bluuebunny/crossref_metadata_2025 huggingface dataset:
The DNA‐binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells
Authors: Bianchi Nicoletta , Osti Fabio , Rutigliano Cristina , Corradini Federica Ginanni , Borsetti Elena , Tomassetti Marina , Mischiati Carlo , Feriotto Giordana , Gambari Roberto
Published: March 2003

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5‐azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo‐fetal globin genes, such as the ζ, ε and γ globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate γ‐globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult β‐globin genes. In this paper we demonstrated that the G + C selective DNA‐binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) γ‐globin mRNA.
Pharmacokinetics and red cell utilization of iron(III) hydroxide–sucrose complex in anaemic patients: a study using positron emission tomography
Authors: Beshara Soheir , Lundqvist Hans , Sundin Johanna , Lubberink Mark , Tolmachev Vladimir , Valind Sven , Antoni Gunnar , Långström Bengt , Danielson Bo G.
Published: March 2003

The pharmacokinetics of a single intravenous injection of 100 mg iron hydroxide–sucrose complex labelled with a tracer in the form of 52Fe/59Fe was followed in six anaemic patients for a period ranging from 6 to 8.3 h using positron emission tomography (PET). Red cell utilization of the labelled iron was followed for 4 weeks. PET data showed radioactive uptake by the liver, spleen and bone marrow. The uptake by the macrophage‐rich spleen demonstrated the reticuloendothelial uptake of this iron preparation, with subsequent effective release of that iron for marrow utilization. Red cell utilization, followed for 4 weeks, ranged from 59% to 97%. The bone marrow influx rate constant was independent of blood iron concentration, indicating non‐saturation of the transport system in bone marrow. This implied that higher doses of the iron complex can probably be used in the same setting. A higher influx rate into the marrow compared with the liver seemed to be consistent with higher red cell utilization. This would indicate that early distribution of the injected iron complex may predict the long‐term utilization.
Multiple myeloma related cells in patients undergoing autologous peripheral blood stem cell transplantation
Authors: Guikema Jeroen E. J. , Vellenga Edo , Veeneman Jorden M. , Hovenga Sjoerd , Bakkus Marleen H. C. , Klip Harry , Bos Nicolaas A.
Published: March 2003

A high incidence of oligoclonal serum M‐components is observed in multiple myeloma (MM) patients treated with autologous stem cell transplantation (ASCT). To determine whether these M‐components are produced by myeloma clonally related cells or caused by an aberrant B‐cell regeneration we analysed by semi‐nested ASO‐RT‐PCR and DNA sequencing the immunoglobulin (Ig) variable genes (VH) obtained from bone marrow samples obtained before and after transplantation and peripheral blood stem cell (PBSC) samples from seven patients.

Myeloma clonally related cells are identifiable by the expression of variant Ig heavy chain isotypes and were detected in two patients at presentation. No myeloma clonally related cells were found in post‐transplantation samples (n = 7) in spite of the appearance of new serum M‐components. However, in two cases we amplified sequences from post‐transplantation bone marrow cells that were able to bind to the B‐cell clone‐specific CDR3 oligonucleotides but showed no further similarity regarding the VDJ rearrangement.

These data indicate that serum oligoclonality post‐transplantation is not caused by myeloma clonally related B cells but rather by the regenerating B‐cell compartment.
Bone marrow CD34+ cells generate fewer T cells in vitro with increasing age and following chemotherapy
Authors: Offner F. , Kerre T. , De Smedt M. , Plum J.
Published: March 2003

To assess the influence of high‐dose chemotherapy and age on the intrinsic capacity of stem cells to generate T cells, CD34+ cells derived from bone marrow used in clinical transplantation were evaluated in an in vitro T‐cell assay using a mouse thymic microenvironment. CD34+ cells were sorted from healthy donor and autologous back‐up bone marrow after density gradient centrifugation and depletion for CD1, 3, 4, 7, 8, 19 and glycophorin A using magnetic beads. CD34+ cells were then introduced in day 14–15 fetal SCID mouse thymus lobes by incubation in hanging drops for 48 h. After transfer to gelfoam rafts they were cultured for variable time periods. The lobes were then homogenized in a tissue grinder for flow cytometric analysis gating on human cells. These were evaluated for CD4, CD8, CD3 and HLA‐DR surface expression. 51 samples were analysed and three patterns of T‐cell precursor development could be observed. In pattern A no human cells could be recovered, in pattern B maturation stopped at the CD4+CD8−CD3− pre‐T‐cell stage, and in pattern C maturation to double‐positive CD4+CD8+ thymocytes was reached. In 25 healthy donors under age 40 three showed pattern A, 12 pattern B and 10 pattern C, whereas in 16 healthy donors over the age 40 there were respectively four with A, seven with B and only five with C (P=0.01). In 10 patients who had previously received chemotherapy, none developed pattern C, five pattern B and five pattern A, in contrast to 15/41 pattern C, 19/41 pattern B and 7/41 pattern A in healthy donors. These data suggest an intrinsic loss of T‐cell generation capacity from adult bone marrow stem cells in comparison to reports on stem cells of fetal origin. This loss correlated weakly with age, irrespective of thymic involution, and may be further reduced by prior chemotherapy.
Relationship between size and thiazole orange fluorescence of platelets in patients undergoing high‐dose chemotherapy
Authors: Balduini  , Noris  , Spedini  , Belletti  , Zambelli  , Da Prada
Published: March 2003

