bluuebunny/crossref_metadata_2025 huggingface dataset:
Biological and molecular characterization of PNH‐like lymphocytes emerging after Campath‐1H therapy
Authors: Fracchiolla Nicola S. , Barcellini Wilma , Bianchi Paola , Motta Marina , Fermo Elisa , Cortelezzi Agostino
Published: March 2003

Campath‐1H, an anti‐CD52 monoclonal antibody, is therapeutically active in lymphoproliferative and autoimmune diseases. After Campath‐1H therapy, lymphocytes with a paroxysmal nocturnal haemoglobinuria (PNH) phenotype have been reported to emerge. We characterized a PNH‐like lymphocyte population emerging after Campath‐1H therapy, in a patient with fludarabine refractory B‐cell chronic lymphocytic leukaemia (B‐CLL). We demonstrated a reduction in PIG‐A mRNA levels compared with controls, and of all cytokines tested [interleukin (IL)‐4, IL‐13, IL‐2, interferon(IFN)‐γ, IL‐6, IL‐10, and tumour necrosis factor (TNF)‐α], except transforming growth factor (TGF)‐β. Given the inhibitory activity of TGF‐β, its elevated levels may contribute to the selective pressure of Campath‐1H, leading to the emergence of PNH‐like lymphocytes.
Erythropoietin synthesis by tumour tissues in a patient with uterine myoma and erythrocytosis
Authors: Suzuki Mitsuaki , Takamizawa Satoru , Nomaguchi Koji , Suzu Shinya , Yamada Muneo , Igarashi Takashi , Sato Ikuo
Published: March 2003

We report a patient with uterine myoma (leiomyoma) and erythrocytosis in whom erythropoietin (Epo) production in the leiomyoma tissue was identified by reverse transcription polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA). A 48‐year‐old Japanese woman with uterine myoma showed marked erythrocytosis (haemoglobin: 20·2 g/dl, haematocrit: 61·1%, red blood cells: 6·51 × 1012/1). After hysterectomy, erythrocytosis rapidly disappeared. In the leiomyoma tissue collected from the patient, Epo mRNA expression was confirmed using RT‐PCR. Furthermore, ELISA showed that the Epo protein level was significantly increased compared with those in control tissues. It is suggested that the pathogenesis of erythrocytosis in patients with uterine myoma involves ectopic Epo production by leiomyoma tissues.
Cytogenetic risk groups in acute myeloblastic leukaemia differ greatly in their semi‐solid colony growth
Authors: Öhler Leopold , Berer Andrea , Aletaha Daniel , Kabrna Eva , Heinze Georg , Streubel Berthold , Fonatsch Christa , Haas Oskar A. , Lechner Klaus , Geissler Klaus
Published: March 2003

We have analysed the results of semi‐solid bone marrow cultures in 296 patients withde novoacute myeloblastic leukaemia (AML) and correlated them with the leukaemic karyotype. A favourable prognostic karyotype was found in 52 patients (group A, 18·3%), an intermediate karyotype in 163 patients (group B, 57·4%), and unfavourable cytogenetics were observed in 69 patients (group C, 24·3%). Median colony growth according to the three risk groups was 2 (range 0–344) in group A, 14·5 (range 0–5000) in group B and 50·0 (0–3000) in group C (A vs. B,P < 0·001; A vs. C,P < 0·001; B vs. C,P < 0·01). Among the patients treated with chemotherapy (n = 257), median colony growth was 10 (range 0–5000) in those who achieved complete remission (CR) compared with 56·5 (range 0–1000) in patients without remission (NR) (P = 0·002). The median colony growth of all patients [13/105bone marrow mononuclear cells (BMMCs); range 0–5000] significantly discriminated between patients regarding survival (OS 11 vs. 7 months,P = 0·044). However, multiple Cox regression analysis revealed cytogenetic risk groups as the most important predictor for achieving CR, disease‐free and overall survival, with colony growth adding no additional prognostic information. In 64 patients, colony growth was also investigated without the addition of exogenous cytokines. Interestingly, none of the patients with a favourable karyotype exhibited autonomous growth, whereas 50% with an intermediate and 73% of patients with an unfavourable karyotype displayed either partial or full autonomous growthin vitro(P = 0·0004). Our data suggest that the growth potential of the leukaemic clone seems to be critically influenced by the molecular changes emerging from chromosomal abnormalities.
Effect of chemotherapy and stem cell transplantation on T lymphocyte clones in familial haemophagocytic lymphohistiocytosis
Authors: Kimura Nobuhiro , Ishii Eiichi , Sako Masahiro , Yoshida Tetsuya , Nagano Mitsuyuki , Takada Hidetoshi , Imashuku Shinsaku , Tamura Kazuo
Published: March 2003

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare disorder in infancy, curative only by an allogeneic stem cell transplantation (SCT). We recently confirmed the clonal evidence of T cells in FHL. To confirm the effect of chemotherapy and SCT in FHL, the change of T‐cell clones was analysed in two patients using inverse reverse transcription‐polymerase chain reaction (RT‐PCR) of the T‐cell receptor variable region (TCR V) gene, followed by PCR for the junctional region (Jβ‐PCR), a single‐strand conformation polymorphism (SSCP) and sequencing analysis at diagnosis, after chemotherapy and after SCT. A high frequency (> 15%) of αβ T‐cell clones and a predominant bias (Jβ1:Jβ2, 85:15) for the Jβ1 subgroup were observed in the two patients at diagnosis. In one patient, however, an inverted predominant bias (Jβ1:Jβ2, 9:91) for the Jβ2 subgroup and oligoclonal expansion were observed at relapse after chemotherapy. In the other patient, correction of both restricted Jβ cluster usage and variation of TCR were observed after chemotherapy and SCT. Using sequence analysis, the clonal T cells detected at diagnosis were found to be substituted at low frequency (< 0.75%) by several new clones after chemotherapy and SCT. These results indicate that any genetic defect could influence the regulation of the T‐cell network, and normalization of both the variation in each Vβ repertoire and the Jβ1/Jβ2 ratio is needed to achieve remission, and might support the rationale that the only acceptable curative therapy of FHL is allogeneic SCT.
Polymorphisms in inflammatory cytokines and Fcγ receptors in childhood chronic immune thrombocytopenic purpura: a pilot study
Authors: Foster Charles B. , Zhu Shaoxian , Erichsen Hans Christian , Lehrnbecher Thomas , Hart Elizabeth S. , Choi Eunhwa , Stein Steven , Smith Michael W. , Steinberg Seth M. , Imbach Paul , Kühne Thomas , Chanock Stephen J. ,   for the Early Chronic ITP Study Group
Published: March 2003

