bluuebunny/crossref_metadata_2025 huggingface dataset:
Allelic distribution of the glycoprotein Ia (α2‐integrin) C807T/G873A dimorphisms among Caucasian venous thrombosis patients and six racial groups
Authors: Dinauer David M. , Friedman Kenneth D. , Hessner Martin J.
Published: March 2003

Two linked silent dimorphisms, 807 C → T (Phe224) and 873 G → A (Thr246) within the glycoprotein Ia (GPIa) gene have been correlated with low and high platelet receptor density, respectively, and associated with vascular disease. A multiplexed allele‐specific PCR assay was used to determine the GPIa 807T/873A allele frequency among 331 Caucasian venous thrombosis patients and 3571 unrelated individuals belonging to six different racial groups. The 807T/873A allele frequencies were 54%, 51%, 39%, 39%, 38%, 34% and 30% among Native Americans, Hispanics, Caucasians, Caucasian venous thrombosis patients, Asian Indians, African‐Americans, and Koreans, respectively. Significant differences in the GPIa allele frequency among racial groups were revealed which emphasized the need for appropriate controls in studies evaluating the association of GPIa genotype to vascular disease.
Intravenous immunoglobulin for adults with autoimmune thrombocytopenic purpura: results of a randomized trial comparing 0.5 and 1 g/kg b.w.
Authors: Godeau Bertrand , Caulier Marie‐Thérèse , Decuypere Laurent , Rose Christian , Schaeffer Annette , Bierling Philippe ,   for the Groupe d'Étude du Traitement du PTAI
Published: March 2003

Since the first reports demonstrating the ability of a total dose of 2 g/kg body weight (b.w.) of intravenous immunoglobulin (IVIg) to increase the platelet count in patients with autoimmune thrombocytopenic purpura (AITP), the optimal dose has remained controversial. We report the results of a randomized study which compared two low doses of IVIg (0.5 g/kg b.w., group A, n = 19, and 1 g/kg b.w., group B, n = 18) in 37 adults with AITP and platelet count <50 × 109/l, in preparation for surgery or in a situation with a risk of bleeding. On day 4 the proportion of responses, defined by a platelet count > 80 × 109/l and at least twice the initial platelet count, was significantly higher in the group receiving 1 g/kg b.w. (12/18 in group B versus 4/19 in group A, P = 0.005). All but one of the day 4 responders had already responded on day 3. The daily changes in the platelet count from the beginning of IVIg treatment were larger in group B, with a significant difference relative to group A on day 3 (92 × 109/l in group B versus 50 × 109/l in group A, P = 0.03) and on day 4 (106 × 109/l in group B versus 55 × 109/l in group A, P = 0.03). Patients who had not responded by day 4 subsequently received 1.5 g IVIg/kg b.w. (group A) or 1 g IVIg/kg b.w. (group B). A response was observed in 11/13 initial non‐responders in group A, and in 2/6 initial non‐responders in group B. Finally, on day 8, the proportion of responders was 78% (29/37) in the entire group and was similar in the two subgroups. In conclusion, (1) initial treatment with 1 g/kg b.w. of IVIg appeared to be more effective than 0.5 g/kg b.w. in adults with AITP; (2) infusion of a low dose of IVIg did not jeopardize the efficacy of IVIg reinfusion; (3) some adults who did not respond to 1 g IVIg/kg b.w. responded to a higher dose.
Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients
Authors: Bagnall Richard D. , Waseem Naushin H. , Green Peter M. , Colvin Brian , Lee Christine , Giannelli Francesco
Published: March 2003