The reticulated platelet count relies upon the assumption that newly formed platelets contain a residual amount of RNA which selectively binds the dye thiazole orange (TO) and greatly enhances its fluorescence signal. It has, however, recently been shown that almost half of the platelet TO‐signal is derived from the labelling of dense‐granule nucleotides. It is therefore possible that the higher TO fluorescence of young platelets partially derives from the higher granule content due to their larger volume. To investigate the relationship between platelet size and TO fluorescence we studied 13 patients with high‐risk breast cancer undergoing high‐dose chemotherapy. Mean platelet volume, platelet distribution width, platelet–large cell ratio, membrane content of glycoprotein Ib and IIb–IIIa and platelet aggregation were significantly greater during resolution than during development of thrombocytopenia, suggesting a prevalence of young and old platelets respectively. Mean TO fluorescence per cell was higher in the platelet population enriched in young cells than in that enriched in old cells, but this difference was no longer observed when the ratio TO signal/platelet size was examined. Moreover, RNase treatment and platelet degranulation reduced TO fluorescence to a similar extent in platelet populations enriched in young or old cells. Therefore our data suggest that the higher TO signal of young platelets is derived, to a significant extent, from their larger volume and granule content.
Contribution of the glycoprotein Ia 807TT, methylene tetrahydrofolate reductase 677TT and prothrombin 20210GA genotypes to prothrombotic risk among factor V 1691GA (Leiden) carriers
Authors: Hessner Martin J. , Dinauer David M. , Luhm Robert A. , Endres Janet L. , Montgomery Robert R. , Friedman Kenneth D.
Published: March 2003

The cooperative effects of the GPIa 807TT, MTHFR 677TT and prothrombin 20210GA genotypes with the FV Leiden 1691GA (FVL) genotype were evaluated by comparing these genotype frequencies in 77 asymptomatic and 156 symptomatic heterozygous FVL carriers. The GPIa 807TT and MTHFR 677TT genotypes did not segregate within the symptomatic FVL carrier group and did not contribute to venous thrombotic risk in this patient cohort. There was no difference in the prothrombin 20210GA genotype frequency between asymptomatic FVL carriers and a random Caucasian control group; however, the prothrombin 20210GA genotype was nearly 5 times as prevalent (19/156 v 2/77; P < 0.02) in the symptomatic FVL carriers (odds ratio 5.21; 95% confidence interval 1.20–47.62), demonstrating that this important prothrombotic risk factor acts synergistically with FVL.
Coil embolization of cerebral aneurysms in patients with sickling disorders
Authors: McQuaker I. G. , Jaspan T. , McConachie N. S. , Dolan G.
Published: March 2003

In patients with sickle cell disease cerebral aneurysm formation is thought to be a complication of recurrent red cell sickling, and multiple aneurysms have been reported in these patients. Management of patients with suspected cerebral aneurysm has traditionally involved cerebral vessel angiography followed by craniotomy and aneurysmal clipping. In patients without sickle cell disease, non‐operative intervention in the form of endovascular coil embolization is increasingly being used to ablate aneurysms, but has not thus far been reported in patients with sickle cell disease. We report two patients with sickling disorders who have undergone coil embolization of cerebral aneurysms with good functional and radiological outcomes. These patients illustrate that endovascular coiling is useful in the treatment of cerebral aneurysms associated with sickling disorders, although, as with surgical intervention, preparation with exchange transfusion is appropriate.
Thrombomodulin with the Asp468Tyr mutation is expressed on the cell surface with normal cofactor activity for protein C activation
Authors: Nakazawa Fumie , Koyama Takatoshi , Saito Takako , Shibakura Misako , Yoshinaga Haruhiko , Chung Dong Hui , Kamiyama Ryuichi , Hirosawa Shinsaku
Published: March 2003

Thrombomodulin (TM) is an endothelial cell glycoprotein that acts as an anticoagulant. Mutation in the TM gene is a potential risk factor for thrombosis. The first TM mutation identified was a heterozygous substitution of T for G at nucleotide position 1456, which predicted Asp468 with Tyr in a Ser/Thr‐rich domain. To evaluate the reported TM gene mutation as a possible cause of thrombosis, we transiently tranfected a vector for TM gene carrying the mutation to mammalian COS7 cells.

TM antigen levels in lysates of cells transfected with variant TM were comparable to those in preparations of normal TM. The TM cofactor activity for protein C (PC) activation on the variant TM‐expressing cells was similar to that of the control. The Michaelis constant Km and Vmax. of variant TM for PC activation were shown to be similar compared to those of normal TM. The affinity of each TM for thrombin in PC activation was also similar. We obtained several stable cell lines expressing normal and variant TM. Lysate of the cell lines with normal and variant TM genes had a similar expression level of TM antigen. Pulse‐chase analysis showed that normal and variant TM were glycosylated and resistant to endoglycosidase H, indicating that the variant TM was expressed on the cell surface in a mature form.