Inflammatory cytokines and low‐affinity Fcγ receptor (FcγR) polymorphisms were investigated in 37 children with chronic immune thrombocytopenic purpura (cITP) and 218 controls. Genotype analysis included common variants in the regulatory regions of cytokines, TNF, LTA, IL1RN, IL1A, IL1B, IL4, IL6 and IL10, and structural variants of the low affinity FcγRs, FCGR2A, FCGR3A and FCGR3B. Associations were observed for TNF (P = 0·0032), LTA (P = 0·019), FCGR3A (P = 0·038) and FCGR3B (P = 0·0034). Two combinations of genotypes (TNF and FCGR3A;P = 0·0003, and LTA and FCGR3B;P = 0·011) were significantly associated with cITP. These results provide preliminary evidence that variant genotypes of FcγRs and cytokines contribute to cITP pathogenesis.
Bcl‐2 and Bcl‐x expression in the CD34+ cells of aplastic anaemia patients: relationship with increased apoptosis and upregulation of Fas antigen
Authors: Ismail M. , Gibson F. M. , Gordon‐Smith E. C. , Rutherford T. R.
Published: March 2003

Aplastic anaemia (AA) is a syndrome of haemopoietic failure involving increased apoptosis in stem cells. AA CD34+ cells often have upregulated Fas antigen, but this does not explain the increased apoptosis in all patients. To examine whether abnormal expression of the apoptotic modulators Bcl‐2 and Bcl‐x is involved in increased apoptosis in the CD34+ cells of patients, we examined cells from 19 AA patients and 18 normal controls by triple staining for CD34, Bcl‐2 or Bcl‐x, together with 7‐amino actinomycin D to determine viability or with staining for Fas antigen. We confirmed increased apoptosis of CD34+ cells in patients. All CD34+ cells in patients and controls expressed Bcl‐2 and Bcl‐x with no significant difference between the groups. In patients, viability of CD34+/Bcl‐2hi cells was similar to that of CD34+/Bcl‐2lo cells, but CD34+/Bcl‐xhi cells were significantly more viable than CD34+/Bcl‐xlo cells. CD34+ cells from AA patients expressed upregulated Fas antigen, but this did not correlate with Bcl‐2 or Bcl‐x expression. These results suggest a more significant role for Bcl‐x as an anti‐apoptotic regulator in CD34+ cells in AA than Bcl‐2. The induction of death by Fas antigen may bypass the anti‐apoptotic effect of Bcl‐2 and Bcl‐x in CD34+ cells in AA.
Hepatic fibrosis plays a central role in the pathogenesis of thrombocytopenia in patients with chronic viral hepatitis
Authors: Adinolfi Luigi Elio , Giordano Maria Grazia , Andreana Augusto , Tripodi Marie‐Francoise , Utili Riccardo , Cesaro Giuseppe , Ragone Enrico , Mangoni Emanuele Durante , Ruggiero Giuseppe
Published: March 2003

The pathogenesis of thrombocytopenia in chronic hepatitis is not well known. This study evaluated the relationship between liver injury, serum thrombopoietin, splenomegaly and thrombocytopenia in chronic viral hepatitis. Two hundred and nine patients were enrolled, 85 with splenomegaly and 124 without. Thrombocytopenia was present in 71% and 23% of patients with or without splenomegaly respectively. In subjects with low platelet count, those with splenomegaly showed significantly lower platelet numbers than those without splenomegaly. The spleen size correlated with portal hypertension. An inverse correlation between spleen size and platelet count was observed (r = −0·54; P < 0·0001). In patients without splenomegaly, thrombocytopenia was associated with the grade of fibrosis; platelet counts were the highest in patients with fibrosis 0–2, lower in those with grade 3 (P < 0·008) and lowest in those with grade 4 (P < 0·05). These findings were independent of demographic and biochemical characteristics, hepatic necroinflammatory activity, portal hypertension and splenomegaly. Patients with normal platelet counts showed higher thrombopoietin levels than those with low platelet counts (P < 0·0001). An inverse correlation between thrombopoietin levels and fibrosis grade was observed (r = − 0·50; P < 0·0001). Median thrombopoietin levels were 58 and 27 pg/ml for fibrosis grade 0–1 and grade 4 respectively (P < 0·001). These data indicate that advanced hepatic fibrosis, causing an altered production of thrombopoietin and portal hypertension, plays the central role in the pathogenesis of thrombocytopenia in chronic viral hepatitis.
Changes in the expression of FGFR3 in patients with chronic myeloid leukaemia receiving transplants of allogeneic peripheral blood stem cells
Authors: Dvorakova Dana , Krejci Pavel , Mayer Jiri , Fajkus Jiri , Hampl Ales , Dvorak Petr
Published: March 2003

