bluuebunny/crossref_metadata_2025 huggingface dataset:
Natural course of HGV infection in haemophiliacs
Authors: Yamada‐Osaki Maki , Sumazaki Ryo , Kajiwara Yasuhiro , Miyakawa Takayuki , Shirahata Akira , Matsui Akira
Published: March 2003

The natural course of hepatitis G virus (HGV) infection was clarified in 70 haemophiliacs by testing for HGV RNA and antibodies against HGV envelope protein (anti‐E2). None of 12 patients treated with only virus‐inactivated coagulation factors were infected with HGV. Of 58 patients who received non‐inactivated products, 28 (48%) were positive for HGV RNA and/or anti‐E2. Of 16 patients with anti‐E2, 14 were negative for the viral RNA, and had recovered from HGV infections. HCV antibodies were detected in 59 patients, and eight patients were successively negative for HCV RNA. Thus, the recovery rate of HGV infection (14/28, 50%) was higher than that of HCV (8/59, 14%) (P < 0.001). Longitudinal study revealed that anti‐E2 developed either during viraemia or some years after seronegativity for HGV RNA. Hence the antibody response itself seemed not to play a major role in the clearance of HGV, though anti‐E2 was associated with the clearance of HGV RNA. In conclusion, HGV and HCV are prevalent in patients treated with unsterilized coagulation factor concentrates. However, in contrast to HCV, spontaneous recovery is frequently observed in HGV infections.
Immunophenotypic analysis enables the correct prediction of t(8;21) in acute myeloid leukaemia
Authors: Ferrara Felicetto , Di Noto Rosa , Annunziata Mario , Copia Carolina , LO Pardo Catia , Boccuni Piernicola , Sebastio Lucia , Del Vecchio Luigi
Published: March 2003

Immunophenotypic findings from 14 patients affected by acute myeloid leukaemia (AML) with t(8;21) were compared to those obtained from 79 AML patients with normal or other aberrant karyotypes. Classic lineage markers, adhesion molecules, surface enzymes, stem‐cell‐ related antigens and HLA‐DR were investigated. Following evaluation by the Mann‐Whitney test, we found that t(8;21) AMLs showed a significantly higher expression of CD19, CD34, CD56, CD45RA and CD54. Conversely, blasts from patients in the control group significantly expressed higher levels of CD45R0, CD33, CD36, CD11b and CD14. In order to split the data at the best cut‐off point to achieve the most homogenous subset with regard to cytogenetic pattern, i.e. t(8;21) or not, the CART (Classification and Regression Trees) method was applied. In the univariate analysis by CART, statistically significant differences were found when CD19 was dichotomized at 10%, CD34 at 37%, CD45RA at 84%, CD54 at 21%, CD56 at 12% , CD36 at 14%, CD45R0 at 25%, CD11b at 18% and CD14 at 12%. Once cut‐off points were established by CART, we applied the logistic regression model to establish which combination of two or more antigens was most predictive for t(8;21). The combination CD19‐CD34 at the cut‐off points indicated above correctly classified 92/93 cases (98.9%). The addition of any other antigen combination to the CD19/CD34 model failed to improve the level of prediction. We conclude that AML with t(8;21) displays an exclusive immunophenotype that is highly predictive of the cytogenetic pattern.
Total body irradiation–high‐dose cytosine arabinoside and melphalan followed by allogeneic bone marrow transplantation from HLA‐identical siblings in the treatment of children with acute lymphoblastic leukaemia after relapse while receiving chemotherapy: a Société Française de Greffe de Moelle study
Authors: Bordigoni Pierre , Esperou Hélène , Souillet Gérard , Pico JosÉ , Michel Gérard , Lacour Brigitte , Reiffers Josy , Sadoun Alain , Rohrlich Pierre , Jouet Jean‐Pierre , Milpied Noël , Lutz Patrick , Plouvier Emmanuel , Cornu Guy , Vannier Jean‐Pierre , Gandemer Virginie , Rubie HervÉ , Gratecos Nicole , Leverger Guy , Stephan Jean‐Louis , Boutard Patrice , Vernant Jean‐Paul
Published: March 2003

We investigated the use of a new conditioning regimen followed by allogeneic bone marrow transplantation (BMT) for treating children with acute lymphoblastic leukaemia (ALL) after relapse within 6 months of the completion of therapy. One hundred and sixteen children with acute lymphoblastic leukaemia in second or subsequent complete remission (CR) underwent allogeneic bone marrow transplantation from HLA‐identical siblings after a preparative regimen comprising total body irradiation (TBI), high‐dose cytosine arabinoside and melphalan (TAM regimen). The Kaplan‐Meier product‐limit estimate (mean ± SE) of disease‐free survival (DFS) at 7 years was 59.5 ± 9% (95% confidence interval). The estimated chance of relapse was 22.5 ± 15% with a median follow‐up of 88.5 months (range 51–132). 26 patients (22.4%) died with no evidence of recurrent leukaemia, mainly from interstitial pneumonitis, veno‐occlusive disease or acute graft‐versus‐host disease (GVHD). Three factors significantly affected DFS: acute GVHD, site of relapse and, for children in second remission after a marrow relapse, the disease status at the time of transplantation. The DFS were 59.02 ± 12.6%, 37.5 ± 19.8% and 77.4 ± 15% among patients in CR2 after a marrow relapse, in CR3 or in untreated partial marrow relapse, and in CR2 after an isolated CNS relapse, respectively. The lowest DFS was seen in children with acute GVHD grades 3–4. Two significant factors were associated with relapse: the marrow status at the time of transplantation and chronic GVHD. The relapse rate was lower among children in CR2 or with chronic GVHD. We conclude that transplantation after the TAM regimen is an effective therapy for this population with acceptable toxicity, particularly for children in second remission after a very early marrow relapse, or those with early isolated CNS involvement.
Myelofibrosis with myeloid metaplasia in young indidviduals: disease characteristics, prognostic factors and identification of risk groups
Authors: Cervantes Francisco , Barosi Giovanni , Demory Jean‐Loup , Reilly John , Guarnone Roberta , Dupriez Brigitte , Pereira Arturo , Montserrat Emilio
Published: March 2003