Variant TM protein is apparently expressed on the cell surface with normal cofactor activity for PC activation. It is unlikely that the TM variant directly causes thrombosis by mechanism of reduced expression or impaired cofactor activity for PC activation, which comprises a major anticoagulant activity of TM.
The GPIa C807T dimorphism associated with platelet collagen receptor density is not a risk factor for myocardial infarction
Authors: Croft S. A. , Hampton K. K. , Sorrell J. A. , Steeds R. P. , Channer K. S. , Samani N. J. , Daly M. E.
Published: March 2003

The platelet collagen receptor, GPIa/IIa, is an important mediator of platelet adhesion to fibrillar collagens at sites of vascular injury. Recently, a dimorphism at nucleotide 807 of the GPIa cDNA (TTC/TTT in codon 224) was shown to be associated with variation in GPIa/IIa receptor density on the platelet surface. We conducted a case–control study to determine if the 807T allele, linked with increased GPIa/IIa density, contributed to risk of myocardial infarction (MI). DNA from 546 acute MI cases and 507 controls, all aged <75 years, was genotyped for the C807T dimorphism using the TaqManTM system of allelic discrimination. The allelic odds ratio (OR) for MI in the complete cohort was 0.88 (95% CI 0.74–1.05, P = 0.17), indicating that the 807T allele was not associated with an increased risk of MI. There was also no increased risk of MI associated with the homozygous 807TT (P = 0.22) or heterozygous 807CT (P = 0.24) genotypes or for carriers of the 807T allele in any cohort subgroup analysed. We conclude that the GPIa 807T allele is not a risk factor for MI in our population either alone or in combination with other major cardiovascular risk factors.
Resolution of immune haemolytic anaemia with allogeneic bone marrow transplantation after an unsuccessful autograft
Authors: De Stefano Piero , Zecca Marco , Giorgiani Giovanna , Perotti Cesare , Giraldi Eugenia , Locatelli Franco
Published: March 2003

Autologous transplantation of lymphocyte‐depleted peripheral blood stem cells (PBSC) has been proposed for treatment of patients with severe autoimmune disease. However, several patients have been reported to achieve only transient remissions. We report on a child with thalassaemia intermedia and immune‐mediated haemolytic anaemia, given an autologous lymphocyte‐depleted PBSC transplant, who relapsed 7 weeks after transplant. A complete remission, lasting 18 months to date, was obtained with allogeneic bone marrow transplantation (BMT) from an HLA‐matched unrelated donor. This experience indicates that, in selected cases, allogeneic BMT may be the treatment of choice for life‐threatening autoimmune disease. A graft‐versus‐autoimmunity effect may favour the eradication of the recipient autoaggressive lymphocytes.
Platelet c‐mpl expression is dysregulated in patients with essential thrombocythaemia but this is not of diagnostic value
Authors: Harrison Claire N. , Gale Rosemary E. , Pezella Francesco , Mire‐Sluis Anthony , Machin Samuel J. , Linch David C.
Published: March 2003

Essential thrombocythaemia (ET) can be difficult to discriminate from an occult case of reactive thrombocytosis (RT). Since thrombopoietin (TPO) is the primary regulator of thrombopoiesis, we have investigated whether levels of TPO and/or its receptor, c‐mpl, are of value in the differential diagnosis of ET. Plasma TPO levels in patients with ET, RT and other myeloproliferative disorders (MPDs) did not differ significantly from normal controls. However, surface c‐mpl expression was significantly reduced in platelets from 18 ET patients, 0–65.5% of controls (P < 0.001). Immunoblots on five of these and five additional patients were consistent with absent or reduced c‐mpl protein levels. The surface c‐mpl expression results were significantly different from those in eight RT patients (21.3–95.5%, P = 0.0015), but there was considerable overlap between the two groups and a reduced level was not restricted to ET. Furthermore, c‐mpl expression in ET patients was not different from eight patients with other MPDs (0–87.6%, P = 0.06), nor could it differentiate between ET patients with monoclonal and polyclonal haemopoiesis. Although a low or absent c‐mpl level is suggestive of a primary rather than a secondary thrombocytosis, it is insufficiently discriminatory to be used as a diagnostic marker for ET.
Long‐term decrease of CD4+CD45RA+ T cells and impaired primary immune response after post‐traumatic splenectomy
Authors: Wolf Hermann M. , Eibl Martha M. , Georgi Erich , Samstag Aysen , Spatz Martin , Uranüs Selman , Passl Rainer
Published: March 2003

Congenital or acquired absence of the spleen and functional hyposplenism are associated with abnormalities of host defence such as an increased susceptibility to infection with encapsulated bacteria. The effects of the lack of the spleen on cell‐mediated immunity are largely unknown. In the present study we have investigated peripheral blood lymphocyte subpopulations in healthy adults who had undergone splenectomy because of severe abdominal trauma > 4 years before the study. The results show a significant reduction in the percentage of CD4+ T cells due to a selective and long‐term decrease in the percentage of CD4+CD45RA+ lymphocytes, the CD4+ T‐cell subset mainly involved in primary immune responses to newly encountered antigens. Levels of the reciprocal CD45RO+CD4+ T‐cell subset were comparable between splenectomized and control individuals, as were lymphoproliferative responses and IFN‐gamma production to recall antigens. Decreased levels of CD4+CD45RA+ cells were accompanied by an impairment in primary immune responsiveness, as assessed by investigating T‐cell proliferation to stimulation with keyhole limpet haemocyanin and by measuring antibody responses following primary immunization with a clinically relevant T‐dependent antigen, hepatitis A vaccine, in vivo. These findings suggest a possible role of the spleen in the generation, maintenance and/or differentiation of naive, unprimed T cells or their precursors, which might have a possible functional relevance for primary immune responses following splenectomy.
Complete remission rate and outcome after intensive treatment of 177 patients under 75 years of age with IgG myeloma defining a circumscribed disease entity with a new staging system
Authors: Sirohi Bhawna , Powles Ray , Mehta Jayesh , Raje Noopur , Kulkarni Samar , Ramiah Veshana , Saso Radovan , Horton Clive , Bhagwati Niyati , Singhal Seema , Treleaven Jennifer
Published: March 2003

Because the presence of IgG paraprotein in the blood is clear cut, it makes IgG myeloma a more circumscribed disease than myeloma as a whole in which to study treatment efficacy, particularly relating to complete remission (CR).