Fibroblast growth factor receptors (FGFR1–4) are implicated in various cellular events, including cell growth and transformation. Here, we showed that patients suffering from chronic myeloid leukaemia (CML) express high levels of FGFR3 mRNA in white blood cells (WBCs). After stem cell transplantation and reconstitution of haematopoiesis, the expression of FGFR3 decreased and was maintained at low levels that are typical of healthy individuals. However, FGFR3 expression became upregulated again in those patients that had accelerated BCR/ABL rearrangement and underwent relapse of leukaemia. Our findings suggest that, in CML, the changing levels of FGFR3 transcripts in WBCs may have prognostic significance.
Post‐menopausal hormone therapy and concentrations of protein C and antithrombin in elderly women
Authors: Cushman Mary , Psaty Bruce M. , Meilahn Elaine N. , Dobs Adrian S. , Kuller Lewis H.
Published: March 2003

The effects of post‐menopausal hormone therapy (HRT) on blood coagulation in elderly women are not well defined. We studied associations of HRT use with levels of natural anticoagulant proteins in a cross‐sectional study of 3393 women ≥ 65 years of age participating in the Cardiovascular Health Study. Protein C antigen and antithrombin were measured in all users (n = 230 unopposed oestrogen; 60 oestrogen/progestin) and a comparison group of 196 age‐ and race‐matched non‐users. Compared with non‐users, oestrogen use was associated with higher protein C (4·80 vs. 4·30 µg/ml, P < 0·01). Results were similar for oestrogen/progestin (P > 0·05). In both user groups, antithrombin was lower than in non‐users (109% for each vs. 115% in non‐users, P < 0·001). Adjustment for factors related to prescription of HRT and to anticoagulant protein levels had little impact on the results. For antithrombin, associations with HRT were larger for thinner Caucasian women and black women. Venous thrombosis from HRT may be mediated partly by alterations in antithrombin, but not protein C concentrations. This study extends previous observations to older women, the group at highest risk of venous thromboembolism. Studies of HRT‐induced alterations in anticoagulant function in relation to the occurrence of thrombosis with HRT are required.
Severe factor VII deficiency with recurrent intracranial haemorrhages owing to double heterozygosity for a splice site mutation of an IVS4 and a novel nonsense mutation in exon 8 (Gln211→Term)
Authors: Takamiya Osamu , Okimoto Yuri
Published: March 2003

Genetic analysis of a 10‐month‐old Japanese baby boy with recurrent intrathoracic bleeding, cerebral haemorrhages and gastrointestinal bleeding secondary to severe factor VII (FVII) deficiency revealed evidence of two distinct mutations of FVII: a splice site mutation of G→A at nucleotide 6071 in the IVS4 splice site and a novel nonsense mutation (Gln211→Term) in exon 8. His bleeding was difficult to control without prophylactic infusion of FVII. We detected a heterozygous splice site mutation of the IVS4 in his mother and a heterozygous nonsense mutation in exon 8 (Gln211→Term) in his father. The parents' FVII levels are both 50% of normal controls. The FVII:C in plasma from the proband was < 1·5% of normal controls. FVII:antigen (Ag) was < 1% of normal controls, using a monoclonal antibody (mAb) hVII‐B101/1 that specifically reacts with FVII epidermal growth factor 1 (EGF‐1), and 5% of normal controls, using a rabbit polyclonal antibody against human FVII. After immunoadsorption with mAb hVII‐B101/B1–Sepharose 4B, FVII levels of both the proband and his mother were 5% of normal controls; after immunoadsorption the FVII levels of normal subjects were < 1%. We hypothesize that secretion of a small amount of dysfunctional FVII lacking EGF‐1 into the circulation accounts for this observation.
Prolonged ex vivo culture of cord blood CD34+ cells facilitates myeloid and megakaryocytic engraftment in the non‐obese diabetic severe combined immunodeficient mouse model
Authors: Rice Alison M. , Wood Julie A. , Milross Christopher G. , Collins Cathryn J. , Case Jamie , Vowels Marcus R. , Nordon Robert E.
Published: March 2003

A clinical goal for ex vivo expansion of cord blood (CB) CD34+ cells is to shorten the period of neutropenia and thrombocytopenia following myeloablative therapy and transplantation. Prolongation of cytokine expansion leads to the production of greater numbers of cells, and should have an impact on neutrophil and platelet recovery. Furthermore, expansion of CD34+ cells should support the continued production of neutrophils and platelets in the 6‐week period following transplantation. We tested these hypotheses by characterization of the kinetics (human CD45+ cells in the blood) and phenotype (CD45, CD34, CD61, CD33, CD19 and CD3) of human engraftment in the non‐obese diabetic severe combined immunodeficient mouse (NOD–SCID) following 7 or 14 d of ex vivo expansion of CB CD34+ cells. Mice transplanted with 14 d cells showed greater percentages of human CD45+ cells in the blood, bone marrow and spleen than mice transplanted with unexpanded cells or 7 d cells. Prolonging cytokine exposure of CD34+ cells and transplantation with increasing numbers of input cells facilitated the production of absolute numbers of CD34+, CD33+, CD61+ and CD19+ cells in vivo. Furthermore, analysis of SCID engrafting potential showed that prolongation of culture duration facilitates in vivo expansion of CD45+, CD34+ and CD19+ cells after transplantation. It is anticipated that prolonged (2 weeks) ex vivo culture of CB will have a beneficial clinical effect.
Transcript profiling of human dendritic cells maturation‐induced under defined culture conditions: comparison of the effects of tumour necrosis factor alpha, soluble CD40 ligand trimer and interferon gamma
Authors: Moschella Federica , Maffei Antonella , Catanzaro Richard P. , Papadopoulos Kyriakos P. , Skerrett Donna , Hesdorffer Charles S. , Harris Paul E.
Published: March 2003

