bluuebunny/crossref_metadata_2025 huggingface dataset:
Dose intensification in acute myloid leukaemia: greater effectiveness at lower cost. Principal report of the Medical Research Council's ATML9 study
Authors: Rees J. K. H. , Gray R. G. , Wheatley K.
Published: October 2003

Between 1984 and 1990, 972 patients aged 1–79 years with acute myeloid leukaemia (AML), from 85 British hospitals, were entered into the MRC's 9th AML trial. Patients were randomized between DAT 1+5 (daunorubicin for 1 d, with cytarabine and 6‐thioguanine for 5 d) and DAT 3+10 (same dose drugs for 3 and 10 d respectively) as induction therapy. The 63% who achieved complete remission (CR) were randomized to receive two courses of DAT 2+7 alternating with two courses of either MAZE (m‐AMSA, 5‐azacytidine, etoposide) or COAP (cyclophosphamide, vincristine, cytarabine, prednisone). Finally, those still in CR were randomized to receive either 1 year of maintenance treatment with eight courses of cytarabine and thioguanine followed by four courses of COAP, or no further cytotoxic therapy.  Resistance to induction therapy was less common with the DAT 3+10 regimen than with DAT 1+5 (13% v 23%; P==0.0001) and hence, despite a 5% increase in the risk of induction death, the CR rate was higher (66% v 61%; P=0.15). Moreover, CR was achieved more rapidly with DAT 3+1 (median 34 v 46 d; P<0.0001) and thus patients required less time in hospital (mean 20 v 29 d) and less blood product support. 5‐year relapse‐free survival (28% v 23%; P=0.05) and survival (23% v 18%; P<0.05) were also better with DAT 3+10. Post‐remission intensification of therapy with MAZE resulted in fewer relapses (66% v 74% at 5 years; P=0.03) but patients allocated MAZE required considerably more supportive care and 14 (4.5%) died following 312 MAZE courses, whereas no deaths occurred following COAP. 5‐year survival was not significantly higher with MAZE (37% v 31%). Finally, although 1 year of out‐patient maintenance treatment appeared to delay, but not prevent, recurrence it did not improve 5‐year survival which was non‐significantly worse for those allocated maintenance treatment (41% v 44%).  We conclude that the more intensive induction regimen, DAT 3+10, is not only more effective than DAT 1+5, even for older patients, but is also less expensive; intensive post‐remission therapy with MAZE achieves better leukaemic control but at the cost of substantial toxicity; whereas low‐level maintenance therapy confers no apparent advantage in survival as well as being inconvenient and costly.
Unrelated donor bone marrow transplantation to treat severe aplastic anaemia in children and young adults
Authors: Margolis David , Camitta Bruce , Pietryga Daniel , Keever‐Taylor Carolyn , Baxter‐Lowe Lee Ann , Pierce Karen , Kupst Mary Jo , French III James , Truitt Robert , Lawton Colleen , Murray Kevin , Garbrecht Frederick , Flomenberg Neal , Casper James
Published: October 2003

Alternative donor bone marrow transplantation (BMT) to treat severe aplastic anaemia (SAA) in children and young adults has been complicated by high rates of graft rejection and severe graft‐versus‐host disease (GVHD). We hypothesized that increased immunosuppression combined with T‐cell depletion of the marrow graft would enable successful use of unrelated donor BMT in this disease. Preconditioning consisted of cytosine arabinoside, cyclophosphamide, and total body irradiation (TBI). T‐cell depletion was with the anti‐CD3 antibody T10B9. GVHD prophylaxis consisted of cyclosporine A. 28 previously transfused patients were transplanted. Nine donor/recipient pairs were HLA matched. As of 1 January 1996, 15/28 (54%) patients are alive, transfusion independent and well with a range of follow‐up of 13 months to 8 years (median 2.75 years). Fatalities include all three patients with non‐engraftment and all three patients with grade III/IV GVHD. Other fatalities were due to infections or therapy‐related toxicity. The incidence ≥ grade II acute GVHD was 28%. These data show that in children with SAA who have failed immunosuppression, unrelated donor BMT offers a reasonable hope of long‐term survival.
Regulation of colony forming cell generation by flt‐3 ligand
Authors: Brashem‐Stein Carolyn , Flowers David A. , Bernstein Irwin D.
Published: October 2003