We have constructed a confidential U.K. database of haemophilia A mutations and pedigrees by characterizing the gene defect of one index patient in each U.K. family. Mutations were identified by screening all coding regions of the factor VIII (FVIII) mRNA, using solid‐phase fluorescent chemical cleavage of mismatch and examining additional non‐coding regions of the gene. Here we report two haemophilia A patients (UK 114 FVIII:C 2% and UK 243 FVIII:C < 1%) with an abnormal FVIII mRNA due to an A to G point mutation, 1.4 kb downstream from exon 1 in the FVIII gene. This mutation creates a new donor splice site in intron 1 and leads to insertion of a 191 bp novel exon in the mRNA. Haplotype analysis suggests that the mutation may have originated in a common ancestor of the two patients, who further illustrate how mRNA analysis allows higher efficiency of haemophilia A mutation detection, because their mutation would not have been identified by direct analysis of the factor VIII gene.
pH‐dependent fibrillogenesis of a VκIII Bence Jones protein
Authors: Rostagno Agueda , Vidal Ruben , Kaplan Batia , Chuba Joseph , Kumar Ashok , Elliott James I. , Frangione Blas , Gallo Gloria , Ghiso Jorge
Published: March 2003

Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma‐cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light‐chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8–5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VκIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post‐mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N‐terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril‐formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro‐environments may contribute to the stabilization of amyloidogenic conformations.
Distribution of cell‐associated prion protein in normal adult blood determined by flow cytometry
Authors: Barclay G. Robin , Hope James , Birkett Christopher R. , Turner Marc L.
Published: March 2003

Leucocyte subpopulations from normally healthy individuals were identified by recognized combinations of fluorochrome‐conjugated antibodies to CD markers and stained by different monoclonal antibodies (MAb) to normal cellular prion protein (PrPC), including the 3F4 MAb. Cell preparations were examined by three‐colour flow cytometry. All mononuclear leucocyte subpopulations and platelets expressed PrPC, but polymorphonuclear leucocytes and red blood cells expressed little or no PrPC. The amounts of PrPC expressed by the different cells were calculated by comparison to bead standards. Mononuclear leucocytes expressed 3000–4000 molecules of antibody‐reactive PrPC per cell. Resting platelets expressed around 1400 molecules of PrPC per cell, whereas activated platelets expressed around 4800 molecules of PrPC per cell. Extrapolation of these values to the amounts of the various cells in whole blood showed that platelet PrPC accounted for at least 96% of cell‐expressed PrPC in blood. The PrPC on mononuclear cells and platelets was sensitive to enzymatic treatment of cells by proteinase k and phosphatidylinositol‐specific phospholipase C. Certain anti‐PrPC MAbs which showed equivalent intensity of staining to MAb 3F4 on fresh cells showed relative reductions of staining compared to MAb 3F4 on stored cells, indicating possible structural alterations of PrPC under these conditions.
Low‐dose melphalan induces favourable responses in elderly patients with high‐risk myelodysplastic syndromes or secondary acute myeloid leukaemia
Authors: Denzlinger  , Bowen  , Benz  , Gelly  , Brugger  , Kanz
Published: March 2003

We treated 21 elderly patients with high‐risk myelodysplasia (n = 14) or secondary acute myeloid leukaemia (n = 7) with 2 mg of melphalan orally once a day until a complete peripheral response was obtained or until there was evidence of treatment failure. We observed seven (30%) complete and two (10%) partial peripheral responses occurring within 4–16 weeks and lasting for 12 + to 55 weeks. In relapse, retreatment was successful in most of the patients. Responses were associated with the absence of complex cytogenetic abnormalities and with a normal or reduced bone marrow cellularity.
Treatment of an acute flush reaction caused by subcutaneous r‐hirudin with pegylated hirudin
Authors: Harenberg  , Hoffmann  , Huhle  , Song  , Wang
Published: March 2003

We report a patient who was treated with recombinant (r)‐hirudin for heparin‐induced thrombocytopenia and developed a flush reaction twice upon re‐exposure to 25 mg of subcutaneous r‐hirudin. Antihirudin IgG antibodies developed. The patient received 50 mg of PEG‐hirudin subcutaneously over 2 days. No side‐effects occurred. The level of IgG antihirudin antibodies increased. Ecarin clotting time and thrombin inhibition S2238 assay were not influenced by the patient's IgG antihirudin antibody. PEG‐hirudin may be used in patients with intolerance to r‐hirudin because of a dissociation of the allergenic and immunogenic properties of the pegylated drug.
CD34+ cell dose predicts relapse and survival after T‐cell‐depleted HLA‐identical haematopoietic stem cell transplantation (HSCT) for haematological malignancies
Authors: Bahçeci  , Read  , Leitman  , Childs  , Dunbar  , Young  , Barrett
Published: March 2003