Using cDNA arrays, we characterized patterns of gene expression in populations of human dendritic cells (DCs) produced for clinical use. Culture and maturation induction of myeloid adherent cells under serum‐free conditions yielded DCs with phenotypes similar to those described in serum‐based systems. Analysis of gene expression in DCs treated with tumour necrosis factor alpha, soluble CD40L trimer or interferon gamma, however, showed specific patterns for each factor examined. Our studies document the expression of several transcripts that have not hitherto been described in DCs and/or differentially regulated according to the differentiation state of the DCs, and suggest important functional differences among the DC populations examined. In addition, DC maturation directs changes in the levels of mRNA specific for transcriptional regulators that effect the production of cytokines (e.g. BCL‐6, c‐rel). Other changes observed, including alteration in the gene expression profile of adhesion molecules and chemokine receptors such as CD44H, CD 49B, Rantes R, CXCR5 and CD37, suggest differences in trafficking potential between the populations studied. This broad‐based description of DC populations, produced under serum‐free conditions, has enabled us to better define intermediate stages of DC maturation as well as the differentiation‐inducing effects of cytokines on these cells.
Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin‐induced differentiation
Authors: Bornstein Rafael , García‐Vela José , Gilsanz Florinda , Auray Claude , Calés Carmela
Published: March 2003

Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2·4 times higher in CB than in PB cells. Importantly, the TPO‐induced ploidy level was notably different: whereas 82·7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.
Intensification of salvage treatment with high‐dose sequential chemotherapy improves the outcome of patients with refractory or relapsed aggressive non‐Hodgkin's lymphoma
Authors: Cortelazzo Sergio , Rambaldi Alessandro , Rossi Andrea , Oldani Elena , Ghielmini Michele , Benedetti Fabio , Tarella Corrado , Zaglio Francesco , Vitolo Umberto , Di Nicola Massimo , Pogliani Enrico , Cavalli Franco , Gianni Alessandro Massimo , Barbui Tiziano
Published: March 2003

The aim of the present study was to retrospectively evaluate whether a high‐dose sequential chemotherapy programme (HDS: cyclophosphamide, methotrexate, etoposide) administered prior to autologous transplantation could optimize the salvage of patients with refractory or relapsed aggressive non‐Hodgkin's lymphoma. Between 1985 and 1999, 103 patients (median age 43 years, range 16–65) from eight Italian centres and one Swiss centre, with refractory (n = 38) or relapsed (n = 65) diffuse large B‐cell and T‐cell lymphoma, were treated using HDS followed by high‐dose regimens with autologous haematopoietic stem cell transplantation. Eighty‐three patients responded to the HDS regimen (81%, 95% C.I., 73– 88%) and 79 eventually achieved a complete response (CR) after autotransplantation (90%, 95% C.I., 81– 96%). None of 20 cases resistant to HDS attained CR. Treatment‐related mortality was 4%. After a median follow‐up of 24 months (range 6–174 months), 3‐year estimates of overall survival, event‐free survival and disease‐free survival were 47% (95% C.I., 36–59%), 44% (95% C.I., 34–54%) and 64% (95% C.I., 50–74%) respectively. Multivariate analysis showed that chemosensitivity to HDS represented the strongest predictor of both CR and survival. This retrospective study shows that salvage treatment using HDS had relatively low toxicity and was associated with remarkable response rates, allowing further effective therapy with high‐dose autograft programmes.
Inhibitory effect of interleukin 3 on early development of human B‐lymphopoiesis
Authors: Miyamoto Koichiro , Tsuji Kohichiro , Maekawa Taira , Asano Shigetaka , Nakahata Tatsutoshi
Published: March 2003

Interleukin 3 (IL‐3) is known as a stimulator of proliferation and differentiation of non‐lymphoid cells, but information about the activity of IL‐3 in lymphopoiesis is limited. In the present study, we examined the effect of IL‐3 on human B‐lymphopoiesis using the co‐culture system on a murine stromal cell line, MS‐5, with stem cell factor (SCF) and granulocyte colony‐stimulating factor (G‐CSF). Although a large number of CD45+CD19+ B cells were generated in the co‐culture of cord blood CD34+ cells, the addition of IL‐3 to the co‐culture suppressed the B‐cell generation in a dose‐dependent manner. In the co‐culture, CD34+IL‐3 receptor α‐chain (IL‐3Rα)+ cells, but not IL‐3Rα− cells, produced B cells, and IL‐3 suppressed the B‐cell generation from CD34+IL‐3Rα+ cells, suggesting that IL‐3 exerts an inhibitory effect through the binding to IL‐3Rα on CD34+ cells. Because the delayed addition of IL‐3 and the short‐term exposure to IL‐3 showed that IL‐3 acted as an inhibitor at an early stage of B‐cell development from CD34+ cells, before the generation of CD19+ B cells, the effect of IL‐3 on the early development of B cells from immature CD34+CD38− cells was examined. Individual colonies, produced from clone‐sorted CD34+CD38− cells by SCF, thrombopoietin and a complex of IL‐6/soluble IL‐6R with or without IL‐3, were divided into two fractions and analysed for B‐cell potential using co‐culture on MS‐5 with SCF and G‐CSF, and haematopoietic potential using methylcellulose clonal culture. Although some colonies cultured without IL‐3 showed both B‐cell and haematopoietic potential, all the colonies cultured with IL‐3 showed only haematopoietic potential. These results indicate that IL‐3 has an inhibitory effect on the B‐cell generation from human lymphohaematopoietic progenitor cells.
Locoregional intrasplenic chemotherapy for hypersplenism in myelofibrosis
Authors: Camba Lionello , Aldrighetti Luca , Ciceri Fabio , Bernardi Massimo , Marktel Sarah , Angeli Enzo , Giacomelli Maurizio
Published: March 2003

