‐initiating cell (LTC‐IC) population was enriched in PB‐derived CD34+ IL‐6R+ cells, but the extended LTC‐IC (ELTC‐IC) population, which represents a less mature class of haematopoietic progenitors, seemed to be equally distributed in the IL‐6R+ and IL‐6R− cell populations. In contrast, the number of LTC‐ICs and ELTC‐ICs was similar in CB‐derived CD34+ IL‐6R+ or IL‐6R− cells. It is noteworthy that the number of LTC‐ICs and ELTC‐ICs in CB‐derived CD34+ cells was markedly higher than that in PB‐derived CD34+ cells regardless of IL‐6R expression. Telomerase activity was consistently lower in PB‐derived CD34+ IL‐6R− cells than in CD34+ IL‐6R+ cells. In contrast, telomerase activity was similar in CB‐derived CD34+ IL‐6R+ or IL‐6R− cells. The pattern of telomerase induction upon cytokine stimulation differed between CB‐ and PB‐derived CD34+ IL‐6R+ or IL‐6R− cells. However, overall telomerase activity per dish was well correlated with the proliferative potential of both cell populations, suggesting that induction of telomerase plays an important role in the escape from replicative senescence of primitive haematopoietic progenitors. Collectively, these results suggest that CB‐derived primitive progenitors are less mature than PB‐derived progenitors and that the expression of IL‐6R by primitive haematopoietic progenitors may have different implications for PB‐ and CB‐derived CD34+ cells.
Lack of interferon‐α‐induced tyrosine phosphorylation of Vav proto‐oncogene in patients with myelofibrosis with myeloid metaplasia
Authors: Micouin Anne , Steunou Virginie , Wietzerbin Juana , Martyré Marie‐Claire
Published: March 2003

Myelofibrosis with myeloid metaplasia (MMM) is an uncommon chronic myeloproliferative disorder characterized by clonal stem cell proliferation and reactive non‐clonal proliferation of bone marrow fibroblasts with fibrosis. In the absence of curative therapy, the current management for the majority of patients is directed towards alleviation of symptoms and improvement in quality of life. A number of experimental therapies have been investigated, among which is the use of type I interferon (IFN)‐α, whose overall results are disappointing. We recently showed that the Vav proto‐oncogene product, p95Vav, which is phosphorylated under IFN‐α treatment, associates with both chains that constitute the functional type I IFN receptor and contributes to the antiproliferative effects of IFN‐α. The involvement of p95Vav in IFN‐α signalling and the frequent non‐responsiveness of patients to IFN‐α led us to investigate for any functional defect(s) of Vav in response to IFN in MMM patients. Our results showed that in the majority of patients Vav is constitutively hyperphosphorylated and that IFN‐α failed to increase substantially such a tyrosine phosphorylation of p95Vav.
Haematopoietic stem cell transplantation for patients with myelo‐dysplastic syndromes and secondary acute myeloid leukaemias: a report on behalf of the Chronic Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT)
Authors: De Witte Theo , Hermans Jo , Vossen Jaak , Bacigalupo Andrea , Meloni Giovanna , Jacobsen Niels , Ruutu Tapani , Ljungman Per , Gratwohl Alois , Runde Volker , Niederwieser Dietger , Van Biezen Anja , Devergie Agnes , Cornelissen Jan , Jouet Jean‐Pierre , Arnold Renate , Apperley Jane
Published: March 2003

Allogeneic stem cell transplantation from an HLA‐identical sibling donor is a curative treatment option for a young patient with myelodysplastic syndrome, limited by age and lack of sibling donors. Alternative stem cell sources have been used more recently, such as unrelated donors, non‐identical family members or autologous transplants. This analysis of 1378 transplants reported to the European Group for Blood and Marrow Transplantation (EBMT) addresses the outcome of the varying procedures according to the known risk factors. The estimated disease‐free survival (DFS) and estimated relapse risk at 3 years were both 36% for 885 patients transplanted with stem cells from matched siblings. In the multivariate analysis, age and stage of disease had independent prognostic significance for DFS, survival and treatment‐related mortality. Patients transplanted at an early stage of disease had a significantly lower risk of relapse than patients transplanted at more advanced stages. The estimated DFS at 3 years was 25% for the 198 patients with voluntary unrelated donors, 28% for the 91 patients with alternative family donors and 33% for the 126 patients autografted in first complete remission. The non‐relapse mortality was 58% for patients with unrelated donors, 66% for patients with non‐identical family donors and 25% for autografted patients. The relapse rate of 18% was relatively low for patients with non‐identical family donors, 41% for patients with unrelated donors and 55% for patients treated with autologous stem cell transplantation. Both allogeneic and autologous stem cell transplantation have emerged as treatment options for patients with myelodysplastic syndromes. Transplantation with an HLA‐identical sibling donor is the preferred treatment option. Patients without an HLA‐identical sibling donor may be treated with either autologous stem cell transplantation or an alternative donor transplantation. Patients younger than 20 years may be treated with an unrelated donor transplantation. Patients older than 40 years, and probably also patients between 20 and 40 years, may benefit most from an autologous stem cell transplantation.
Prospective evaluation of oral mucositis in patients receiving myeloablative conditioning regimens and haemopoietic progenitor rescue
Authors: Wardley A.M. , Jayson G.C. , Swindell R. , Morgenstern G.R. , Chang J. , Bloor R. , Fraser C.J. , Scarffe J.H.
Published: March 2003