Seventy‐eight patients with haematological malignancies, received T‐cell‐depleted stem cell transplants and cyclosporin followed by delayed add‐back of donor lymphocytes to prevent leukaemia relapse. The source of stem cells was bone marrow in 50 patients and granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood in 28 patients. In univariate analysis, only the CD34+ cell dose (but not the stem cell source or the T lymphocyte dose) and disease status were predictive for transplant‐related mortality, relapse and survival. Patients receiving ≥ 3 × 106 CD34+ cells/kg had an overall actuarial survival of 68% compared with 52%, 35% and 10%, respectively, for cell doses of 2–2.99, 1–1.99 and < 1 × 106/kg. Multivariate analysis of risk factors for relapse identified disease risk and CD34+ cell dose as the only factors. Relapse was 62.5% in 38 patients at high risk of relapse vs. 25% for 40 patients at intermediate or low risk. CD34+ cell doses of ≥ 3 × 106/kg were associated with a 13.5% relapse vs. 48% for recipients of lower doses. This favourable effect of CD34+ cell dose on relapse was apparent in both high‐ and intermediate‐ plus low‐risk groups. Our results support the potential benefit of a high stem cell dose in lowering transplant‐related mortality (TRM) and in reducing relapse after allogeneic marrow or blood stem cell transplants.
The diagnosis of low‐grade peripheral B‐cell neoplasms in bone marrow trephines
Authors: Pezzella  , Munson  , Miller  , Goldstone  , Gatter
Published: March 2003

The aim of this study was to establish how effective is the use of immunohistochemistry on formalin‐fixed bone marrow in diagnosing low‐grade B‐cell neoplasms. We investigated a series of 41 consecutive patients with bone marrow involvement for whom no other diagnostic tissues were available. The sections were stained with the following antibodies: CD3, CD20, CD79a, CD5, CD10, CD23, anti‐cyclin D1 and kappa and lambda light chains. Antigen retrieval was performed using either a microwave oven or a pressure cooker. Labelling was performed with an avidin–biotin–peroxidase labelling system. A final diagnosis was reached in 37 out of 41 cases (90%): B‐chronic lymphocytic leukaemia (15 cases), follicular lymphoma (10 cases), mantle‐cell lymphoma (eight cases) and lymphoplasmacytoid lymphoma/immunocytoma (four cases). In the remaining four cases, a generic diagnosis of low‐grade B‐cell neoplasm was made. The immunophenotyping of formalin‐fixed marrow is a useful technique for diagnosing most of the low‐grade B‐cell neoplasms.
Immunoplatelet counting: a proposed new reference procedure
Authors: Harrison  , Horton  , Grant  , Briggs  , Machin
Published: March 2003

Given the high degree of interoperator error and poor precision of manual platelet counting, it has recently been proposed that an immunoplatelet counting method could become the new reference procedure. Platelets are identified immunologically with a suitable monoclonal antibody, and the platelet count is derived from the ratio of fluorescent platelet events to collected red blood cell (RBC) events that are also counted by a reliable and calibrated standard impedance counter (RBC ratio). In this study, we have set up a rapid and simple method for immunoplatelet counting and simultaneously compared the RBC ratio with the bead ratio derived from two different preparations of commercial calibration beads (Trucount and FlowCount beads). Comparison of the level of imprecision of the RBC ratio with either the manual count or bead ratios revealed a superior coefficient of variation of < 5% even in samples with a platelet count < 20 × 109/l. The RBC ratio correlated extremely well with the existing manual phase reference method (r2 = 0.93) and especially well with three different commercial impedance counters and a dual‐angle optical counter (r2 = 0.98–0.99). However, at < 100 × 109/l, the correlation of the RBC ratio with the dual‐angle optical count (ADVIA 120) (r2 = 0.96) was superior to all impedance counters. This suggests that automated optical counting methods may be more accurate at determining platelet counts in thrombocytopenic samples. As the RBC ratio is rapid, cheap and relatively easy to perform, we propose that this method could replace the manual count as a new international reference method.
Defective fibrinogen polymerization associated with a novel γ279Ala→Asp mutation
Authors: Brennan  , Wyatt  , Ockelford  , George
Published: March 2003