A 79‐year‐old patient with post‐polycythaemic myelofibrosis presented with severe hypersplenism. After splenic artery catheterization, cytosine arabinoside was given intrasplenically from November 1999 to March 2000 for 5 d/month at 10 mg/m2 and increased each month by 10 mg/m2. It was then administered by continuous infusion until June 2000, starting at 20 mg/m2/d and tapering by 5 mg/m2 every 2 weeks to a final daily dose of 5 mg/m2/d. The drug was then stopped. The spleen had decreased to one third of the initial volume. Clinical conditions and haematological indices improved substantially. Intrasplenic therapy could be a new therapeutic tool for hypersplenism in chronic idiopathic and post‐myeloproliferative myelofibrosis.
Cytokine production and T‐cell activation by macrophage–dendritic cells generated for therapeutic use
Authors: Coronel Agnès , Boyer Aurélie , Franssen Jean‐Denis , Romet‐Lemonne Jean‐Loup , Fridman Wolf Herman , Teillaud Jean‐Luc
Published: March 2003

Clinical grade ex vivo‐generated antigen‐presenting cells, macrophage–dendritic cells (MAC–DCs) or macrophage‐activated killers (MAKs) were derived from peripheral blood mononuclear cells (PBMCs). Cultures (7 d) were performed in non‐adherent conditions in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and either interleukin 13 (IL‐13) or dihydroxy‐vitamin D3 respectively. MAKs were activated during the last 24 h with interferon γ (IFNγ). Reverse transcription polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA) analyses indicated that IL‐1β and tumour necrosis factor α (TNFα) were produced by both cells. Higher pro‐inflammatory cytokine (IL‐1β and TNFα) amounts were detected on average in MAK supernatants. In contrast, IL‐12 p40 was found only in MAC–DC supernatants, but the biologically active IL‐12 form (p70) was never detected. T‐cell cytokines (IL‐2, IL‐4, IL‐10) were not produced in culture conditions in which T cells were nevertheless present. At d 7, TNFα or lipopolysaccharide (LPS) upregulated IL‐12 p40 production by MAC–DCs, while IL‐12 p70 remained undetectable. LPS stimulation also increased TNFα production by these cells. Allogeneic mixed lymphocyte reactions (MLR) showed that MAKs are poor stimulatory cells compared with MAC–DCs. The MAC–DC stimulatory capacity was enhanced by LPS, although the expression of HLA class II, CD83, CD80 and CD86 was unmodified. Thus, MAC–DCs represent a tool for triggering adaptative immunity, while MAK should be primarily used as effector killer cells.
Increased lactate production follows loss of mitochondrial membrane potential during apoptosis of human leukaemia cells
Authors: Tiefenthaler Martin , Amberger Albert , Bacher Nicole , Hartmann Bernd L. , Margreiter Raimund , Kofler Reinhard , Konwalinka Günther
Published: March 2003

Acute tumour‐lysis syndrome (ATLS) is a frequently fatal complication after cytoreductive leukaemia therapy. Lactic acidosis is associated with ATLS and its extent is correlated with the severity of ATLS. In the course of cytoreductive therapy, apoptosis is induced in tumour cells, which results in loss of mitochondrial function. We hypothesize that loss of mitochondrial function leads to compensatory glycolysis, which is the main cause of lactate accumulation and acidosis. We tested this hypothesis using the model of glucocorticoid‐induced apoptosis in the human acute lymphoblastic leukaemia cell line CCRF‐CEM. After induction of glucocorticoid‐induced apoptosis, a biphasic course of lactate production was observed. Prior to the onset of apoptosis, i.e. prior to the loss of membrane potential, lactate production was reduced. However, subsequent to loss of mitochondrial membrane potential a massive increase in lactate production was observed (15·5 ± 0·5 versus 10·17 ± 0·09 mmol/106 cells, P = 0·001). We also demonstrated that inhibition of respiratory chain activity by antimycin A resulted in excess lactate production. In the model cell line used, conditional bcl‐2 expression delayed glucocorticoid‐induced apoptosis by protecting against loss of mitochondrial membrane potential; bcl‐2 expression delayed the increase in lactate production and had no effect on the pre‐apoptotic drop in lactate production. Apoptosis‐induced lactate production was also observed in other cell lines (HL60, THP1 and OPM2) with various cytotoxic agents [doxorubicin, gemcitabine and vumon (VM26)]. Thus, the data suggest that lactate acidosis can be caused by apoptotic loss of mitochondrial function and massive apoptosis of a tumour mass via lactic acidosis may be the essential pathological event in ATLS.
Unrelated donor marrow transplantation in children with severe aplastic anaemia using cyclophosphamide, anti‐thymocyte globulin and total body irradiation
Authors: Kojima Seiji , Inaba Jun , Yoshimi Ayami , Takahashi Yoshiyuki , Watanabe Nobuhiro , Kudo Kazuko , Horibe Keizo , Maeda Naoko , Kato Koji , Matsuyama Takaharu
Published: March 2003

We report a favourable outcome in 15 patients with severe aplastic anaemia (SAA) who were < 20 years of age and who underwent bone marrow transplantation (BMT) from a human leucocyte antigen (HLA)‐matched unrelated donor. All patients were non‐responders to intensive immunosuppressive therapy (IST) and were multiply transfused. The conditioning regimen consisted of cyclophosphamide (60 mg/kg/d, on d −4 and −3), anti‐thymocyte globulin (2·5 mg/kg/d, on d −5 to −2) and total body irradiation (2·5 Gy × 2/d, on d −2 and −1). Patients received cyclosporine and methotrexate for prophylaxis of graft‐versus‐host disease (GVHD), except for the last four who received tacrolimus instead of cyclosporine. Donor/recipient pairs were identical for HLA class I and II antigens by serological typing, but four pairs were found to have a mismatch at the HLA‐A, ‐B or ‐DRB1 locus by high‐resolution typing. All patients achieved rapid engraftment and are alive at 2–86 months after transplantation (median follow‐up, 51 months). Moderate to severe acute GVHD occurred in 5 out of 15 patients (33%); only one patient developed extensive chronic GVHD. Considering our encouraging results, unrelated donor transplantation for SAA is recommended as a salvage therapy in non‐responders to IST.
Differentiation stage of natural killer cell‐lineage lymphoproliferative disorders based on phenotypic analysis
Authors: Mori Kiyoshi L. , Egashira Motoki , Oshimi Kazuo
Published: March 2003