The recently cloned ligand for the flt‐3/flk‐2 receptor was examined for its effect on colony formation by subpopulations of CD34+ cells including the least mature CD34+lin−CD38− small‐medium lymphocyte‐sized cell population. Flt‐3 ligand (flt‐3l) had little or no effect when added alone to cells. Isolated CD34+lin+ cells formed increased numbers of colony‐forming cells (CFC) when flt‐3l was added together with IL‐3, IL‐6, G‐CSF, GM‐CSF or c‐kit ligand (KL), or with the combination of IL‐3 and KL. Significant increases in CFC formation from CD34+lin− cells were consistently seen when flt‐3l was added to the IL‐3 and KL combination, with variable effects observed when it was added to individual growth factors. Studies of the generation of CFC from CD34+lin− cells in liquid cultures showed that cultures containing IL‐3 and KL continued to produce CFC after 3 weeks of culture, whereas cultures with IL‐3, KL and flt‐3l produced few CFC past 2 weeks of culture. Flt‐3l alone or the combination of IL‐3 and KL did not stimulate significant growth of CD34+lin−CD38− small‐medium lymphocyte‐sized cells, although these cells reproducibly generated CFC when grown in the combination of IL‐1β, IL‐3, IL‐6, G‐CSF, GM‐CSF and KL. Addition of flt‐3l to either IL‐3 and KL or to a combination of growth factors induced increased CFC in three of four experiments. These data therefore demonstrate a role for flt‐3l in the induction of myelopoiesis by haemopoietic precursors, including the least mature subpopulation population of CD34+ cells.
Phenotype, genotype and clonality of Reed‐Sternberg cells in nodular sclerosis Hodgkin’s disease: results of a single‐cell study
Authors: D elabie Jan , Tierens Anne , Gavriil Theodora , Wu Gang , Weisenburger Dennis D. , Chan Wing C.
Published: October 2003

The genotype and clonality of Reed‐Sternberg (RS) cells in Hodgkin’s disease (HD) has remained a controversial issue, largely due to the scarcity of RS cells in tissues and the limitations of the techniques used to resolve this issue. Southern hybridization and polymerase chain reaction (PCR) assays using DNA extracted from tissues can only document clonal gene rearrangements, but do not indicate which cellular population is responsible for such rearrangements. To overcome the limitations of these previous techniques for studying the genotype and clonality of RS cells, we analysed single RS cells with a single‐cell PCR assay to detect immunoglobulin heavy chain gene (IgH) rearrangements and X‐chromosome inactivation. Six cases of nodular sclerosis (NS) HD were studied. The RS cells displayed a B‐cell phenotype in three cases and a null‐cell phenotype in the other three cases. IgH rearrangements were detected in the RS cells of the three cases with a B‐cell phenotype, but not in the other cases. In these three cases the IgH rearrangements in the RS cells were polyclonal, although a subpopulation of clonal RS cells was documented in one case. The finding that the RS cells with IgH rearrangements were not monoclonal was supported in one case by studying the pattern of X‐chromosome inactivation in single RS cells by a single‐cell human androgen receptor gene (HUMARA) PCR assay. Our results indicate that NSHD begins as a polyclonal process in which a clonal RS cell population may arise; and that the RS cells in a subset of NSHD show evidence of B‐lineage differentiation.
A role of GAGs in ECM on morphogenesis of megakaryocytes
Authors: Tajika Kenji , Ikebuchi Kenji , Dan Kazuo , Asano Shigetaka
Published: October 2003

The morphogenesis of megakaryocytes that results in the formation of cytoplasmic processes is thought to be the final maturation step before liberation of platelets. We studied the in vitro effects of glycosaminoglycans (GAGs) which are abundant in the bone marrow extracellular matrices, on the morphogenesis of murine megakaryocytes and compared them with those of thrombopoietic cytokines. Heparin, heparan sulfate, chondroitin‐6 sulfate, and dermatan sulfate promoted the formation of megakaryocytic processes. Hyaluronic acid failed to support this phenomenon, suggesting that sulfated GAGs in extracellular matrices are involved in the morphogenesis of megakaryocytes. Sulfated GAGs began to act on megakaryocytes with a higher ploidy (16N–32N) from 6 to 24 h after incubation, whereas neither rhIL‐6 nor rhIL‐11 affected this early phase. Our findings indicate that sulfated GAGs promote the morphogenesis of murine megakaryocytes and participate in thrombopoiesis in a different manner from that of cytokines such as rhIL‐6 and rhIL‐11.
The bone marrow aspirate of healthy subjects
Authors: Bain Barbara J.
Published: October 2003

Bone marrow aspirates were obtained from the right or left posterior superior iliac spine of 50 healthy volunteers, 30 men and 20 women. Reference ranges were derived for each cell type and for the myeloid : erythroid (M : E) ratio. The M : E ratio and the percentage of neutrophils were significantly higher in the women and the erythroid component significantly lower. All 28 evaluable men and 11/17 evaluable women had storage iron present in more than trace amounts. The percentage of erythroblasts with detectable iron granules varied very widely, from 3% to 69% in those with more than a trace of storage iron. Minor dyserythropoietic features were present in a high percentage of subjects and 19/50 subjects had one or two dysplastic megakaryocytes. Granulocytic dysplasia was not detected.
Parathyroid hormone‐related protein in hypercalcaemia associated with haematological malignancy
Authors: Firkin Frank , Seymour John F. , Watson Anne‐Marie , Grill Vivian , Martin T. John
Published: October 2003