Myelofibrosis with myeloid metaplasia (MMM) is an uncommon disorder in young individuals, for whom haemopoietic stem cell transplantation offers the only possibility of cure. However, although the latter procedure is associated with significant morbidity and mortality, the clinical course of MMM is variable, with some patients surviving for less than a year and others showing an indolent course. Selection of young MMM patients for transplantation or other newer therapies is currently difficult since no prognostic data exists for this subgroup. In the present collaborative study a number of initial clinical and laboratory parameters have been evaluated for prognosis in 121 MMM patients aged 55 years or less. Median survival of the series was 128 months (95% CI 90–172). In the Cox proportional hazard regression model three initial variables were independently associated with shorter survival: Hb <10 g/dl (P < 0.0001), the presence of constitutional symptoms (fever, sweats, weight loss) (P = 0.001), and circulating blasts ≥ 1% (P = 0.003). Based on the above three criteria, of the 116 patients with complete data, two groups were identified: a ‘low‐risk’ group, characterized by 88 patients with up to one adverse prognostic factor, in whom MMM had an indolent course (median survival 176 months, 95% CI 130–188), and a ‘high‐risk’ group, including 28 patients with two or three factors, who had a more aggressive disease (median survival 33 months, 95% CI 20–42). The above prognostic scoring system showed a high positive predictive value, sensitivity and specificity to predict survival in the series, and could be of help in making treatment decisions in young patients with MMM.
Cytomegalovirus infection in recipients of related and unrelated donor bone marrow transplants: no evidence of increased incidence in patients receiving unrelated donor grafts
Authors: Foot  , Pamphilon  , Caul  , Roome  , Hunt  , Cornish  , Oakhill
Published: March 2003

Recent reports suggest an increased incidence of cytomegalovirus (CMV) infection in recipients of unrelated donor (UD) bone marrow transplantation (BMT).

In this study we have collated the incidence of CMV infection and disease in sequential UD (n = 119) and related donor (RD; n = 79) BMT performed in a single institution over a 7‐year period. Low‐risk patients (CMV seronegative recipient and donor) accounted for 51% of UD BMT (n = 61) and 62% of RD BMT (n = 49), with CMV excretion documented in one RD BMT only. The remaining high‐risk patients received identical prophylaxis regimens with aciclovir and intravenous immunoglobulin (IVIG). Two groups consisting of 58 UD BMT (median age 9.0 years, range 0.7–45.3 years) and 30 RD BMT (median age 13.6 years, range 1.6–47.6 years) were analysed. CMV reactivation/re‐infection was documented in 15 UD BMT (26%) and 10 RD BMT (33%) (P = 0.72), and CMV disease in four UD BMT (8%) and four RD BMT (13%) (P = 0.533). In this series the risk of CMV excretion and disease following UD BMT was similar to that following RD BMT.
Expression of the PAX5/BSAP transcription factor in haematological tumour cells and further molecular characterization of the t(9;14)(p13;q32) translocation in B‐cell non‐Hodgkin's lymphoma
Authors: Hamada  , Yonetani  , Ueda  , Maesako  , Akasaka  , Akasaka  , Ohno  , Kawakami  , Amakawa  , Okuma
Published: March 2003

The PAX5 gene encodes the BSAP (B‐cell‐specific activator protein) which is a key regulator of B‐cell development and differentiation. A recurring translocation t(9;14)(p13;q32) in non‐Hodgkin's lymphoma moves the PAX5 on 9p13 within close proximity of the immunoglobulin heavy chain gene (IGH). KIS‐1 cell line was established from a patient with diffuse large cell lymphoma of B‐cell type carrying t(9;14). We analysed PAX5/BSAP expression by Northern and Western blotting in a panel of haematological tumour cell lines with other chromosome abnormalities in comparison with that of KIS‐1. PAX5 mRNA and BSAP expression were detected in all B‐cell lines tested, and the high level in KIS‐1 was confirmed. However, a diffuse large B‐cell lymphoma cell line and an acute B‐lymphoid/myeloid leukaemia cell line expressed the PAX5/BSAP at levels comparable with KIS‐1. PAX5 transcripts were readily detectable in clinical materials with a wide variety of B‐cell neoplasms by reverse transcriptase‐mediated polymerase chain reaction (PCR). Thus, PAX5/BSAP activation in haematological tumour cells is not necessarily associated with t(9;14). Although binding sites for BSAP have been identified in the promoters of CD19, this study failed to find clear correlation between the level of PAX5/BSAP expression and that of CD19. In contrast to KIS‐1 in which the Eμ enhancer of IGH was juxtaposed to PAX5, cloning of t(9;14) from another case by long‐distance PCR revealed that the PAX5 promoter was linked to a Cγ constant region in divergent orientation, suggesting that the mechanism of PAX5 activation through recombination with IGH varies among individual cases. Breakpoints on 9p13 of the two translocations were clustered upstream of PAX5, leaving the PAX5 coding region intact.
VLA‐4 is involved in the engraftment of the human pre‐B acute lymphoblastic leukaemia cell line NALM‐6 in SCID mice
Authors: Filshie Robin , Gottlieb David , Bradstock Kenneth
Published: March 2003