In the normal developmental pathway of natural killer (NK) cells, pre‐NK cells express CD161, immature NK cells express CD161 and CD56, and mature NK cells express CD161, CD56 and CD94. To identify the normal counterpart of NK cells from which neoplastic cells originate, surface antigens were analysed. Blastic NK‐cell lymphoma/leukaemia lacked CD94 and CD161 but had CD56. Aggressive NK‐cell leukaemia/lymphoma and nasal NK‐cell lymphoma, although morphologically immature, expressed both CD56 and CD94 and strong NK activity. Cells from chronic NK lymphocytosis expressed CD56 and CD94.
The effect of treatment with Campath‐1H in patients with autoimmune cytopenias
Authors: Willis F. , Marsh J. C. W. , Bevan D. H. , Killick S. B. , Lucas G. , Griffiths R. , Ouwehand W. , Hale G. , Waldmann H. , Gordon‐smith E. C.
Published: March 2003

We describe 21 patients with severe and life‐threatening autoimmune cytopenias resistant to standard immunosuppression who were treated with the monoclonal antibody Campath‐1H. Four patients had autoimmune neutropenia, four had autoimmune haemolytic anaemia, four had pure red cell aplasia, one had immune thrombocytopenia purpura (ITP), three had autoimmune haemolytic anaemia and ITP (Evan's syndrome), three had autoimmune pancytopenia (ITP, autoimmune neutropenia and autoimmune haemolytic anaemia), one had ITP (associated with acquired Glanzmann's disease) and autoimmune neutropenia, and one had ITP and red cell aplasia. Campath‐1H was administered at a dose of 10 mg/d as an intravenous infusion for 10 d. Responses were seen in 15 patients, which were sustained in six. Relapse occurred in eight patients after Campath‐1H treatment. Patients entering the study later, received cyclosporine after Campath‐1H in an attempt to reduce the incidence of relapse. Three patients received a second course of Campath‐1H; all responded but later relapsed. Fourteen patients are alive at a median of 12 months (range 4–61) after Campath‐1H. Campath‐1H represents an alternative therapeutic option for severe, refractory autoimmune cytopenias.
The role of heterocellular hereditary persistence of fetal haemoglobin in β0‐thalassaemia intermedia
Authors: Chang Yen‐Pei C. , Littera Roberto , Garau Raffaela , Smith Kirby D. , Dover George J. , Iannelli Sergio , Cacace Enrico , Contu Licinio
Published: March 2003

β0‐thalassaemia intermedia (β0‐TI) describes patients who lack β‐globin synthesis yet manifest a non‐transfusion‐dependent form of β‐thalassaemia. Co‐inheritance of α‐thalassaemia, certain variants of the β‐like globin gene cluster and elevated fetal haemoglobin (HbF) production are all associated with β0‐TI. However, the mild phenotypes of many β0‐TI patients are unexplained. Genetically determined HbF levels in β‐thalassaemia are difficult to assess because erythrocytes containing HbF (F cells) preferentially survive over erythrocytes without HbF. To evaluate the importance of genetically elevated HbF in β‐thalassaemia, F‐cell levels of 19 TI patients' relatives were compared with relatives of transfusion‐dependent β‐thalassaemia major patients and those of β‐globin genotype‐matched controls. The β‐globin and α‐globin genotypes, as well as their Gγ promoter were also examined. Using this approach, in all but one patient the mild phenotype was attributable to either α‐globin genotype, γ‐globin promoter polymorphism or inherited elevated F‐cell levels. The findings of this study establish the F‐cell levels required to modify the degree of disease severity significantly and demonstrate that F‐cell level is a crucial parameter in the understanding of phenotypic variation in β‐thalassaemia.
Prognostic significance of risk group stratification in elderly patients with acute myeloid leukaemia
Authors: Wahlin Anders , Markevärn Berit , Golovleva Irina , Nilsson Marie
Published: March 2003

Prognostic factors were studied in a series of 211 acute myeloid leukaemia (AML) patients over 60 years of age, treated at a single centre. The patients were allocated into three risk groups based on cytogenetics, occurrence of antecedent haematological disorder and leucocyte count. Only 3% had low‐risk features, 39% had intermediate‐ and 58% had adverse‐risk features. Complete remission (CR) was achieved in 43% of all patients. In multivariate analyses, the number of cycles needed to achieve CR and the risk group were significantly associated with the duration of CR. Median survival time for the entire cohort of patients was only 107 d. Advanced age, low induction treatment intensity, treatment during earlier years and adverse‐risk group were associated with shorter overall survival times. Risk group classification may help selection of elderly patients with a good chance of benefiting from intensive treatment to actually receive such treatment, while sparing others with a low probability of survival benefit from toxic treatment. Low intensity induction treatment reduces the chance of obtaining complete remission, produces inferior survival times and should consequently be avoided when the aim is to obtain complete remission. In elderly AML patients, introducing age and re‐evaluation of intermediate and good prognosis patients regarding response to induction treatment may improve the risk group classification.
β‐Thalassaemia intermedia in a Turkish girl: homozygosity for G→A substitution at +22 relative to the β‐globin cap site
Authors: Öner R. , Öner C. , Birben E. , Sözen M. , Gümrük F. , Gürgey A. , Altay Ç.
Published: March 2003