The incidence of hypercalcaemia and its association with humoral mechanisms involving parathyroid hormone‐related protein (PTHrP), parathyroid hormone (PTH), or 1,25(OH)2 vitamin D were assessed in a prospective study of patients admitted to a clinical haematology unit. Hypercalcaemia was detected in 18/165 patients, and was due to primary hyperparathyroidism in 3/17 patients in whom results of humoral mediator assessments were obtained. In the other patients, hypercalcaemia was associated in nine instances with myeloma, in five with B‐cell non‐Hodgkin's lymphoma (NHL), and in one with myeloid neoplasia. No evidence was obtained of a humoral mechanism involving 1,25(OH)2 vitamin D, but elevated circulating levels of PTHrP, comparable with those in humoral hypercalcaemia of malignancy, were present in 2/4 patients with NHL, and in 3/9 with myeloma. The relationship between presence or absence of elevated circulating PTHrP, and presence or absence of hypercalcaemia during the course of treatment, indicated PTHrP was involved in the production of hypercalcaemia. Such an association raises the possibility that PTHrP released by neoplastic cells in these disorders acts in a paracrine manner to produce local bone resorption, and when produced in greater amounts causes elevated circulating levels which make an additional humorally‐mediated contribution to the development of hypercalcaemia.
The purification of CD34+ cells from human cord blood: comparison of separation techniques and cytokine requirements for optimal growth of clonogenic progenitors
Authors: CHARBORD PIERRE , NEWTON ISOBEL , VOILLAT LAURENT , SCHAAL JEAN PATRICK , HERVE PATRICK
Published: October 2003

This study was performed to assess the methods which gave maximal recovery of purified CD34/45+ cells from a cord blood specimen and optimal growth of progenitors cultured from the purified cells. Cord blood samples were separated using Percoll gradients (either one (1.080) or two successive (1.080 and 1.068) gradient(s)) and commercially available devices for CD34+ cell isolation (affinity columns as manufactured by CellPro Inc. or immunomagnetic separation procedure as devised by Baxter Inc.). ‘CellPro’ or ‘Baxter’ techniques gave similar results in terms of nucleated, CD34/45+ and progenitor cell concentration; however, the yield of CD34/45+ cells in the CD34+ enriched fraction was significantly higher when using the ‘CellPro’ technique. We also found significantly higher numbers of CD34/45+ cells in the CD34+ enriched final fraction when using only one, 1.080, Percoll density gradient in the first separation step. Using one density separation step followed by the ‘CellPro’ technique, we obtained an average of 3×106 purified CD34/45+ cells from samples containing 8.5×108 nucleated cells. Granulomonocytic progenitors (CFU‐GM) and mixed progenitors (CFU‐GEMM) cells from light‐density and purified CD34/45+ cell fractions were evaluated. We found that 20–30% of the light‐density cells and the purified CD34/45+ cells, yielded a granulomonocytic colony in serum free medium in the presence of interleukins 3 and 6, erythropoietin, granulomonocytic and granulocytic colony‐stimulating factors and stem cell factor. The addition of tumour‐necrosis factor α to the cocktail significantly improved the growth of CFU‐GEMM allowing 10% of the purified CD34/45+ cells to yield a mixed colony, which confirms the role of this cytokine on CD34+ cells from cord blood. This study provides an improved method for the recovery of CD34/45+ purified cells and their colony formation. These methods may serve as a basis for studies on CD34/45+ cell amplification and gene transfer.
The use of rh‐G‐CSF in chronic autoimmune neutropenia: reversal of autoimmune phenomena, a case history
Authors: Kuijpers Taco W. , DeHaas Masja , DeGroot CeesJ. , Von Dem Borne Albert E. G. Kr , Weening Ron S.
Published: October 2003

An 8‐year‐old boy had been suffering from chronic autoimmune neutropenia for more than 5 years. The neutropenia proved to be resistant to high‐dose steroids and intravenous (either low‐ or high‐dose) immunoglobulin (Ig) therapy. The chronic autoimmune thrombocytopenia and recurrent phases of autoimmune haemolytic anaemia did, however, respond to high‐dose prednisone. Other signs of immune dysregulation in this patient consisted of insulin‐dependent diabetes mellitus type I (IDDM) and an acquired hypogammaglobulinaemia, most compatible with common variable immunodeficiency (CVI). Prior to rhG‐CSF therapy the child had suffered for more than 2 years from recurrent life‐threatening bacterial infections.

Anti‐neutrophil autoantibodies had pan‐FcγRIII (CD16, NA1/NA2) specificity. The neutropenia as well as the anti‐neutrophil autoantibodies disappeared when subcutaneous rhG‐CSF therapy was started. Upon tapering rhG‐CSF, anti‐FcγRIII antibodies reappeared together with an absolute neutropenia. Renewed administration resulted again in the normalization of symptoms.