A woman with menorrhagia was investigated for a suspected fibrinogen mutation when coagulation tests revealed prolonged thrombin (55 s) and reptilase (43 s) times together with a functional and an antigenic fibrinogen concentration of 0.7 and 2.8 mg/ml respectively. Heterozygosity for a γ‐chain mutation was suggested by a doublet γ band on SDS–PAGE and an increased negative charge was observed on isoelectric focusing of HPLC‐isolated γ‐chains. Electrospray ionization mass spectrometry revealed a γ‐chain mass of 48 411 Da, which was 20 Da more than the control value of 48 391 Da. Because the normal and variant γ‐chains were not resolved, this implied a 40‐Da increase in 50% of the γ‐molecules. An increased negative charge and a 44‐Da increase in mass was verified when DNA sequencing showed heterozygosity for an Ala (GCC)→Asp (GAC) substitution at codon 279 of the γ‐gene. Fibrin polymerization curves indicated a delay in the onset, and a decrease in the rate, of polymerization. Examination of crystal structures showed that the adjacent Tyr‐γ280 side chain is involved in bonding across the D–D interface, and from the proximity of the γ279Ala→Asp mutation it would appear that this perturbs the end‐to‐end DD interactions between fibrin units of the growing polymer.
Effect of ex vivo cytokine treatment on human cord blood engraftment in NOD‐scid mice
Authors: Ballen Karen , Becker Pamela S. , Greiner Dale , Valinski Helen , Shearin Danielle , Berrios Virla , Dooner Gerri , Hsieh Chung‐Cheng , Wuu Joanne , Shultz Leonard , Cerny Jan , Leif Jean , Stewart F. Marc , Quesenberry Peter
Published: March 2003

Umbilical cord blood transplantation is considered an alternative to traditional bone marrow transplantation for patients who do not have matched sibling donors. In this study, we examined the effects of ex vivo treatment of human cord blood cells with cytokine mixtures and assessed the ability of treated cells to engraft in NOD‐scid mice. We incubated the cord blood with a four‐factor cytokine mixture of interleukin (IL)‐3, IL‐6, IL‐11 and stem cell factor, or with a two‐factor cytokine mixture of thrombopoietin and flt‐3. Incubation of cord blood for 48 h with either cytokine mixture did not affect progenitor cell number or proliferative potential as measured by the high proliferative potential (HPP) assay. Cytokine‐treated cord blood injected into irradiated NOD‐scid mice resulted in multilineage human engraftment. Overall, incubation with cytokines resulted in variable levels of engraftment with different cord blood samples. Incubation of cord blood with the four‐factor cytokine mixture resulted in increased survival of irradiated NOD‐scid recipients. These results demonstrate that short‐term ex vivo treatment of human progenitor cells gives variable results on in vivo multipotential capabilities.
Incidence, characterization and prognostic significance of chromosomal abnormalities in 640 patients with primary myelodysplastic syndromes
Authors: Solé  , Espinet  , Sanz  , Cervera  , Calasanz  , Luño  , Prieto  , Granada  , Hernández  , Cigudosa  , Diez  , Bureo  , Marqués  , Arranz  , Ríos  , Martínez Climent  , Vallespí  , Florensa  , Woessner
Published: March 2003