Between May 1989 and December 1997, 177 consecutive patients with IgG myeloma who were <75 years old were seen, of whom 153 entered a sequential therapy (ST) programme of initial courses of C‐VAMP infusional chemotherapy (IC), high‐dose treatment (with or without stem cell rescue) (119 patients) and maintenance interferon (87 patients).

74/153 (48.4%) patients entered CR. Median overall survival (OS) and event‐free survival (EFS) were 4.9 and 2.1 years, respectively. Multivariate analysis at presentation showed OS was significantly prolonged for β2M <2.7 mg/l and age <median 52, whilst β2M <2.7 mg/l and Hb >8.5 g/dl predicted for longer EFS. For CR patients, age <median 51 years, absence of Bence‐Jones proteinuria (BJP), male sex and white blood cells (WBC) <7 × 109/l predicted for a longer OS. Longer length of first CR was predicted by absence of BJP at presentation (P = 0.03) and fewer than five courses of IC (P = 0.02) to attain CR.

We have therefore been able to refine the use of ST in IgG myeloma, redefine CR as a ‘macro’ endpoint, and propose a new staging system for IgG myelomas. Analysis of 41 comparable IgA patients showed IgG to be a distinct entity.
Neutrophil hypersegmentation in iron deficiency anaemia: a case–control study
Authors: Westerman  , Evans  , Metz
Published: March 2003

Neutrophil hypersegmentation (NH) is an important haematological feature of cobalamin or folate deficiency. As iron deficiency and folate deficiency often occur in the same target groups it is important to establish whether iron deficiency alone is a cause of NH. We report a case–control study which addresses this issue. Two groups of hospital patients were studied. Group 1 comprised 50 patients with iron deficiency anaemia (IDA). Group II comprised 50 control age‐ and sex‐matched patients who were haematologically normal without evidence of iron deficiency from the iron studies. Patients with other factors which could affect the degree of neutrophil segmentation (cobalamin/folate deficiency, renal failure, infection and drug exposures) were excluded from the study. A total of 10 000 neutrophils were examined, 100 from each patient. NH was defined as the presence of five or more five‐lobed neutrophils per 100, or any neutrophils with six or more lobes. The results were as follows: IDA, mean neutrophil lobe count 3.36; number of patients with NH 31/50 (62%): controls, mean neutrophil lobe count 2.96, number of patients with NH 2/50 (4%). These differences were statistically significant. We conclude that NH is common in IDA. The mechanism whereby iron deficiency results in NH is not clear.
The immunosuppression and potential for EBV reactivation of fludarabine combined with cyclophosphamide and dexamethasone in patients with lymphoproliferative disorders
Authors: Lazzarino Mario , Orlandi Ester , Baldanti Fausto , Furione Milena , Pagnucco Guido , Astori Cesare , Arcaini Luca , Viglio Alessandra , Paulli Marco , Gerna Giuseppe , Bernasconi Carlo
Published: March 2003

Fludarabine is effective in chronic lymphocytic leukaemia (CLL) and low‐grade non‐Hodgkin's lymphoma (NHL). A major side‐effect of this purine analogue is immunosuppression which may favour opportunistic infections. Additionally, impairment of immunosurveillance might promote Epstein‐Barr virus (EBV) reactivation and possibly favour transformation to high‐grade malignancy. The aim of this study was to evaluate the immunosuppression‐related effects of the fludarabine‐based combination Flucyd in advanced low‐grade NHL or CLL by serially monitoring T‐lymphocyte subsets, opportunistic infections, EBV‐reactivation, and histologic transformation. 24 patients with advanced NHL (n = 21) or CLL (n = 3) received fludarabine 25 mg/m2/d + cyclophosphamide 350 mg/m2/d + dexamethasone 20 mg/d in 3 d courses for a maximum of six courses. The overall response rate was 79% (eight CR, 11 PR, five failures); 11 patients relapsed or progressed between 3 and 19 months from response, and eight are in CR or PR at 3–27 months. The CD4+ lymphocyte counts decreased significantly during therapy from a median of 484/μl pre‐treatment (range 142–1865) to a median of 198/μl (71–367). In 19 responders monitored off therapy every 3 months until relapse/progression, CD4+ counts were persistently low with minimal recovery over time. During treatment, 16 infections occurred in 11/24 patients. No delayed opportunistic infections occurred in responders while off therapy. The circulating EBV DNA load serially measured in 19 patients by a quantitative PCR assay showed an increase in four patients during treatment. A lymph node biopsy performed in two of these was PCR positive for EBV DNA, whereas LMP1 and EBERs were negative. Six NHL patients evolved into high‐grade B‐cell NHL. In conclusion, fludarabine combined with cyclophosphamide and dexamethasone is an effective therapy for recurrent indolent lymphoma. This combination produces prolonged T‐lymphocytopenia and has the potential to reactivate a latent EBV infection. T‐cell dysfunction, however, is not associated with higher incidence of clinical opportunistic infections and does not adversely influence clinical outcome.
Age of onset evidence for anticipation in familial non‐Hodgkin's lymphoma
Authors: Wiernik Peter H. , Wang Steven Q. , Hu Xiao‐Ping , Marino Patricia , Paietta Elisabeth
Published: March 2003