Soluble FcγRIII (sFcγRIII) antigen levels in plasma increased dramatically during rhG‐CSF treatment. These high levels of sFcγRIII together with increased numbers as well as decreased apoptotic reactions of neutrophils apparently result in adsorption of the autoantibodies in vivo, contributing to the normalization of autoimmune‐mediated neutropenia upon rhG‐CSF treatment. Long‐term administration of rhG‐CSF represents an alternative in the treatment of autoimmune neutropenia.
Rapid quantitative analysis of protein tyrosine residue phosphorylation in defined cell populations in whole blood and bone marrow aspirates
Authors: Müller C. , Kremerskothen J. , Zühlsdorf M. , Cassens U. , Büchner Th , Barnekow A. , Koch O. M.
Published: October 2003

We developed a rapid method for quantification of tyrosine phosphorylation in immunophenotypically defined cell populations in specimens of whole blood and unprocessed bone marrow. Samples were formaldehyde‐fixed and cells were permeabilized. Phosphotyrosine residues and surface antigens were simultaneously stained by monoclonal antibodies and visualized by flow cytometry. The accuracy of the method was confirmed by demonstration of an increase of phosphotyrosine levels in pp60v‐src transformed fibroblasts. In blood of healthy donors, monocytes and granulocytes showed higher levels of phosphotyrosine than lymphocytes. CD34+ peripheral blood stem cells showed slightly increased tyrosine phosphorylation compared to autologous lymphocytes. Significantly elevated levels of phosphotyrosine were demonstrated in leukaemic blasts compared to lymphocytes (P = 0.01).
Hb Utrecht [α2 129(H12)Leu → Pro], a new unstable α2‐chain variant associated with a mild α‐thalassaemic phenotype
Authors: Harteveld C. L. , Giordano P. C. , Losekoot M. , Heister J. G. A. M. , Batelaan D. , Van Delft P. , Bruin M. C. A. , Bernini L. F.
Published: October 2003

We describe a new α2‐globin gene point mutation found in six individuals of a three‐generation Dutch family. The mutant, which is associated with a mild α‐thalassaemic phenotype, is not detectable at the protein level. The α2 cd129 (CTG → CCG) transition was found by molecular analysis using denaturing gradient gel electrophoresis (DGGE) and single‐strand conformation analysis (SSCA) followed by direct sequencing of the α2‐globin gene. Southern analysis revealed a triplication of the ζ‐gene in cis with the mutant α‐globin gene.
Incidence and implication of additional chromosome aberrations in acute promyelocytic leukaemia with translocation t(15;17)(q22;q21): a report on 50 patients
Authors: Schoch Claudia , Haase Detlef , Haferlach Torsten , Freund Mathias , Link Hartmut , Lengfelder Eva , Löffler Helmut , Büchner Thomas , Fonatsch Christa
Published: October 2003

Acute promyelocytic leukaemia (APL) is characterized by the translocation t(15;17)(q22;q21). Usually t(15;17) is the sole cytogenetic abnormality, but some patients show other chromosome aberrations in addition to t(15;17). The influence of additional chromosome aberrations on the clinical outcome of patients with t(15;17) is unclear. We have analysed 50 cases of APL carrying the translocation t(15;17). Additional chromosome aberrations were observed in 17/47 patients (36%) studied at initial diagnosis and in all three patients studied at relapse. In nine cases (18%) an additional chromosome 8 and in six cases (12%) an isochromosome of the long arm of the derivative chromosome 17 was observed. Various structural rearrangements in addition to t(15;17) were detected in nine patients (18%). Clinical follow‐up data were available for 44 patients studied at diagnosis. A complete remission (CR) was achieved in 34 patients (77%). 10 patients (23%) died within 1 month after diagnosis due to infection or bleeding, eight (24%) relapsed within 10–18 months after initial diagnosis. 28 patients are alive 2–93 months after diagnosis (25 in first CR, two in second and one in third CR) (median follow‐up 18.5 months). Bone marrow transplantation was performed in six patients (three in first CR, two in second CR, one in third CR), all are alive and in CR. An influence of secondary chromosome anomalies on prognosis was not observed. However, if a higher rate of long‐term remission can be reached, specific secondary chromosome aberrations might turn out to be of prognostic value.
Growth stimulatory effect of thrombopoietin on the blast cells of acute myelogenous leukaemia
Authors: Motoji Toshiko , Takanashi Minoko , Motomura Sayuri , Yan‐Hua Wang , Shiozaki Hiroko , Aoyama Masako , Mizoguchi Hideaki
Published: October 2003