Attachment of human pre‐B leukaemic cells to human or murine bone marrow stromal cells in vitro is largely mediated by the β1 integrin VLA‐4 binding to VCAM‐1. Cells subsequently migrate within the stroma, a process also involving VLA‐4. A variant of the pre‐B acute lymphoblastic leukaemia cell line NALM‐6, designated 4A1, lacking expression of VLA‐4, was generated by radiation‐induced mutagenesis followed by several rounds of negative selection with immunomagnetic beads, fluorescence activated cell sorting and clonal expansion. In vitro assays using 4A1 cells showed reduced binding to, and migration under, the murine stromal line M2‐10B4. Sublethally irradiated mice (n = 19) with severe combined immunodeficiency were injected intravenously with NALM‐6 cells. Animals developed signs of leukaemia with hind‐limb paralysis at a median of 30 d (95% confidence interval 28–30). Although there were no gross abnormal findings at autopsy, histological analysis revealed extensive marrow replacement and focal liver infiltration with leukaemic blasts, which were confirmed to be of human origin by flow cytometry. 12 mice were injected with a similar number of cells from the VLA‐4‐negative variant cell line 4A1. Six mice developed signs of leukaemia after 43–74 d, with the remaining six being free of signs of disease after > 100 d (P < 0.001). Mice in this group with leukaemia had a lower incidence of hind‐limb paralysis and less leukaemic infiltration in the marrow, but in some cases had large tumour nodules elsewhere. After a single 500 μg intraperitoneal injection of anti‐murine VCAM‐1 monoclonal antibody (MK2.7), five additional mice were injected with an identical number of wild‐type (VLA‐4+) NALM‐6. All animals developed signs of leukaemia after a similar period to those injected with wild‐type NALM‐6 only. These results demonstrate that the β1 integrin VLA‐4 is involved in the engraftment of the pre‐B‐cell leukaemic cell line NALM‐6 in SCID mice, although the interaction with VCAM‐1 is unlikely to be the sole explanation.
The expression of co‐stimulatory molecules and their relation‐ ship to the prognosis of human acute myeloid leukaemia: poor prognosis of B7‐2‐positive leukaemia
Authors: Maeda Akinori , Yamamoto Kokichi , Yamashita Kouhei , Asagoe Kohsuke , Nohgawa Masaharu , Kita Kenkichi , Iwasaki Hiromichi , Ueda Takanori , Takahashi Atsushi , Sasada Masataka
Published: March 2003

We examined the expression of co‐stimulatory molecules on leukaemic cells of 52 adult patients with acute myeloid leukaemia (AML) (34 men and 18 women) and analysed the relationship between these expressions and the patient's prognosis. B7‐1 was not expressed in any of the 23 patients investigated, whereas B7‐2 was expressed in 26/52 patients (50.0%). B7‐2 was expressed in all AML patients with monocytic morphology (M4 or M5) and in 16/42 cases without monocytic morphology. CD54 was expressed in 28/37 patients examined (75.7%), and CD58 was expressed in all of the AML patients except one (M7). The overall survival of the 26 B7‐2‐positive leukaemia patients (1–24 months, median survival 11.5 months) was significantly shorter than that of the 26 B7‐2‐negative leukaemia patients (1–71+ months, median 35.1 months) (P = 0.0080). In addition, the B7‐2‐positive patients exhibited significantly shorter disease‐free survival periods compared to the B7‐2‐negative patients (P = 0.021). There was no significant difference in age, sex, haematological data and complete remission rate between the B7‐2‐positive and B7‐2‐negative patients. Our results indicated that B7‐2 is one of the most crucial factors in the prognosis of adult acute leukaemia and can be expected to have an important role in tumour immunity.
Effect of granulocyte‐macrophage colony‐stimulating factor treatment on phenotype, cytokine release and cytotoxicity of circulating blood monocytes and monocyte‐derived macrophages
Authors: Hennemann  , Kreutz  , Rehm  , Andreesen
Published: March 2003

We studied phenotype, function and differentiation of mononuclear phagocytes in 11 cancer patients treated subcutaneously with 10 μg/kg recombinant human (rhu) GM‐CSF for 7 d. The rhuGM‐CSF treatment induced (1) a 5.9‐fold increase in the number of blood monocytes (MO), (2) a decrease of CD14bright/CD16bright cells with a diminution of the mean fluorescence intensity (MFI) of CD14, and (3) a decrease of MO cellular cytotoxicity. In patients' sera, tumour necrosis factor (TNF)‐α, interleukin (IL)‐10, IL‐12, neopterin, macrophage‐colony‐stimulating factor (M‐CSF), and IL‐1 receptor antagonist (IL‐1RA) increased, whereas GM‐CSF and granulocyte‐colony‐stimulating factor (G‐CSF) decreased after an initial peak. In whole blood samples the lipopolysaccharide (LPS)‐stimulated release of TNF‐α, IL‐6 and IL‐1RA increased initially, whereas IL‐1β, IL‐10 and IL‐12 decreased. During differentiation from MO to macrophages (MAC), interferon (IFN)‐γ‐stimulated tumour cytotoxicity increased, but both MO and MAC were less cytotoxic upon rhuGM‐CSF treatment. The differentiation‐associated increase of LPS‐induced TNF‐α, IL‐1RA and IL‐10 secretion was reduced by the rhuGM‐CSF treatment, and the expression of CD14 on MAC as well as the proportion of CD14+/CD16+, CD14+/MAX.1+ and CD14+/CD71+ cells in 7‐d cultured MAC declined. We interpret these findings as (1) an increase of immature MO upon rhuGM‐CSF therapy, (2) a priming effect on the LPS‐induced proinflammatory cytokine repertoire of MO, and (3) an impact of rhuGM‐CSF on the capacity of MO to differentiate to MAC in vitro.
Effect of 1‐β‐ D‐arabino‐furanosyl‐cytosine (ara‐C) induction of K562 cells on expression of Rh and other blood group active proteins
Authors: Wiener Edith , Shiels Allan , Wickramasinghe  , Avent
Published: March 2003