Anticipation, a phenomenon in which an inherited disease is diagnosed at an earlier age in each successive generation of a family, has been demonstrated in certain neurological and haematological disorders. This study was conducted to determine whether anticipation occurs in familial non‐Hodgkin's lymphoma (NHL). Eleven published reports of multigenerational familial NHL were analysed for evidence of anticipation, together with 18 previously unreported families with familial NHL. Differences in disease‐free survival between generations were determined. The difference between age at onset for each affected parent–child pair was tested against the null hypothesis that there was no difference in age at onset. These analyses were also performed separately using only parent–child pairs with age of onset > 25 years to avoid ascertainment bias. In addition, the age at onset distribution of the studied cases was compared with that of the Surveillance Epidemiology and End Results (SEER) Program using data for 1973–98. The median ages at onset in the child and parent generations of all families (48.5 and 71.3 years respectively) and in the selected pairs (52.5 and 71.5 years respectively) were significantly different (P < 0.000002 and P < 0.000001 respectively). The null hypothesis was rejected for all (P < 0.000001) as well as selected pairs (P < 0.000003). A significant difference was observed between the ages of onset of the child generation and the SEER population (P < 0.009), but not between the parent generation and the SEER population. Anticipation occurs in familial NHL, which suggests a genetic basis for it.
Local and systemic effects after adenoviral transfer of the murine granulocyte–macrophage colony‐stimulating factor gene into mice
Authors: Burger Jan A. , Baird Stephen M. , Powell Henry C. , Sharma Sanjai , Eling David J. , Kipps Thomas J.
Published: March 2003

Vectors encoding immunostimulatory genes are under investigation for their use as adjuvants for immunotherapy. Granulocyte–macrophage colony‐stimulating factor (GM‐CSF) is a prominent candidate gene for this approach because this cytokine can prime immune responses to ‘self’ tumour or other weak antigens. Prior studies suggested that GM‐CSF induces accumulation and differentiation of antigen‐presenting cells, particularly dendritic cells that can initiate immunity. To evaluate this model in vivo, we performed i.m. and i.p. injections of an adenovirus vector encoding murine GM‐CSF (Ad‐mGM‐CSF) and evaluated local and systemic effects. After intramuscular injection, local changes were characterized by the accumulation of myeloid cells, a subsequent infiltration of lymphocytes and then myonecrosis. Intraperitoneal injection also induced an accumulation of myeloid cells, an increase in CD3‐positive T and a decrease in B220‐positive B lymphocytes. Expression of the dendritic cell marker CD11c on 48 ± 9% of the peritoneal cells (n = 6) along with high levels of surface MHC class II, a characteristic morphology, and endocytosis of FITC‐dextran suggested in vivo differentiation of dendritic cells after i.p. injection of Ad‐mGM‐CSF. Systemic effects were observed after i.m. and i.p. injection of Ad‐mGM‐CSF. All mice developed hepatosplenomegaly resulting from extramedullary haematopoiesis. These changes were specific to GM‐CSF as they were not seen in mice injected with an adenovirus vector without a transgene. Our observations indicate that adenoviral transfer of GM‐CSF is a powerful tool for inducing local and systemic expansion of haematopoietic cells. The local expansion of myeloid cells displaying signs of dendritic cell differentiation, as characterized for the peritoneal cell compartment, can explain the potency of GM‐CSF when used as an adjuvant in genetic immunotherapy.
Combined alpha interferon and ribavirin for the treatment of hepatitis C in patients with hereditary bleeding disorders
Authors: Shields  , Mutimer  , Muir  , Skidmore  , Britnell  , Roberts  , Wilde
Published: March 2003

Patients with hereditary bleeding disorders who received non‐virally inactivated plasma‐derived clotting factor concentrates before the mid‐1980s invariably became infected with hepatitis C virus (HCV). Therapy with interferon alpha (IFN‐α) alone has been disappointing in this group. We conducted an open‐label study, using a combination of IFN‐α2b (3 million units three times per week) and ribavirin 1–1.2 g/d in 28 patients with hereditary bleeding disorders. Twenty‐one of the 28 patients had liver biopsy‐confirmed chronic hepatitis (median histological activity index 5; range 1–10) and all patients were HCV RNA positive by PCR. Virological response rate to therapy at 3 months was 82% (23 out of 28). Three HIV co‐infected patients showed an early virological response with loss of HCV RNA, but two subsequently relapsed after 3 and 6 months of therapy. Four patients stopped treatment early (one at 4, one at 7 and two at 9 months) because of treatment‐related side effects, although three of these have maintained a virological response. Seventeen patients completed the 48‐week course. Twenty of the 28 (71%) treated have had a durable virological response with a median follow‐up of 16 months (range 1–24). Combination therapy represents a significant advance in the treatment of hepatitis C in patients with hereditary bleeding disorders.
Differential constitutive and activation‐dependent expression of prion protein in human peripheral blood leucocytes
Authors: Dürig Jan , Giese Armin , Schulz‐Schaeffer Walter , Rosenthal Christoph , Schmücker Ute , Bieschke Jan , Dührsen Ulrich , Kretzschmar Hans A.
Published: March 2003