Thrombopoietin stimulated blast colony formation in 11/20 acute myelogenous leukaemia (AML) patients studied. The FAB subtypes of the blasts responding to thrombopoietin were not restricted to those of the megakaryocyte lineage, but also included M1–M5 AML blasts. The morphology of colony cells produced by megakaryocytic blasts showed megakaryocytoid features, whereas colony cells produced by M1–M5 AML blasts remained myeloblasts. An increase in CD41 was observed in the cells of colonies produced by blasts from the megakaryocyte lineage involving leukaemia and chronic myeloid leukaemia in blastic crisis. Thrombopoietin receptor was observed on leukaemic blasts which formed colonies following incubation with thrombopoietin.
Enhanced production of osteopontin in multiple myeloma: clinical and pathogenic implications
Authors: Saeki Mima Yukihiko, Toru , Ishii Taeko , Ogata Atsushi , Kobayashi Hideyuki , Ohshima Shiro , Ishida Tetsushi , Tabunoki Yuichiro , Kitayama Hitoshi , Mizuki Masao , Katada Yoshinori , Asaoku Hideki , Kitano Masayasu , Nishimoto Norihiro , Yoshizaki Kazuyuki , Maeda Masahiro , Kon Shigeyuki , Kinoshita Naokazu , Uede Toshimitsu , Kawase Ichiro
Published: October 2003

Summary. In this study, we examined osteopontin (OPN) production in myeloma cells and plasma OPN levels in multiple myeloma (MM) patients. We assessed OPN production in bone marrow cells (BMCs) by immunocytochemistry and enzyme‐linked immunosorbent assay (ELISA). We also assessed OPN production in various B‐cell malignant cell lines, including three myeloma cell lines by reverse transcription polymerase chain reaction (RT‐PCR) and Western blotting. In addition, we measured plasma OPN concentrations by ELISA in 30 MM patients, 21 monoclonal gammopathy of undetermined significance (MGUS) patients and 30 healthy volunteers. As a result, in an immunocytochemical study, abundant OPN was detected in BMCs from overt MM patients, whereas no OPN was detected in BMCs from patients with other haematological diseases, including MGUS. Cultured BMCs from overt MM patients produced more OPN than those from patients with either smouldering MM or MGUS. Myeloma cell lines spontaneously produced OPN. Plasma OPN levels of MM patients were significantly higher than those of MGUS patients and healthy volunteers (P < 0·05). Moreover, they correlated with both progression and bone destruction of the disease (P < 0·05). These suggest that myeloma cells actively produce OPN, which possibly contributes to osteoclastic bone resorption in MM. Plasma OPN levels may be a useful biomarker for assessing bone destruction in MM and distinguishing MM from MGUS or smouldering MM.
Granulomatous slack skin disease – disease features and response to pentostatin
Authors: Osuji Nnenna , Fearfield Louise , Matutes Estella , Wotherspoon Andrew C. , Bunker Christopher , Catovsky Daniel
Published: October 2003

Summary. Granulomatous slack skin disease (GSSD) is a rare condition characterized clinically by redundant skin folds, which show a predilection towards flexural areas, and histologically by a granulomatous T‐cell infiltrate and loss of elastic fibres. The disease is often indolent, although rapid progression and transformation have been described. There is much debate as to whether this condition is a subset of mycosis fungoides or a separate disease entity in itself. We describe a case of GSSD with unique manifestations including granulomatous bone marrow involvement and hypercalcaemia. The patient has twice achieved a good response to pentostatin after failure of combination chemotherapy. This is the first report of the successful use of the purine analogue pentostatin in the management of GSSD.
T‐cell alloreactivity dominates natural killer cell alloreactivity in minimally T‐cell‐depleted HLA‐non‐identical paediatric bone marrow transplantation
Authors: Lowe Eric J. , Turner Victoria , Handgretinger Rupert , Horwitz Edwin M. , Benaim Ely , Hale Gregory A. , Woodard Paul , Leung Wing
Published: October 2003

Summary. Natural killer (NK) cell alloreactivity resulting from killer immunoglobulin‐like receptor (KIR) ligand incompatibility improves outcomes in patients receiving extensively T‐cell‐depleted bone marrow (BM) grafts. Patients with KIR ligand incompatibility are at risk for donor T‐cell alloreactivity. We investigated the relative significance of NK‐cell and T‐cell alloreactivity in 105 paediatric patients who received a minimally T‐cell‐depleted human leucocyte antigen‐non‐identical BM transplantation. Donor NK‐cell incompatibility did not improve patient outcome [engraftment, graft‐versus‐host disease (GVHD), relapse or overall survival]. In contrast, donor T‐cell incompatibility was a risk factor for acute GVHD, chronic GVHD and death. Thus, T‐cell alloreactivity dominated that of NK cells in minimally T‐cell‐depleted grafts.
Clinical significance of residual disease during treatment in childhood acute myeloid leukaemia
Authors: Coustan‐Smith Elaine , Ribeiro Raul C. , Rubnitz Jeffrey E. , Razzouk Bassem I. , Pui Ching‐Hon , Pounds Stanley , Andreansky Martin , Behm Frederick G. , Raimondi Susana C. , Shurtleff Sheila A. , Downing James R. , Campana Dario
Published: October 2003