K562 cells undergoing differentiation induced by 1‐β‐ D‐arabino‐furanosyl‐cytosine (ara‐C) were examined as a model for studying the biosynthesis and regulation of Rh and other blood group active membrane proteins. Untreated and ara‐C‐induced K562 cells were analysed for the expression of these proteins using monoclonal antibodies in combination with flow cytometry. The major membrane proteins glycophorins A and C remained unaltered upon induction by ara‐C. The display of LFA‐3 (CD58) and DAF (CD55) by uninduced K562 was one order of magnitude lower than that of the glycophorins; following ara‐C treatment there was a 50% rise in LFA‐3 but a modest decrease in the level of DAF expression. The expression by untreated K562 cells of Rh, Lutheran and Kell proteins as well as the Rh D antigen was low, whereas that of CD44 and band 3 protein was negligible. Following induction by ara‐C the levels of Rh and Kell proteins rose up to 7‐ and 3.5‐fold respectively, and there was an increase in RhD‐antigen expression. In contrast, ara‐C induction of K562 cells failed to augment their display of Lutheran, CD44 and band 3 proteins.

Analysis of Rh transcripts following the purification and RT‐PCR analysis of K562 mRNA showed that uninduced K562 cells contain two distinct mRNAs corresponding to Rh Ce (1.8 kb) and Rh D (3.5 kb). The apparent concentration of each mRNA increased following induction with ara‐C. K562 plasma membranes also contained Rh polypeptides as determined by immunoblot analysis using anti‐Rh polypeptide rabbit polyclonal sera raised to Rh synthetic peptides. A novel hybrid Rh transcript corresponding to exons 1–4 of RHD and exons 5–10 of RHCE has been cloned and sequenced from ara‐C induced K562 cells, and may have arisen by general recombination between the RHD and RHCE genes.
Activating point mutations in the βC chain of the GM‐CSF, IL‐3 and IL‐5 receptors are not a major contributory factor in the pathogenesis of acute myeloid leukaemia
Authors: Freeburn Robin W. , Gale Rosemary E. , Linch David C.
Published: March 2003

A number of mutant growth factor receptors have been described which are constitutively activated and confer factor independence on growth factor dependent cells, possibly through constitutive dimerization in the absence of ligand or induction of a conformational change. Mutations in receptor chains may therefore contribute to the pathogenesis of haemopoietic malignancies, for example by causing constitutive receptor activation or uncontrolled downstream signalling. Since most of the activated mutants reported for the βC chain of the GM‐CSF/IL‐3/IL‐5 receptor involve point mutations or truncations of the extracellular domain, we have analysed the coding sequence of this region using RT‐PCR‐SSCP of RNA from blast cells of 31 patients with acute myeloid leukaemia (AML). Two point mutations detected were silent, C301 → T (Cys91) and C1306 → T (Ser426). The latter had previously been identified with an allele frequency of 0.13 in the general population. Two further point mutations detected led to amino acid substitutions, G773 → C (Glu249Gln), which is equivalent to the mouse sequences, and G962 → A (Asp312Asn), both of which were found at similar frequencies in normal controls. Activating mutations of the βC chain which might contribute to the pathogenesis of the disease are therefore rare in AML.
Successful adoptive immunotherapy for relapse of AML 9 years after T‐cell‐depleted BMT
Authors: Bertz Hartmut , Kunzman Regine , Bunjes Donald , Finke JÜrgen
Published: March 2003

Relapse is one of the main problems which can occur following allogeneic transplantation for haematological malignancies. In this situation the enhancement of the graft‐versus‐leukaemia effect by transfusion of donor buffy coats with or without cytokines may lead to complete remission especially in myeloid leukaemias. FISH (fluorescence in situ hybridization) is a sensitive method to monitor chimaerism in gender‐different transplantation.

We report a case of successful buffy coat transfer therapy > 9 years after bone marrow transplanation. This is the longest interval reported, to our knowledge, between transplantation to relapse, which was treated by adoptive immunotherapy. Complete donor chimaerism was confirmed by FISH.
The susceptibility of Philadelphia chromosome positive cells to FAS‐mediated apoptosis is not linked to the tyrosine kinase activity of BCR‐ABL
Authors: Gora‐Tybor Joanna , Deininger Michael W. M. , Goldman John M. , Melo Junia V.
Published: March 2003

We investigated whether inhibition of the BCR‐ABL tyrosine kinase by the CGP57418B compound would render chronic myeloid leukaemia (CML) cells susceptible to Fas (CD95, Apo‐1)‐mediated cell death. Only two (AR230 and SD1) out of 10 BCR‐ABL positive cell lines were found to express the CD95 protein. No change in Fas expression was observed in any of the 10 cell lines after 48 h exposure to CGP57418B. AR230 cells were resistant and SD1 cells were partially resistant to Fas‐mediated apoptosis induced by ligation of the Fas receptor to an anti‐Fas IgM antibody. Pre‐incubation with 1 μM CGP57418B did not change the susceptibility of these cell lines to Fas‐mediated cell death. Similar results were observed in experiments with CD34+ cells from CML patients and from normal individuals. The data suggest that, in contrast to some cytotoxic drugs, the CGP57148B tyrosine kinase inhibitor utilizes a pathway other than the CD95 system in order to induce apoptosis in CML cells.
Acute liver failure induced by alcohol and paracetamol in an HCV‐infected haemophiliac
Authors: Leach  , Makris  , Gleeson  , Preston
Published: March 2003