The cellular isoform of the prion protein (PrPC) is a cell surface glycoprotein that has recently been shown to play a role in haemopoietic cell activation and proliferation. We have characterized the constitutive expression of PrPC on human peripheral blood (pB) cell populations, using PrP‐specific antibodies in a multiparameter flow cytometry approach. We found that T cells, NK cells and monocytes exhibit similar PrPC levels, whereas PrPC surface staining on B cells was significantly lower and was virtually absent on granulocytes. Within the T‐cell compartment, CD8+ cells showed a significantly higher PrPC expression than CD4+ cells. Similarly, CD3+ cells co‐expressing the activation marker CD56 (N‐CAM) exhibited significantly higher PrPC expression levels than their CD56− counterparts. Culture of CD14+ pB monocytes for 12–48 h in the presence of interferon γ (IFN‐γ) resulted in a significant increase in PrPC expression in a time‐ and concentration‐dependent manner. This effect was partially abrogated by the addition of the metabolic inhibitor cycloheximide, indicating the role of protein synthesis in this process. Our results show that PrPC expression on human haemopoietic cells correlates with the activation and developmental status of these cells, suggesting an important functional role of PrPC in the haemopoietic system.
Constitutive levels of cAMP‐dependent protein kinase activity determine sensitivity of human multidrug‐resistant leukaemic cell lines to growth inhibition and apoptosis by forskolin and tumour necrosis factor alpha
Authors: Yin Yuzhi , Allen Paul D. , Jia Li , Macey Marion G. , Kelsey Stephen M. , Newland Adrian C.
Published: March 2003

The cyclic adenosine monophosphate (cAMP)‐dependent protein kinase (PKA) signal pathway regulates cell proliferation, differentiation and cell death. It may also regulate the multidrug resistance (MDR) phenotype in leukaemic cells. These data showed that MDR1+ K/Dau600 cells exhibited a higher basal level of PKA activity than MDR− parental cells. The significance of this on tumour necrosis factor α (TNFα)‐induced apoptosis and cytostasis was investigated further. In comparison with MDR1− parental cells, K/Dau600 cells had a higher expression of PKA regulatory subunit RIα and nuclear catalytic subunit PKAcα. They were also more susceptible to inhibition of proliferation and induction of apoptosis by TNFα and/or forskolin, but this could be attenuated by H89. An increase in cAMP was associated with the apoptosis in the K/Dau600 cell line. Forskolin inactivated NF‐κB in K/Dau600 cells but not in K562 cl.6 cells, whereas TNF activated NF‐κB in K562 cl.6 cells but not in K/Dau600 cells. 8‐Cl‐cAMP exhibited similar inhibitory effects on the proliferation of all of the cell lines used via its metabolite 8‐Cl‐adenosine, which indicates that these effects were independent of residual PKA or cAMP. Therefore, the differential sensitivity to apoptosis and/or growth inhibition could be mediated via cAMP, partly through PKA via NF‐κB and partly by PKA‐independent pathways.
Enrichment of lineage−CD34− cells using a newly developed filter system
Authors: Nakamura Y. , Ando K. , Muguruma Y. , Sumita M. , Terashima S. , Yoshida M. , Yamashita Y. , Hotta T. , Kato S.
Published: March 2003

Recent studies have demonstrated the presence of human lineage− (lin−)CD34− cells that are capable of differentiating into CD34+ cells in xenogeneic transplantation systems. We developed a new filter system that can enrich lin−CD34− cells, a precursor cell population of CD34+ cells. The filter consists of polyethylene terephthalate non‐woven fabrics coated with hydrophilic polymer. The frequency of lin−CD34− cells in the cell population after filtration was 7.45 ± 4.41%, a 16.8 ± 8.81‐fold enrichment compared with 0.49 ± 0.31% in the cell population before filtration. The mean recovery of lin−CD34− cells was 48.57 ± 13.59% (n = 15). Purified lin−CD34− cells, obtained by sorting the filtrated cell population, acquired CD34 expression and colony‐forming activity after 7 d of culture. Our results indicate that this filter system is useful for isolating lin−CD34− cells, including precursors of CD34+ cells, and will help further the study of lin−CD34− cells.
Vaccination of multiple myeloma patients with idiotype‐pulsed dendritic cells: immunological and clinical aspects
Authors: Titzer Sandra , Christensen Olaf , Manzke Oliver , Tesch Hans , Wolf Jürgen , Emmerich Berthold , Carsten Cornelia , Diehl Volker , Bohlen Heribert
Published: March 2003

Multiple myeloma (MM) is characterized by a clonal proliferation of malignant plasma cells in the bone marrow secreting a monoclonal immunoglobulin (paraprotein) with specific antigenic determinants, the idiotype (Id), which can be regarded as a tumour‐associated antigen (TAA). In order to analyse the impact of a dendritic cell (DC)‐based vaccine, 11 patients with advanced MM were treated with CD34 stem cell‐derived dendritic cells that were pulsed with Id peptides. Subsequently, the patients received three boost immunizations every other week with a combination of Id and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (nine patients) or with Id peptide‐pulsed dendritic cells again (two patients). The treatment was well tolerated with no side‐effects. The present clinical study was a proof of concept analysis of dendritic cell‐based vaccines in MM. The capacity of the dendritic cells to activate idiotype‐specific T cells was verified by in vitro stimulation experiments before the vaccination therapy. Immunological effects of the Id vaccination were analysed by monitoring changes in anti‐idiotype antibody titres and idiotype‐specific T‐cell activity. After vaccination, three out of 10 analysed patients showed increased anti‐idiotype antibody serum titres, indicating the induction of an idiotype‐specific humoral immune response. The idiotype‐specific T‐cell response analysed by ELISpot was increased in four out of 10 analysed patients after vaccination, and one patient had a decreased plasma cell infiltration in the bone marrow. In conclusion, five out of 11 patients showed a biological response after vaccination. Thus, our data indicate that immunotherapy with Id‐pulsed DCs in MM patients is feasible and safe. DC generated from CD34+ progenitor cells can serve as a natural adjuvant for the induction of clinically relevant humoral and cellular idiotype‐specific immune responses in patients suffering from advanced MM.
γ‐Ray irradiation induces B7.1 expression in myeloid leukaemic cells
Authors: Vereecque Rodolphe , Buffenoir Geraldine , Gonzalez Ricardo , Cambier Nathalie , Hetuin Dominique , Bauters Francis , Fenaux Pierre , Quesnel Bruno
Published: March 2003