Summary. In children with acute myeloid leukaemia (AML), morphological and karyotypic studies cannot precisely assess response to treatment, and less than one‐third of patients have genetic markers for molecular studies of residual disease. We determined the usefulness of a four‐colour flow cytometric strategy developed in our laboratory to study residual disease. We first compared the immunophenotypes of AML cells obtained from 54 children at diagnosis with those of cells from 59 normal or regenerating bone marrow samples. Forty‐six of the 54 AML cases (85·2%) had immunophenotypes that allowed detection of 0·1–0·01% residual leukaemic cells. Of 230 bone marrow samples obtained from those 46 patients during and off treatment, 61 (26·5%) had ≥ 0·1% AML cells by flow cytometry. We found that core binding factor‐associated AML had a significantly better early treatment response. Mean (± standard error) 2‐year survival estimate was 33·1 ± 19·1% for patients with ≥ 0·1% AML cells by flow cytometry after induction therapy, but 72·1 ± 11·5% for those with < 0·1% AML cells (P = 0·022); overt recurrence of AML within the subsequent 6 months was significantly more likely in the former group. The assay described here holds promise for guiding the choice of post‐remission treatment options in children with AML.
The role of haploidentical stem cell transplantation in the management of children with haematological disorders
Authors: Veys Paul , Amrolia Persis , Rao Kanchana
Published: October 2003

The broader application of stem cell transplantation (SCT) for paediatric diseases has been limited by a lack of human leucocyte antigen (HLA)‐matched donors. Virtually all children, however have at least one haploidentical parent who could serve as a donor. Such a donor is immediately available and the considerable costs of additional HLA typing, registry and banking expenditures that are necessary to procure an unrelated donor, could be reduced. Recent technological advances appear to have overcome the historical problems of graft rejection and severe graft versus host disease in the haploidentical setting, and in the latest studies the overall survival for children undergoing haploidentical SCT for leukaemia is now comparable with that following unrelated donor bone marrow or cord blood transplantation. Post‐transplant infectious complications and leukaemia relapse remain the most important barriers yet to overcome, and new directions in the use of adoptive cellular immunity appear to be promising in this respect. Haploidentical SCT is now a viable option for those children who do not have an HLA compatible sibling or fully matched unrelated donor. The relative merits of a haploidentical family donor versus mismatched unrelated bone marrow or cord blood donation needs to be assessed in prospective, randomized clinical trials.
Mainstream or margin? The current use of family group conferences in child welfare practice in the UK
Authors: Brown Louise
Published: October 2003

A decade has passed since family group conferences were initially introduced into the UK by Family Rights Group. Ten years on, this paper examines the extent to which family group conferences have developed and become embedded into current social work practice. Despite the initial interest by social work practitioners and the picture often painted of a growing radical movement, the degree to which family group conferencing has become part of mainstream practice has until now remained fairly anecdotal. A number of difficulties have been identified with implementing the model, including fitting it into an existing system and the challenge it poses to professionals to hand over power. Two surveys, the first undertaken in 1999 and the second in 2001, describe the current use of the model in the UK by Councils with Social Services Responsibilities (Councils). The surveys reveal the areas of practice within which family group conferences are being used, the size and capacity of projects and why some Councils have adopted the model whilst others remain hesitant. It concludes by considering why family group conferences remain on the margins of practice.
Services for troubled adolescents: exploring user variation
Authors: Berridge David , Beecham Jennifer , Brodie Isabelle , Cole Ted , Daniels Harry , Knapp Martin , MacNeill Virginia
Published: October 2003

This paper outlines some of the main findings from an exploratory study of a sample of 257 adolescents living in children's homes, foster homes and residential special schools ‘for pupils with emotional and behavioural difficulties’ (‘EBD’). It focuses on the characteristics of service users, particularly contrasting the looked after and EBD groups. Though there are some similarities, there are also important differences between the populations in terms of age, gender, ethnicity, reasons for separation, legal status, family background and education. An attempt was made systematically to compare groups by creating a subsample of ‘difficult’ adolescents, based on involvement in anti‐social behaviour. Unexpectedly, this revealed that half of the teenagers were not ‘difficult’. Factors associated with being identified as ‘difficult’ were explored.
From Habermas's communicative theory to practice on the internet
Authors: Heng Michael S. H. , De Moor Aldo
Published: October 2003