Paracetamol (acetaminophen) is frequently used by haemophiliacs in the management of acute or chronic pain, primarily due to its lack of adverse effects when taken at therapeutic doses. We describe acute hepatic and renal toxicity of paracetamol in a 36‐year‐old patient with haemophilia B, chronic hepatitis C and chronic alcohol abuse. Moderate doses of paracetamol (6 g/d for 4 d), taken with therapeutic intent, resulted in life‐threatening organ dysfunction which gradually recovered with full supportive care.
Anticipation in familial plasma cell dyscrasias
Authors: Deshpande Hari A. , Hu Xiao‐Ping , Marino Patricia , Jan Naveed A. , Wiernik Peter H.
Published: March 2003

Familial myeloma was described as early as 1925; however, the causative factors are unknown. Studies of families with other familial haematological malignancies demonstrate anticipation. Five new families are described in which plasma cell dyscrasias occurred in parent and child generations (six such pairs), and data were pooled with those of 16 other families (with 20 parent–child pairs affected) recorded in the literature. Disease‐free survival for parent and child generations were each estimated and differences in the disease‐free survival between generations were tested by the log‐rank and signed rank methods. In all six previously unreported parent–child pairs with plasma cell dyscrasia and in 18/20 such pairs found in the literature, the disease occurred at an earlier age in the child generation. The median age of onset of myeloma in the parent and child generations of all 26 pairs was 71 years (95% CI 67–78 years) and 50 years (95% CI 45–55 years), respectively (P < 0.0001). The ages of onset of malignant plasma cell dyscrasias in the parent and child generations of these families compared with patients in the general population were significantly different for the child generation (P < 0.005) but not for the parent generation. It would appear that anticipation occurs in familial myeloma.
Primordial role of CD34+38− cells in early and late trilineage haemopoietic engraftment after autologous blood cell transplantation
Authors: HÉnon  , Sovalat  , Becker  , Arkam  , Ojeda‐Uribe  , Raidot  , Husseini  , Wunder  , Bouderont  , Audhuy
Published: March 2003

In order to better define which cell subset contained in graft products might be the most predictive of haemopoietic recovery following autologous blood cell transplantation (ABCT), the relationships between the amounts of reinfused mononuclear cells (MNC), CFU‐GM, total CD34+ cells and their CD33 and CD38 subsets, and the successive stages of trilineage engraftment kinetics, were studied in 45 cancer patients, using the Spearman correlation test, a linear regression model and a log‐inverse model. No relationship was found between the infused numbers of MNC, CD33+ and CD33− subsets observed and the numbers of days to reach predetermined absolute neutrophil (ANC), platelet and reticulocyte counts. The infused numbers of CFU‐GM, CD34+ and CD34+38+ cells correlated inconstantly with haemopoietic recovery parameters. The strongest and the most constant correlations were significantly observed between the infused numbers of CD34+38− cells and each trilineage engraftment parameter. The log‐inverse model determined a threshold dose of 0.05 × 106 (= 5 × 104) CD34+38− cells/kg, below which the trilineage engraftment kinetics were significantly slower and unpredictable. Post‐transplant TBI‐conditioning regimens increased the low cell dose‐related delay of engraftment kinetics whereas post‐transplant administration of haemopoietic growth factors (HGF) seemed to abrogate this delay. This would justify clinical use of HGF only in patients transplanted with CD34+38− cell amounts lower than the proposed threshold value.  This study suggests that the CD34+38− subpopulation, although essentially participating in late complete haemopoietic recovery, is also composed of committed progenitor cells involved in early trilineage engraftment.
Efficacy of high‐dose methylprednisolone as a first‐line therapy in adult patients with idiopathic thrombocytopenic purpura
Authors: Alpdogan Önder , Budak‐Alpdogan Tülin , Ratip Siret , Firatli‐Tuglular Tülin , Tanriverdi Serpil , Karti Sami , Bayik Mahmut , Akoglu Tevfik
Published: March 2003

Fifty‐seven adult patients with idiopathic thrombocytopenic purpura (ITP) were treated with either conventional‐dose prednisolone (CDP) (1 mg/kg/d, 36 patients) or high‐dose methylprednisolone (HDP) (30 mg/kg/d, 21 patients), as first‐line treatment. Patients in the HDP arm responded more rapidly (4.7 v 8.4 d), with a higher response rate (80% v 52.7%), and without severe side‐effects. One quarter of the patients (3/12) who were non‐responsive to CDP achieved complete remission when they were treated with HDP. The findings suggest that HDP may be a more effective first‐line treatment than CDP for adult ITP, and it may also be preferred for life‐threatening cases of ITP. However, these results must be confirmed by a randomized study prior to any change in the current practice of employing CDP as first‐line treatment for adult ITP.
Dyskeratosis Congenita (DC) Registry: identification of new features of DC
Authors: Knight Stuart , Vulliamy Tom , Copplestone Adrian , Gluckman Eliane , Mason Philip , Dokal Inderjeet
Published: March 2003

Dyskeratosis congenita (DC) is an inherited disorder characterized by skin pigmentation, nail dystrophy and mucosal leucoplakia. In 1995 a Dyskeratosis Congenita Registry was established at the Hammersmith Hospital. In the 46 families recruited, 76/83 patients were male, suggesting that the major form of DC is X‐linked. As well as a variety of noncutaneous abnormalities, the majority (93%) of patients had bone marrow (BM) failure and this was the principal cause (71%) of early mortality. In addition to BM hypoplasia, some patients also developed myelodysplasia and acute myelod leukaemia. Pulmonary abnormalities were present in 19% of patients. In affected females the phenotype was less severe. Some female carriers of X‐linked DC had clinical features. Carriers of X‐linked DC showed skewed X‐chromosome inactivation patterns (XCIPs), suggesting that cells expressing the normal DC allele have a growth/survival advantage over cells that express the mutant allele. Linkage analysis in multiplex families confirmed that the DKC1 gene, responsible for the X‐linked form of DC, is located within Xq28 and facilitated its positional cloning. The high incidence of BM failure in association with a wide range of somatic abnormalities together with the ubiquitous expression of DKC1 suggest that, as well as having a critical role in normal haemopoiesis, this gene has a key role in normal cell biology.
Pre‐treatment of peripheral blood progenitor cells with macrophage inflammatory protein‐1α induces prolonged survival of early progenitor cells over 6 weeks of long‐term culture
Authors: Egger, Dietmar , Günther Christine , Helbig Werner , Schulze Elisabeth
Published: March 2003