Four hundred and twenty‐nine patients received myeloablative chemotherapy for solid and haematological malignancies in a bone marrow transplantation unit. Regimens appropriate to the tumour type were administered and haemopoietic reconstitution was achieved with peripheral blood progenitor cells (PBPC; n = 275), autologous bone marrow (auto‐BMT; n = 69) or allogeneic bone marrow (allo‐BMT; n = 85). World Health Organization (WHO) oral mucositis scores were collected prospectively from the start of chemotherapy (d 1) until d 28 or discharge. Oral mucositis (OM) was experienced by 425 (99%) patients and in 289 (67·4%) this was grade III or IV. Strong opiate analgesia was prescribed for a median of 6 d to 47% of patients. Univariate analysis suggested that the area under the OM curve (AUC; sum of daily mucositis grades, d 1–28) was associated with the myeloablative regimen, haemopoietic progenitor source (PBPC > allo‐BMT > auto‐BMT), use of myeloid growth factors and age. Multivariate analysis showed that the only independent risk factor for mucositis was the conditioning regimen (P < 0·00005). The mean OM AUC for high‐dose melphalan (HDM) regimens (52 grade–days) exceeded busulphan (41), busulphan–cyclophosphamide (35), cyclophosphamide–total body irradiation (TBI) (34), cyclophosphamide–carmustine (BCNU) (20) and cyclophosphamide–etoposide–carmustine (CVB) (19). HDM regimens resulted in the highest mean peak OM (3·6), followed by busulphan regimens (2·6), cyclophosphamide/TBI (2·3) and cyclophosphamide–carmustine and CVB (1·4). Busulphan produced significantly delayed OM (median 3 d; P < 0·00005). There was a linear association between the area under the OM curve for each treatment group and the time to reach grade 3 OM (P < 0·00005), but no association with the time to reach grade 4 neutropenia (P = 0·24) or thrombocytopenia (P = 0·73), implying that haematological and mucosal toxicity are not associated. The cytotoxic regimen is the most significant determinant of OM. Studies investigating agents to ameliorate mucosal toxicity should be stratified according to cytotoxic regimen.
Novel BCR–ABL transcript containing an intronic sequence insert in a patient with Philadelphia‐positive acute lymphoblastic leukaemia
Authors: Hirota Masako , Hidaka Eiko , Ueno Ichiro , Ishikawa Masayo , Asano Naoko , Yamauchi Kazuyoshi , Ishida Fumihiro , Tozuka Minoru , Katsuyama Tsutomu
Published: March 2003

In a patient with Philadelphia chromosome‐positive acute lymphoblastic leukaemia (ALL), a novel variant of the chimaeric BCR–ABL mRNA transcript was detected by reverse transcription polymerase chain reaction (RT‐PCR). Sequencing revealed the novel transcript to be a chimaeric mRNA produced by fusion of the BCR exon 14 (b3) to the ABL exon a2 with a 49‐base pair (bp) insertion of an ABL intron 1b sequence between them. The insertion of the 49 bp introduced a stop codon. These data show that this variant of the chimaeric mRNA would not be translated into the p210 BCR–ABL protein. This could be one of the explanations as to why clinically the patient has responded well to therapy and continues to follow a mild clinical course.
Determination of helper T‐cell precursor frequencies against non‐haemopoietic cells: comparison of co‐stimulation provided by anti‐CD28 antibody versus the cellular ligand B7‐1
Authors: Van Dijk Astrid M. C. , De Gast Gijsbert C. , Kessler Floortje L. , Stadhouders‐Keet Simone A. E. , Van Lier Rene A. W. , Verdonck Leo F. , Otten Henny G.
Published: March 2003

Helper T‐cell precursor frequency assays (HTLp‐assays) are commonly used in transplantation to examine the frequency of T cells reactive against donor or host alloantigens. In these assays, peripheral blood mononuclear cells (PBMCs) are most often used as stimulator cells. However, cells targeted after transplantation do not always belong to the haematopoietic lineage and may express different alloantigens, especially minor histocompatibility antigens (mHags). Non‐haematopoietic cells lack expression of the B7 co‐stimulatory molecules needed to activate primary T cells that can be supplied by anti‐CD28 (αCD28) antibodies or transfection with B7‐1 coding sequences. At present, it is not known how these two ways of supplied co‐stimulation compare in HTLp assays. B7‐1‐transfected A431 keratinocytes (A431B7‐1) induced higher proliferative responses in allogeneic primary T cells and more interleukin (IL) 2 production than that induced by A431 cells plus αCD28, whereas the kinetics of proliferation and IL‐2 production were similar. Neither cross‐linking of αCD28 bound to T cells nor prevention of IL‐2 resorption by the anti‐IL‐2 receptor resulted in improved proliferation or IL‐2 production. Results of HTLp assays indicated that A431B7‐1 activated on average 7·5 times more alloreactive IL‐2‐producing T cells than A431 cells plus αCD28. We conclude that primary T‐cell alloresponses against major histocompatibility complexes (MHCs) and mHags expressed on non‐haematopoietic cells can be measured in HTLp assays using supplied co‐stimulation, although αCD28 yields an intrinsic underestimation of actual frequencies.