Abstract.  Communication plays a crucial role in influencing our social life. However, communication has often been distorted by unequal opportunities to initiate and participate in it. Such conditions have been criticized by Habermas who argues for an ideal speech situation, i.e. a situation of democratic communication with equal opportunities for social actors to communicate in an undistorted manner. This ideal situation is partially being realized by the advent of the internet. The paper describes how an internet‐based tool for collaborative authoring was conceptualized, developed and then deployed with Habermas's Critical Social Theory as a guiding principle. The internet‐based electronic forum, known by its acronym GRASS (Group Report Authoring Support System), is a web tool supporting the production of concise group reports that give their readers an up‐to‐date and credible overview of the positions of various stakeholders on a particular issue. Together with people and procedures, it is a comprehensive socio‐technical information system that can play a role in resolving societal conflicts. A prototype of GRASS has been used by an environmental group as a new way in which to create a more equal exchange and comparison of ideas among various stakeholders in the debate on genetically modified food. With the widespread use of the internet, such a forum has the potential to become an emergent form of communication for widely dispersed social actors to conduct constructive debate and discussion. The barriers to such a mode of communication still remain – in the form of entrenched power structures, and limitations to human rationality and responsibility. However, we believe that the support provided by the comprehensive system of technological functionality as well as procedural checks and balances provided by GRASS may considerably reduce the impact of these obstacles. In this way, the ideal speech situation may be approximated more closely in reality.
Disruptive information system innovation: the case of internet computing
Authors: Lyytinen Kalle , Rose Gregory M.
Published: October 2003

Abstract. Information system (IS) innovation can be defined as a novel organizational application of digital computer and information communication technologies (ICT). This paper discusses how modalities of applying ICT technologies in their form and scope exhibit radical breaks, which are introduced herein as ‘disruptive IS innovations’. This notion of disruptive IS innovation is developed by drawing upon and extending Swanson's (1994) theory of IS innovation as well as the concept of radical innovation. Disruptive innovations strongly influence the future trajectory of the adoption and use of ICT in organizational contexts and make the trajectory deviate from its expected course. In doing so, these disruptive innovations distinctly define what an IS is and how it is deployed in order to address current and future organizational and managerial prerogatives. Such changes are triggered breakthroughs in the capability of ICT that lead to the revision and expansion of associated cognitive models (frames) of computing. Disruptive IS innovations are those that lead to changes in the application of ICT that are both pervasive and radical. The pervasive nature implies that innovative activity spans all innovation subsets of the quad‐core model of IS innovation introduced herein. Innovation types include: IS use and development processes; application architecture and capability; and base technologies. Radical in nature, disruptive is innovations depart in significant ways from existing alternatives and lead to deviation from expected use and diffusion trajectory. This paper demonstrates the importance of a concept of disruptive IS innovation by investigating how changes triggered by internet computing (Lyytinen et al., 1998) meet the conditions of a disruptive IS innovation defined herein. The analysis also affirms both the pervasive and radical nature of internet computing and explains how internet computing has fundamentally transformed the application portfolio, development practices and IS services over time. The analysis demonstrates that, with the concept of disruptive IS innovation, we can fruitfully analyse ‘long’ waves of ICT evolution – an issue that has largely been overlooked in the IS community. On a theoretical plane, the paper advocates the view that we need to look beyond linear, unidirectional, and atomistic concepts of the diffusion of IS innovations where innovative activity takes places in a linear fashion by oscillating between small technological innovations and small organizational innovations. In contrast, IS innovation can exhibit fundamental discontinuity; we need to theoretically grasp such disruptive moments. The recent influx of innovation, spurred by internet‐based technology, offers one such moment.
Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human αB‐crystallin
Authors: Valdez Melissa M. , Clark John I. , Wu Gabrielle J. S. , Muchowski Paul J.
Published: October 2003

Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection. MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein aggregation in vitro, and it contains the core ‘α‐crystallin’ domain found in all sHsps. In this study we characterized the chaperone activity of recombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human αB‐crystallin, a well characterized member of the sHsp family. Recombinant MTB HSP 16.3 was expressed in Escherichia coli and purified to apparent homogeneity. Similar to αB‐crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 mm ATP the chaperone activity was enhanced by twofold. ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPγS, a nonhydrolyzable analog of ATP. Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E. coli at 48 °C. While the sequence similarity between human αB‐crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core ‘α‐crystallin’ domain are much closer.
Binding of hemolin to bacterial lipopolysaccharide and lipoteichoic acid
Authors: Yu Xiao‐Qiang , Kanost Michael R.
Published: October 2003