It has previously been shown that a combination of macrophage inflammatory protein‐1α (MIP‐1α) and interleukin (IL)‐3 maintained human bone marrow (BM)‐derived long‐term culture‐initiating cells (LTC‐IC) for at least 8 weeks in vitro. We investigated colony‐ and cobblestone area‐formation potential of peripheral blood progenitor cells (PBPC) at week 6 of long‐term culture (LTC) in the absence of exogenous MIP‐1α, but using cells which had been pre‐incubated in the presence of MIP‐1α for 40 h in liquid culture. The recovery of colony‐forming cells (CFC) and cobblestone‐area‐forming cells (CAFC) after pre‐incubation of PBPC with MIP‐1α was up to threefold higher (P < 0.05, n =5) than that of untreated controls. These results demonstrate that short‐term pre‐treatment of PBPC with MIP‐1α induces long‐lasting survival effects on early PBPC‐derived progenitors in vitro.
Human neonatal blood: stem cell content, kinetics of CD34+ cell decline and ex vivo expansion capacity
Authors: Li Karen , Liu Jie , Fok Tai Fai , Wan Yau Fung , Wong Annie , Kong Li Chi , Yang Mo , Woon So King , Chik Ki Wai , Tsang Kam Sze , Kong Shing Matthew Ming , Man Pan Yuen Patrick
Published: March 2003

Haemopoietic stem cells are present in fetal blood but their levels decline rapidly in the peripheral circulation of the infant after birth. We previously reported a case of stem cell transplant in a β‐thalassaemia boy using a combination of the cord blood (CB) and neonatal blood (NB) of his sister. This transplant resulted in a successful engraftment. To investigate the possibility of using NB to supplement CB for related transplants, we further characterized stem and progenitor cells and lymphocyte subsets in 20 NB samples, comparing the findings with those in 20 CB samples. Our data showed that NB contained substantial levels of CD34+ cells, CD34+CD38− cells, colony‐forming units‐granulocyte macrophage (CFU‐GM), colony forming units‐erythroid (CFU‐E), burst forming units‐erythroid (BFU‐E) and long‐ term culture initiating cells (LTCIC). NB was similar to CB in the levels of T lymphocytes, but the amounts of B lymphocytes and natural killer cells were higher in CB (P = 0.033, P = 0.001, respectively). The kinetics of CD34+ cells in NB was investigated in serial blood samples obtained from 10 full‐term infants at 2, 4, 6, 8, 24 and 48 h after birth. CD34+ cells decreased rapidly after birth, declining to only 30% of the 2 h level at 48 h (P < 0.012). The rate of decline was greatest in the first 4 h of life. NB from four infants was expanded by culturing the blood samples in the presence of thrombopoietin (Tpo), interleukin 1β (IL‐1β), IL‐3, IL‐6, flt‐3 ligand and stem cell factor (SCF) for 7 d. This resulted in the increase of CD34+ cells, CFU‐GM and CFU‐MK by 271 ± 179, 556 ± 385 and 113 ± 75 fold respectively. Three of the five samples expanded for 7 d contained LTCIC. These findings suggest that NB might be a supplementary or alternative source of stem cells to CB for transplant. The ethics and practicality of this approach deserve further exploration.
The breakpoints of a constitutional inversion of chromosome 9 associated with haemophagocytic lymphohistiocytosis are not linked to the disease gene
Authors: Aricò Maurizio , Dellavecchia Claudia , Piantanida Mauro , Clementi Rita , Hasle Henrik , Conter Valentino , D'Angelo Paolo , Varotto Stefania , Santoro Nicola , Danesino Cesare
Published: March 2003

Haemophagocytic lymphohistiocytosis (HLH) is a severe disorder of early infancy consistent with an autosomal recessive inheritance. Neither the genetic locus nor the biochemical defect is known for the disease. A constitutional pericentric inversion of chromosome 9, with breakpoints in bands 9p23 and 9q31, has been reported in a case of HLH, suggesting a possible relationship between this chromosome abnormality and the disease. We investigated such an association, performing a genetic linkage analysis in a set of five consanguineous HLH families. 27 polymorphic markers on chromosome 9 were studied, excluding most of chromosome 9 as a putative site for the HLH gene.
Kinetic analysis of 52Fe‐labelled iron(III) hydroxide–sucrose complex following bolus administration 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

Kinetic analysis of a single intravenous injection of 100 mg iron(III) hydroxide–sucrose complex (Venofer®) mixed with 52Fe(III) hydroxide–sucrose as a tracer was followed for 3–6 h in four generally anaesthetized, artificially ventilated minipigs using positron emission tomography (PET). The amount of injected radioactivity ranged from 30 to 200 MBq.

Blood radioactivity, measured by PET in the left ventricle of the heart, displayed a fast clearance phase followed by a slow one. In the liver and bone marrow a fast radioactivity uptake occurred during the first 30 min, followed by a slower steady increase. In the liver a slight decrease in radioactivity uptake was noted by the end of the study. A kinetic analysis using a three‐compartment (namely blood pool, reversible and irreversible tissue pools) model showed a fairly high distribution volume in the liver as compared with the bone marrow.