We provide the first description of a homozygote patient for the G→A substitution in the 5′ UTR of the β‐globin gene. The proband was a 17‐year‐old girl with β‐thalassaemia intermedia who had never received a blood transfusion. The physical examination revealed a well‐developed women with no facial or bony abnormalities. There was mild paleness and mild splenomegaly which was 2 cm below the costal margin. The haemoglobin (Hb) was 7·6 g/dl, Hb A2 5·4% and Hb F 14·6% of the total Hb. The Hb A2 of both parents was 3·5%. The Hb F level in the mother and father were 0·9, 1·2% and the mean cell volume (MCV) value was 70 and 72 fl respectively. DNA analysis of the β‐gene region of the propositus revealed homozygosity for a G→A substitution at nucleotide +22 relative to the β‐gene cap site, within a functional downstream region that was referred to as the DCE (downstream core element). In addition to the data obtained previously from in vitro transcription assays, clinical findings and in vivo expression studies gave some valuable clues about the effect of +22 G→A mutation on the expression of β‐gene. Phenotypic expression of this homozygous patient is highly suggestive that G→A substitution at nt +22 confers a relatively mild (silent) β+‐thalassaemia phenotype.
Favourable response to therapy with the anti‐CD20 monoclonal antibody rituximab in primary chronic cold agglutinin disease
Authors: Berentsen Sigbjørn , Tjønnfjord Geir E. , Brudevold Robert , Gjertsen Bjørn T. , Langholm Ruth , Løkkevik Erik , Sørbø Jon Hjalmar , Ulvestad Elling
Published: March 2003

The ‘primary’ form of chronic cold agglutinin disease is a clonal B‐cell lymphoproliferative disorder that is notoriously difficult to treat with drugs, including corticosteroids, alkylating agents, alpha‐interferon and purine analogues. We performed a small, open, uncontrolled, prospective study to evaluate the effect of therapy with the monoclonal anti‐CD20 antibody rituximab. Six patients with clonal CD20+κ+B‐cell proliferation received seven courses of rituximab 375 mg/m2, d 1, 8, 15, and 22. One patient achieved a complete response. Four partial responses were observed, including a response to re‐treatment in one patient. Two patients were categorized as non‐responders. Haemoglobin levels increased by a median of 4·1 g/dl in the total group and 4·7 g/dl in the responders, who also experienced a substantial improvement of clinical symptoms. The treatment was well tolerated. We discuss the effect of rituximab therapy compared with other treatment options, and try to explain why two individual patients did not respond. Despite the small numbers, the results are very encouraging. Further studies of rituximab therapy for chronic cold agglutinin disease are warranted.
Procalcitonin‐reduced sensitivity and specificity in heavily leucopenic and immunosuppressed patients
Authors: Svaldi M. , Hirber J. , Lanthaler A.I. , Mayr O. , Faes S. , Peer E. , Mitterer M.
Published: March 2003

Procalcitonin (PCT) has proven to be a very sensitive marker of sepsis for non‐leucopenic patients. Little is known about its relevance in immunosuppressed and leucopenic adults. Four hundred and seventy‐five PCT determinations were carried out in 73 haematological patients: on 221 occasions the white blood cell (WBC) count was < 1·0 × 109/l and on 239 occasions it was > 1·0 × 109/l leucocytes. Patients were classified as: non‐systemic infected controls (n = 280), patients with bacteraemia (n = 32), sepsis (n = 30), severe sepsis (n = 3), septic shock (n = 3) and systemic inflammatory response syndrome (SIRS) (n = 62). When the WBC count was > 1·0 × 109/l, gram‐negative bacteria induced higher PCT levels (median 9·4 ng/ml) than gram‐positives (median 1·4 ng/ml). In cases with a WBC < 1·0 × 109/l, PCT levels were similar for gram‐negative and gram‐positive bacteria (1·1 ng/ml versus 0·85 ng/ml). Regardless of the leucocyte count, the median PCT level in bacteraemia cases always remained < 0·5 ng/ml. In heavily leucopenic situations, PCT levels were never > 2 ng/ml even in the sepsis and severe sepsis/septic shock groups, whereas a WBC count > 1·0 × 109/l resulted in median PCT values of 4·1 ng/ml and 45 ng/ml respectively. The positive predictive value for sepsis (cut‐off 2 ng/ml) was 93% in cases of WBC count > 1·0 × 109/l, but only 66% in leucopenic conditions. The negative predictive value (cut‐off 0·5 ng/ml) was 90% when the WBC count was > 1·0 × 109/l and 63% in leucopenic conditions. Procalcitonin is an excellent sepsis marker with a high positive‐ and negative‐predictive value in patients with WBC count > 1·0 × 109/l, but it does not work satisfactorily below this leucocyte count.
Prospective monitoring of the Epstein–Barr virus DNA by a real‐time quantitative polymerase chain reaction after allogenic stem cell transplantation
Authors: Hoshino Yo , Kimura Hiroshi , Tanaka Naoko , Tsuge Ikuya , Kudo Kazuko , Horibe Keizo , Kato Koji , Matsuyama Takaharu , Kikuta Atsushi , Kojima Seiji , Morishima Tsuneo
Published: March 2003

Epstein‐Barr virus (EBV)‐related lymphoproliferative disorder (LPD) is a serious complication of haematopoietic stem cell transplantation (HSCT). To clarify the frequency, natural course and risk factors for LPD, we prospectively monitored 38 allogeneic (allo)‐HSCT patients, focusing on the use of anti‐thymocyte globulin (ATG). We used a recently developed real‐time polymerase chain reaction assay to monitor EBV genome load. The subjects consisted of 19 patients given ATG for conditioning and 19 patients not given ATG. Of the 19 patients given ATG, 47·4% (nine patients) had a significant increase in EBV genome load (102·5copies/µg DNA). Of these nine patients, two developed LPD. Therefore, 10·5% of the patients receiving allo‐HSCT with ATG developed LPD. In contrast, none of the 19 patients without ATG had a significantly increased EBV load. The increases in viral load were observed in the second or third month after HSCT. We found that the peak viral loads of LPD patients were > 104·0copies/µg DNA. On the other hand, the viral loads of most patients with no symptoms were < 102·5copies/µg DNA. In conclusion, routine monitoring of EBV load during the second and third months after transplantation may benefit patients undergoing HSCT with ATG. We propose that an EBV load > 102·5copies/µg DNA is the reactivation of EBV, and that an EBV load > 104·0copies/µg DNA is indicative of developing LPD.
Involvement of Fcγ receptor polymorphism in the therapeutic response of idiopathic thrombocytopenic purpura
Authors: Fujimoto Tetsuro‐Takahiro , Inoue Maki , Shimomura Takeshi , Fujimura Kingo
Published: March 2003