Expression of B7 molecules provides co‐stimulatory signals to T lymphocytes, which prevent the induction of anergy. It has been previously reported that B7.1 gene transfer in a murine leukaemia model induced a potent antileukaemic immunity and that relative expression of B7.1 and B7.2 in human acute myeloid leukaemia (AML) had prognostic significance. As ex vivo engineering of leukaemic cells for immunotherapy protocols would require prior irradiation of these cells before reinjection to the patient, we investigated in murine and leukaemic cell lines and in 20 ex vivo primary cultured acute myeloid leukaemic cells the effect of γ‐irradiation on the expression of B7 molecules. We observed that γ‐irradiation enhanced B7.1 molecule expression in murine leukaemic cell lines and in B7.2 molecules in human HL60 and K562 cell lines. γ‐Irradiation induced B7.1 molecule expression in 90% AML samples but only 21% showed B7.2 molecule expression enhancement. B7.1 expression was increased both at the protein and RNA level in human AML cells but only at the protein level in the DA1‐3b murine cell line. Oxidative stress increased B7.1 expression in the murine DA1‐3b cell line but human cell lines and AML samples remained unaffected both by heat shock and oxidative stress, suggesting different pathways of B7.1 induction between mouse and human cells. Our data show that B7.1 expression can be induced by ex vivo irradiation of AML cells, indicating that these cells can express co‐stimulatory molecules without gene transfer.
Lack of BLV and PTLV DNA sequences in the majority of patients with large granular lymphocyte leukaemia
Authors: Perzova Raisa N. , Loughran Thomas P. , Dube Syamalima , Ferrer Jorge , Esteban Eduardo , Poiesz Bernard J.
Published: March 2003

The primate T‐cell lymphoma/leukaemia viruses (PTLV) and bovine leukaemia virus (BLV) comprise a unique genus of retroviruses, infection with which induces seroreactivity in the host against conserved epitopes in their p24 gag and gp21 env cognate proteins. Herein, we have confirmed this serocrossreactivity. Patients with large granular lymphocyte (LGL) leukaemia have frequent seroreactivity to the p24 and gp21 env proteins of human T‐cell lymphoma/leukaemia virus I (HTLV‐I), one of the species in the genus. However, only a small minority of patients are actually infected with prototypic HTLV‐I or HTLV‐II, another species within the group. In an attempt to determine whether LGL leukaemia might be associated with other members of the PTLV/BLV genus, we examined the peripheral blood mononuclear cell DNA of 22 HTLV p24 and/or gp21 seropositive LGL leukaemia patients via PCR using degenerate and specific primer pair/probe systems capable of detecting all known members of the PTLV/BLV genus. None of the samples was positive. These data indicate that although HTLV‐II may be associated with some cases of LGL leukaemia most patients are not infected with a PTLV or BLV virus.
Interferon α2b directly induces fibroblast proliferation and transforming growth factor β secretion of macrophages
Authors: Wickenhauser Claudia , Schmitz Beate , Selbach Barbara , Brockbals Carina , Manske Oliver , Thiele Jürgen
Published: March 2003

To elucidate the effects of interferon α2b (IFN‐α) on normal human bone marrow, fibroblasts from patients without haematopoietic pathology were cultivated and used in stimulation experiments. Further, co‐cultures with highly enriched fractions of megakaryocytes and bone marrow macrophages were analysed. In this context, the influence of cell‐to‐cell interactions and humoral factors was determined in transwell and neutralization studies. Finally, secretion of platelet‐derived growth factor (PDGF) and transforming growth factor β1 (TGF‐β1) by single megakaryocytes and macrophages was examined by using the reverse haemolytic plaque assay (RHPA). Following these experimental designs, a direct proliferative activity of IFN‐α on bone marrow fibroblasts could be demonstrated. In the unstimulated co‐cultures, the megakaryocyte‐ but not the macrophage‐enriched fraction induced fibroblast growth and [3H]‐thymidine uptake. This effect was dependent on cell‐to‐cell contact and also on the influence of TGF‐β and PDGF. In the megakaryocyte‐enriched co‐cultures, the fibroblast proliferation was not altered by IFN‐α, but in the macrophage fibroblast cultures addition of IFN‐α enhanced fibroblast growth and [3H]‐thymidine uptake was distinctively higher than in the monocultures. This effect was not obvious in the transwell or neutralization experiments. Finally, IFN‐α treatment exerted a significantly elevated TGF‐β1 secretion in single macrophages. Our findings are in keeping with the assumption that several pathomechanisms participate in IFN‐α‐induced myelofibrosis, including direct and indirect effects.
Detection of prognostic significant translocations in childhood acute lymphoblastic leukaemia by one‐step multiplex reverse transcription polymerase chain reaction
Authors: Cerveira Nuno , Ferreira Susana , Dória Sofia , Veiga Isabel , Ferreira Fátima , Mariz José Mário , Marques Margarida , Castedo Sérgio
Published: March 2003