In conclusion, the pharmacokinetics of the injected complex was clearly visualized with the PET technique. The organs of particular interest, namely the heart (for blood kinetics), liver and bone marrow could all be viewed by a single setting of a PET tomograph with an axial field of view of 10 cm. The half‐life (T1/2) of 52Fe (8.3 h) enables a detailed kinetic study up to 24 h. A novel method was introduced to verify the actual 52Fe contribution to the PET images by removing the interfering radioactive daughter 52mMn positron emissions. The kinetic data fitted the three‐compartment model, from which rate constants could be obtained for iron transfer from the blood to a pool of iron in bone marrow or liver to which it was bound during the study period. In addition, there was a reversible tissue pool of iron, which in the liver slowly equilibrated with the blood, to give a net efflux from the liver some hours after i.v. administration. The liver uptake showed a relatively long distribution phase, whereas the injected iron was immediately incorporated into the bone marrow. Various transport mechanisms seem to be involved in the handling of the injected iron complex.
Syndecan‐1 expression suppresses the level of myeloma matrix metalloproteinase‐9
Authors: Kaushal Gur P. , Xiong Xuede , Athota Anupama B. , Rozypal Tricia L. , Sanderson Ralph D. , Kelly Thomas
Published: March 2003

ARH‐77 human myeloma cells invade into type I collagen gels but become non‐invasive when engineered to express syndecan‐1, a heparan sulphate proteoglycan that promotes cell adhesion to collagen. To determine if syndecan‐1 expression influences the activity of proteases that may facilitate invasion, we analysed media harvested from syndecan‐1 expressing and non‐expressing cells. High levels of a 92 kD gelatinase accumulated in serum‐free growth medium of both parental and control‐transfected ARH‐77, but much less 92 kD gelatinase accumulated in the medium of ARH‐77 transfectants expressing syndecan‐1. The gelatinase was identified as matrix metalloproteinase (MMP)‐9 because its activity was immunoprecipitated with a MMP‐9‐specific monoclonal antibody. Gelatinase activity and Western blot analyses revealed 2–3‐fold less MMP‐9 in medium from syndecan‐1 transfected cells than in medium from parental cells. Decreased MMP‐9 was not due to increased association of MMP‐9 with cells expressing syndecan‐1. An inverse correlation between the syndecan‐1 level and the level of MMP‐9 accumulation in the media was observed using a panel of ARH‐77 transfectants expressing syndecan‐1. Investigation of six unrelated human myeloma cell lines confirmed that high gelatinase levels were recovered from conditioned media of those that did not express syndecan‐1 (ARH‐77, Mer and Col) and one line that expressed a low level of syndecan‐1 (RPMI‐8226), but low gelatinase levels were recovered from media of lines that expressed high levels of syndecan‐1 (ARK and clone 2+). Therefore syndecan‐1 may play a dual role in inhibiting the metastasis of tumour cells by promoting cell adhesion to the extracellular matrix and suppressing the proteolytic activity needed for invasion.
A rapid single‐laser flow cytometric method for discrimination of early apoptotic cells in a heterogenous cell population
Authors: HERAULT OLIVIER , COLOMBAT PHILIPPE , DOMENECH JORGE , DEGENNE MICHEL , BREMOND JEAN‐LOUIS , SENSEBE LUC , BERNARD MARIE‐CHRISTINE , BINET CHRISTIAN
Published: March 2003

A recently reported cytometric method described the possibility of discriminating apoptotic from necrotic cells using FITC‐labelled annexin V and propidium iodide (PI). Nevertheless, the brightness of PI‐staining and its extensive spectral emission overlap with phycoerythrin (PE) does not permit the study of a subset of a heterogenous cell population with single laser instrumentation. The surface staining of a subset with PE in a heterogenous cell population therefore requires another exclusion dye to detect necrotic cells. We used 7‐amino‐actinomycin D (7‐AAD) that can be excited by the 488 nm argon laser line. 7‐AAD emits in the far red range of the spectrum and 7‐AAD spectral emission can be separated from the emissions of FITC and PE. The fluorescence parameters allow characterization of necrotic (7‐AAD+ annexin V‐FITC+ cells), apoptotic (7‐AAD− annexin V‐FITC+ cells) and viable cells (7‐AAD− annexin V‐FITC− cells) in a subset of PE+ cells. The value of this method was demonstrated by measuring apoptosis and necrosis in a model of HL‐60 cells exposed to different inducers of cell death. The method was validated by fluorescent cell sorting in combination with morphologic examination of the sorted cells. The technique we present is particularly valuable in a clinical setting because it enables rapid multiparameter analysis of necrosis and early apoptosis in combination with cell surface phenotyping with a single laser. We present the effects of haemopoietic growth factor deprivation on myeloid progenitor CD34+ cells as an example of its application.
Linear reduction of clonal cells in stem cell enriched grafts in transplanted multiple myeloma
Authors: THUNBERG U. , BÅNGHAGEN M. , BENGTSSON M. , CHRISTENSEN L. D. , GEISLER C. H. , GIMSING P. , LENHOFF S. , MORTENSEN B. T. , OLOFSSON T. , SIMONSSON B. , SMEDEGAARD ANDERSEN N. , SUNDSTRÖM C. , SWEDIN A. , SÄLLSTRÖM J. F. , THURESSON B. , WESTIN J. , CARLSON K.
Published: March 2003