Clearance of autoantibody‐sensitized platelets through Fcγ receptors on phagocytic cells is one of the main mechanisms of thrombocytopenia in idiopathic thrombocytopenic purpura (ITP). We examined the FcγRIIA‐131R/H and FcγRIIIA‐158V/F polymorphisms in 104 adult chronic ITP patients, and in 59 healthy control subjects using polymerase chain reaction‐based allele‐specific restriction analysis. The frequency of FcγRIIA genotypes (131H/H, H/R, R/R) was not significantly different between patients and controls, and did not correlate with the responsiveness to treatment. In contrast, among FcγRIIIA genotypes, frequency of 158F/F homotype was smaller in ITP (P < 0·05). Furthermore, in FcγRIIIA‐158V/V homotype, the complete remission (CR) rate with medication (treatment with corticosteroid or other immunosuppressive agents) was significantly higher (60%) than that in 158V/F (10%) or 158V/F plus 158F/F, (P < 0·01, P < 0·05). Conversely, the CR rate after splenectomy in 158F/F and 158V/F types (64·3% and 54·6%) was higher than in 158V/V (25%). Our results indicate that the polymorphism of FcγRIIIA, but not FcγRIIA, influences the response to treatment in ITP.
Rituximab, a chimaeric anti‐CD20 monoclonal antibody, in the treatment of hairy cell leukaemia
Authors: Hagberg H. , Lundholm L.
Published: March 2003

The introduction of first α‐interferon and later the purine analogues has revolutionized the treatment of hairy cell leukaemia (HCL). However, there are still some patients that initially or eventually fail to respond and, thus, there is a need for alternative treatment modalities. We have treated 11 HCL patients (eight relapsing and three newly diagnosed) with a chimaeric monoclonal antibody, rituximab, in a dose of 375 mg/m2 once a week for 4 weeks. The response rate was seven out of eleven (64%) with six complete remissions and one partial remission, all which have lasted between 0 and 34 months (median 14 months). Rituximab appears promising in the treatment of HCL and warrants further studies.
Prognostic value of positron emission tomography in the evaluation of post‐treatment residual mass in patients with Hodgkin's disease and non‐Hodgkin's lymphoma
Authors: Naumann Ralph , Vaic Astrid , Beuthien‐Baumann Bettina , Bredow Jan , Kropp Joachim , Kittner Thomas , Franke Wolf‐Gunter , Ehninger Gerhard
Published: March 2003

The prognostic value of 18F‐fluorodeoxyglucose (FDG) positron emission tomography (PET) in the assessment of post‐treatment residual masses in patients with Hodgkin's disease (HD) or non‐Hodgkin's lymphomas (NHL) was evaluated. We prospectively studied 58 patients with HD (n = 43) or NHL (n = 15) who had post‐therapeutic complete remission with residual masses (CRu) indicated by computerized tomography. Analysis of 62 residual locations by FDG‐PET was performed separately for HD and NHL. Patients with a PET‐positive residual mass [standardized uptake value (SUV) > 3] had a recurrence rate of 62·5% (5/8 patients), whereas patients with PET‐negative residual mass (SUV ≤ 3·0) showed a recurrence rate of 4% (2/50 patients, P = 0·004). A positive FDG‐PET study correlated with a significantly poorer progression‐free survival (P < 0·00001). No recurrence occurred in any of the 39 HD patients with a negative PET scan (negative predictive value, 100%). Four out of four NHL patients with a positive PET study relapsed (positive predictive value, 100%). In conclusion, FDG‐PET is a suitable non‐invasive method with a high degree of accuracy in the prediction of early recurrence in lymphoma patients with CRu.
Polymorphisms of P‐selectin glycoprotein ligand‐1 are associated with neutrophil–platelet adhesion and with ischaemic cerebrovascular disease
Authors: Lozano María L. , González‐Conejero Rocío , Corral Javier , Rivera José , Iniesta Juan A. , Martinez Constantino , Vicente Vicente
Published: March 2003

P‐selectin glycoprotein ligand (PSGL‐1) shares common features with platelet glycoprotein Ibα. A recently described polymorphism in this receptor that results in a variable number of tandem repeats (VNTR) sequence present either 16, 15 or 14 times (alleles A, B or C) could, similar to GPIbα, be functionally relevant. The allelic frequency of this polymorphism was investigated in 469 individuals from the south of Spain, and was similar to that previously described in other Caucasian populations: 85% A, 14% B and 1% C alleles. We identified two new polymorphisms genetically linked to the C isoform, resulting in the Ser273Phe and Met274Val changes. To assess the functional consequence of the polymorphisms in the receptor, we performed flow cytometric analysis of P‐selectin dependent neutrophil–platelet interaction. Neutrophils carrying the shortest C allele and the amino acid variations in residues 273 and 274 exhibited a significantly lower capacity to bind activated platelets than A/B and A/A samples (mean fluorescence intensity of CD42b+ neutrophils 262 versus 303 and 319 respectively, P < 0·05). The distribution of the VNTR was analysed in three case–control studies including 104 cerebrovascular (CVD), 101 coronary heart disease (CHD) and 150 deep venous thrombosis (DVT) patients. The results showed that smaller (B and C) alleles seem to be associated with a lower risk of developing CVD (P = 0·008) but not to be related to CHD or DVT. In conclusion, polymorphisms of the PSGL‐1 receptor may influence the neutrophil–platelet binding, and represent a risk factor for CVD.