The search for chromosomal translocations in de novo cases of childhood acute lymphoblastic leukaemia (ALL) is crucial for the selection of the appropriate therapeutic protocol. In this work, we describe a new method – one‐step multiplex reverse transcription polymerase chain reaction (RT‐PCR) – to screen for prognostic significant translocations in childhood ALL. Our approach involves a single PCR reaction for the simultaneous detection of the molecular rearrangements resulting from the t(9;22), t(12;21), t(4;11) and t(1;19), with a turnaround time of less than 24 h. This assay proved to be highly sensitive, specific, reproducible and easy to implement in a routine genetics laboratory.
Lack of BCL10 mutations in Hodgkin's disease‐derived cell lines
Authors: Re Daniel , Benenson Elena , Wolf Jürgen , Diehl Volker , Staratschek‐Jox Andrea
Published: March 2003

The pathogenetic events leading to the malignant transformation of Hodgkin–Reed–Sternberg cells are unknown. As Hodgkin–Reed–Sternberg cells are resistant to CD95‐mediated apoptosis and chromosomal aberrations involving the 1p22 region harbouring the proapoptotic BCL10 gene represent a recurrent event in Hodgkin's disease‐derived cell lines, analysis of the BCL10 gene and its transcripts was performed. As transcription of wild‐type BCL10 was detected in all Hodgkin's disease‐derived cell lines analysed, alterations of the coding sequence of the BCL10 gene are unlikely to contribute to the malignant transformation of the Hodgkin–Reed–Sternberg cell.
Functional effects of replacing human α‐ and β‐globins with their embryonic globin homologues in defined haemoglobin heterotetramers
Authors: He Zhenning , Lian Lurong , Asakura Toshio , Russell J. Eric
Published: March 2003

Embryonic‐ and adult‐stage globin subunits assemble into haemoglobin (Hb) heterotetramers that are expressed at low levels throughout human intrauterine development. These haemoglobins differ from adult Hb A (α2β2) by the substitution of embryonic ζ for adult α globin (Hb ζ2β2), or embryonic ε for adult β globin (Hb α2ε2). Several key physiological properties of these ‘semiembryonic’ haemoglobins remain undefined, as ethical and methodological considerations have limited their availability from both human sources and conventional expression systems. The current study attempts to estimate how the physiological properties of semiembryonic and adult haemoglobins may differ, by determining whether the O2‐binding characteristics of hybrid human/mouse haemoglobins change when human α‐ or β‐globin subunits are replaced by human embryonic ζ‐ or ε‐globin subunits respectively. Each of the four human globins is expressed in transgenic mice that are nullizygous for either the endogenous mouse α‐ or β‐globin genes, resulting in the high‐level expression of haemoglobins that can be studied either in situ in intact erythrocytes or in vitro. We showed that the exchange of human ζ‐globin for human α‐globin chains increased haemoglobin O2 affinity, both in the presence and in the absence of 2,3‐bisphosphoglycerate (2,3‐BPG), and reduced the pH‐dependent shift in its oxygen equilibrium curve (Bohr effect). By comparison, hybrid haemoglobins containing either human ε‐globin or human β‐globin exhibited nearly identical O2‐binding properties, both in situ and in vitro, regardless of 2,3‐BPG levels or ambient pH. Neither the ζ‐for‐α nor the ε‐for‐β substitutions substantially altered binding affinity for 2,3‐BPG or cooperativity between globin subunits. These studies suggest that semiembryonic haemoglobins that assemble entirely from human subunits may exhibit properties that are similar to those of human Hb A.
CCNU (lomustine), idarubicin and dexamethasone (CIDEX): an effective oral regimen for the treatment of refractory or relapsed myeloma
Authors: Parameswaran R. , Giles Chrissy , Boots M. , Littlewood T. J. , Mills M. J. , Kelsey S. M. , Samson Diana
Published: March 2003

We report the results of a non‐randomized pilot study of an oral regimen comprising CCNU (lomustine; 25 or 50 mg/m2 on day 1), idarubicin (4‐demethoxydaunorubicin) (10 mg/m2 on days 1–3) and dexamethasone (10 mg b.d. on days 1–4) in patients with relapsed or refractory myeloma. Treatment was given every 28 d for a maximum of six courses. Sixty patients were entered of whom 57 were evaluable. Overall response rate (partial or minor response) was 49% with 30% of patients achieving a partial response (50% tumour reduction). Response rates were higher in patients with untested relapse than in those with refractory disease (overall response rates 56% vs. 31%). The major toxicity was neutropenia and the regimen was otherwise well tolerated. The median survival from entry of all patients was 15 months, with 30% of patients alive at 2 years. This regimen represents a useful addition to available treatment options.
Restriction of T‐cell receptor repertoires in idiopathic CD4+ lymphocytopenia
Authors: Signorini Simona , Pirovano Silvia , Fiorentini Simona , Stellini Roberto , Bianchi Valeria , Albertini Alberto , Imberti Luisa
Published: March 2003

We report that α/β and γ/δ T‐cell repertoires of three patients with idiopathic CD4+ lymphocytopenia, who showed different clinical manifestations and outcomes over time, were highly restricted. The disruption of T‐cell repertoires does not influence the susceptibility to infections: the first patient was unable to attain a protective response to mycobacterium, the second showed clinical improvement and the third did not develop opportunistic infections. These results indicate that idiopathic CD4+ lymphocytopenia could give rise to mono‐/oligoclonal T‐cell expansions, but the degree of repertoire disturbance is not indicative of the severity of disease progression.