In 30 patients with multiple myeloma who were scheduled for peripheral blood stem‐cell transplantation, a quantitative analysis of the stem cells following enrichment by anti‐CD34 was carried out. To detect the cells of the specific myeloma clone, polymerase chain reaction (PCR) was performed using unique allele‐specific oligo primers for the immunoglobulin heavy chain rearrangement. The clonogenic cells before and after stem‐cell enrichment, were quantified by a limiting dilution assay and a highly sensitive semi‐nested PCR combined with a real‐time quantitative PCR. In order to accomplish a statistically adequate end‐point analysis, a large number of PCR analyses (40 per sample) were performed. By this technique the lowest detection limit observed was one myeloma cell per 106 cells. Myeloma cells were detected in 29/30 samples from the CD34‐enriched fraction. The CD34 selection procedure resulted in a median 28‐fold enrichment of CD34+ haemopoietic precursor cells. The stem‐cell selection reduced the median concentration of clonal cells per million total cells by half, with a highly significant linear relationship between the number of myeloma cells before and after stem cell enrichment. The median depletion of clonal cells by the overall procedure was 2.15 log units, corresponding to a reduction of the total quantity of clonal cells reinfused into the patients by at least 99.3%. We conclude that CD34+ cell enrichment led to a reliable tumour cell depletion of the order of 2 log, which may not be sufficient since the total number of tumour cells in the leukapheresis product was 7.2 log (median).
Three major G6PD‐deficient polymorphic variants identified among the Mauritian population
Authors: Kotea R. , Kaeda J. S. , Yan S. L. K. , Sem Fa N. , Beesoon S. , Jankee S. , Ramasawmy R. , Vulliamy T. , Bradnock R. W. , Bautista J. , Luzzatto L. , Krishnamoorthy R. , Mason P. J.
Published: March 2003

We report the results of the first epidemiological study investigating glucose 6‐phosphate dehydrogenase (G6PD) deficiency among the heterogenous Mauritian population. Mauritius has a population of approximately 1 million, and of these 66.8% are Indo‐Mauritian (of Indian origin), 27.9% are Creoles (of African ancestry) and 2.1% are Sino‐Mauritian, predominantly of Chinese origin. Of the 1435 Mauritian males tested, 73 (5.1%) were G6PD deficient. However, the prevalence varied considerably between the two major ethnic groups: 35/1157 (3.0%) for Indo‐Mauritians and 37/267 (13.9%) for Creoles. Molecular analysis revealed three major deficient poymorphic variants; G6PD Orissa, G6PD Mediterranean and G6PD A−. G6PD Orissa (nt 131 G→C; residue 44 Ala→Gly) was found to be the most common variant among Indo‐Mauritians; this deficient variant was recently identified to be highly characteristic of the tribal groups in central India. In Creoles the most common deficient variant was G6PD A− (27/37). These data are consistent with the different ancestral contributions to the present gene pool of the Mauritian population. This study has provided further information as to the precise nature of G6PD deficiency at the molecular level among Indians, about whom previously there was scant information. The data presented suggest that G6PD Orissa is widespread in central and southern states of India. Additionally, the identification and frequency of G6PD‐ deficient alleles in Mauritius is of public‐health importance.
Prolonged release and c‐kit expression of haemopoietic precursor cells mobilized by stem cell factor and granulocyte colony stimulating factor
Authors: Roberts M. M. , Swart B. W. , Simmons P. J. , Basser R. L. , Begley C. G. , To L. B.
Published: March 2003

Mobilization of haemopoietic precursor cells into the circulation by the combination of cytokines, stem cell factor (SCF) and G‐CSF in previously untreated patients with carcinoma of the breast resulted in increased yield of collected peripheral blood precursor cells (PBPC). This mobilization of PBPC by SCF with G‐CSF lasted several days after ceasing the cytokines in comparison to the rapid fall of PBPC after ceasing G‐CSF. Possible mechanisms for this increased and prolonged mobilization were investigated. Immunological phenotyping with CD38, Thy‐1 and MDR‐1 of the CD34‐positive mobilized PBPC detected no difference in maturity compared to PBPC mobilized by G‐CSF alone. However, the down‐regulation of c‐kit, which is associated with the mechanism of mobilization, was much greater in the PBPC mobilized by SCF and G‐CSF. The potential clinical implication of increased and prolonged mobilization is increased yield, allowing transplantation of heavily pretreated patients, transplantation with PBPC from a single apheresis, or PBSC support for multiple courses of high‐dose therapy from one mobilization procedure.
The role of plasminogen activator inhibitor‐1 as inhibitor of platelet and megakaryoblastic cell adhesion
Authors: Wohn Karl‐Dieter , Schmidt Thomas , Kanse Sandip M. , Yutzy Barbara , Germer Matthias , Morgenstern Eberhard , Preissner Klaus T.
Published: March 2003

In the present study the ability of plasminogen activator inhibitor type‐1 (PAI‐1) to interfere with platelet and megakaryoblastic cell adhesion was investigated. Both cell types exhibited integrin‐dependent adhesion in a static system, mediated by αIIbβ3 on platelets and αv‐integrins on different megakaryoblastic cell lines, even though they also expressed αIIbβ3. In a concentration‐dependent manner, active, but not latent or complexed, PAI‐1 abrogated cell adhesion onto vitronectin but not onto fibrinogen or other matrix substrata. Urokinase as well as thrombin neutralized the anti‐adhesive effect of active PAI‐1. The direct binding of vitronectin, but not of other matrix proteins, to integrin αIIbβ3 was blocked by active PAI‐1 in a purified system. Since activated platelets release active and latent PAI‐1 as well as structurally and functionally distinct forms of vitronectin, the described interactions appear to be physiologically significant. Co‐distribution of vitronectin and PAI‐1 at sites of fibrin polymers within platelet thrombi was demonstrated by transmission electron microscopy, suggesting an extracellular functional relationship of both release products with regard to cell adhesion. Our data emphasize the regulatory role of active PAI‐1 in platelet adhesion to provisional matrix proteins as found during wound healing independent of its anti‐proteolytic activity. Furthermore, megakaryocyte maturation may depend on the intact vitronectin–integrin adhesion system that is influenced by PAI‐1, thereby proposing a regulatory role for the inhibitor in cellular differentiation.