Hemolin, a plasma protein from lepidopteran insects, is composed of four immunoglobulin domains. Its synthesis is induced by microbial challenge. We investigated the biological functions of hemolin in Manduca sexta. It was found to bind to the surface of bacteria and yeast, and caused these micro‐organisms to aggregate. Hemolin was demonstrated to bind to lipopolysaccharide (LPS) from Gram‐negative bacteria and to lipoteichoic acid from Gram‐positive bacteria. Binding of hemolin to smooth‐type forms of LPS was competed for efficiently by lipoteichoic acid and by rough mutant (Ra and Rc) forms of LPS, which differ in polysaccharide length. Binding of hemolin to LPS was partially inhibited by calcium and phosphate. Hemolin bound to the lipid A component of LPS, and this binding was completely blocked by free phosphate. Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the O‐specific antigen and the outer‐core carbohydrates of LPS. The binding properties of M. sexta hemolin suggest that it functions as a pattern‐recognition protein with broad specificity in the defense against micro‐organisms.
Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2)
Authors: Nies Anne T. , König Jörg , Cui Yunhai , Brom Manuela , Spring Herbert , Keppler Dietrich
Published: October 2003

The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C‐proximal MRP2 and N‐proximal MRP1 localized to the apical membrane of polarized Madin–Darby canine kidney cells (MDCKII) indicating involvement of the carboxy‐proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven‐amino‐acid extension at its C‐terminus with the last three amino acids (TKF) comprising a PDZ‐interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C‐termini. These constructs were fused via their N‐termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver‐derived human HepG2 cells. Quantitative analysis showed that full‐length GFP–MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C‐terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP–MRP2 was C‐terminally truncated by 15 or more amino acids. Thus, neither the PDZ‐interacting TKF motif nor the full seven‐amino‐acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C‐terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells.
Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom
Authors: Huys Isabelle , Dyason Karin , Waelkens Etienne , Verdonck Fons , van Zyl Johann , Plessis Johan du , Müller Gert J. , van der Walt Jurg , Clynen Elke , Schoofs Liliane , Tytgat Jan
Published: October 2003

A novel peptidyl inhibitor of voltage‐gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called ‘α‐KTx1.1’). PBTx3 is the tenth member (called ‘α‐KTx1.10’) of subfamily 1 of K+ channel‐blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir‐type and ERG‐type channels. The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 µm, 547 nm and 492 nm. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose‐binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The three‐dimensional structure of PBTx3 is proposed by homology modelling to contain a double‐stranded antiparallel β sheet and a single α‐helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other α‐KTx scorpion toxins.
Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes
Authors: Biel Simone , Simon Jörg , Gross Roland , Ruiz Teresa , Ruitenberg Maarten , Kröger Achim
Published: October 2003

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Δψ = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Δψ and H+/e ratio was associated with the reduction of 2,3‐dimethyl‐1,4‐naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Δψ. Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Δψ of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1.

The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Δψ generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Δψ generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.
Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola
Authors: Kanao Tadayoshi , Kawamura Mineko , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki
Published: October 2003

Isocitrate dehydrogenase (IDH) catalyzes the reversible conversion between isocitrate and 2‐oxoglutarate accompanied by decarboxylation/carboxylation and oxidoreduction of NAD(P)+ cofactor. While this enzyme has been well studied as a catabolic enzyme in the tricarboxylic acid (TCA) cycle, here we have characterized NADP‐dependent IDH from Chlorobium limicola, a green sulfur bacterium that fixes CO2 through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO2‐fixation ability of the enzyme. The gene encoding Cl‐IDH consisted of 2226 bp, corresponding to a polypeptide of 742 amino acid residues. The primary structure and the size of the recombinant protein indicated that Cl‐IDH was a monomeric enzyme of 80 kDa distinct from the dimeric NADP‐dependent IDHs predominantly found in bacteria or eukaryotic mitochondria. Apparent Michaelis constants for isocitrate (45 ± 13 µm) and NADP+ (27 ± 10 µm) were much smaller than those for 2‐oxoglutarate (1.1 ± 0.5 mm) and CO2 (1.3 ± 0.3 mm). No significant differences in kinetic properties were observed between Cl‐IDH and the dimeric, NADP‐dependent IDH from Saccharomyces cerevisiae (Sc‐IDH) at the optimum pH of each enzyme. However, in contrast to the 20% activity of Sc‐IDH toward carboxylation as compared with that toward decarboxylation at pH 7.0, the activities of Cl‐IDH for both directions were almost equivalent at this pH, suggesting a more favorable property of Cl‐IDH than Sc‐IDH as a CO2‐fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a competitive inhibitor (Ki = 0.35 ± 0.04 mm) for 2‐oxoglutarate in the carboxylation reaction by Cl‐IDH, a feature not found in Sc‐IDH.
NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1)
Authors: Matthey Ulrich , Braun Daniel , Dimroth Peter
Published: October 2003

The subunit c from the ATP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/water (4 : 4 : 1). In this solvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G29 to N82, respectively. On comparing the secondary structure of subunit c from P. modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the α helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the prominent interruption of the α helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the α helix after the sodium ion‐binding Glu65 residue. The folding of subunit c of P. modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that subunit c of P. modestum did not adopt a stable hairpin structure like subunit c of E. coli.