Recently, a consensus International Prognostic Scoring System (IPSS) for predicting outcome and planning therapy in the myelodysplastic syndromes (MDS) has been developed. However, the intermediate‐risk cytogenetic subgroup defined by the IPSS includes a miscellaneous number of different single abnormalities for which real prognosis at present is uncertain. The main aims of this study were to evaluate in an independent series the prognostic value of the IPSS and to identify chromosomal abnormalities with a previously unrecognized good or poor prognosis in 640 patients. In univariate analyses, cases with single 1q abnormalities experienced poor survival, whereas those with trisomy 8 had a higher risk of acute leukaemic transformation than the remaining patients (P = 0.004 and P = 0.009 respectively). Patients with single del(12p) had a similar survival to patients with a normal karyotype and showed some trend for a better survival than other cases belonging to the IPSS intermediate‐risk cytogenetic subgroup (P = 0.045). Multivariate analyses demonstrated that IPSS cytogenetic prognostic subgroup, proportion of bone marrow blasts and haemoglobin level were the main prognostic factors for survival, and the first two characteristics and platelet count were the best predictors of acute leukaemic transformation risk. A large international co‐operative study should be carried out to clarify these findings.
High‐dose busulphan alone as cytoreduction before allogeneic or autologous stem cell transplantation for chronic myeloid leukaemia: a single‐centre experience
Authors: Olavarria E. , Kanfer E. , Szydlo R. , O'brien S. , Craddock C. , Apperley J. , Goldman J.
Published: March 2003

Transplant‐related morbidity and mortality remain a major problem following stem cell transplantation (SCT). The use of high‐dose single‐agent busulphan before allogeneic or autologous SCT may be a reasonable compromise between maintaining cytoreductive efficacy and minimizing toxicity in patients with chronic myeloid leukaemia (CML). Seventy patients with CML have received busulphan as the only cytoreductive therapy before SCT on 79 occasions. The probability of survival of the 14 allogeneic recipients (all of whom were undergoing a second transplant from the original donor) was 78% at 5 years. Sixty‐five autologous SCT were performed in 56 patients, of whom 40 were in late chronic phase. The actuarial 3‐year post‐autograft survival was 54% for these 56 first autografts. For patients in chronic phase, the 3‐year survival was 76% compared with 30% at 2 years for those with advanced phase disease. Busulphan therapy was well tolerated, and except for mild mucositis little toxicity was experienced. None of these patients developed hepatic veno‐occlusive disease (VOD). Nine patients (one allogeneic and eight autologous recipients) received several doses of an intravenous formulation of busulphan with very low toxicity. High‐dose busulphan alone appears sufficient to provide adequate cytoreduction and immunosuppression in second allogeneic transplants and is also effective as cytoreduction before autologous SCT in patients with CML.
Monocyte‐derived dendritic cells do not proliferate and are not susceptible to retroviral transduction
Authors: Ardeshna  , Pizzey  , Thomas  , Orr  , Linch  , Devereux
Published: March 2003

Dendritic cells may be generated ex vivo from CD34+ progenitor cells or peripheral blood mononuclear cells. Initial reports suggested that monocyte‐derived dendritic cells (MoDCs) arise from a proliferating precursor and several groups subsequently reported successful retroviral transduction of these cells, again implying that cell division occurs. As this is of importance in the development of immunotherapy protocols, we investigated whether monocytes proliferate as they differentiate into MoDCs and also their susceptibility to retroviral transduction. During MoDC differentiation, there was a 51 ± 12% reduction in cell number, 98% of cells were in G0/G1, no DNA synthesis was detectable and the cell cycle regulatory proteins pRb and p130 were in the hypophosphorylated forms observed in non‐cycling cells. As expected from these results, MoDCs were refractory to transduction with a GALV1 pseudotyped Moloney murine leukaemia virus (MoMLV)‐based retroviral vector. In contrast, generation of DCs from purified CD34 progenitors was accompanied by rapid entry into the cell cycle and a 41.1‐fold cell expansion at the end of 14 d culture.
A UK multicentre phase II study of rituximab (chimaeric anti‐CD20 monoclonal antibody) in patients with follicular lymphoma, with PCR monitoring of molecular response
Authors: Foran James M. , Gupta Rajnish K. , Cunningham David , Popescu Razvan A. , Goldstone Anthony H. , Sweetenham John W. , Pettengell Ruth , Johnson Peter W. M. , Bessell Eric , Hancock Barry , Summers Karin , Hughes Julia , Rohatiner Ama Z. S. , Lister T. Andrew
Published: March 2003

Follicular lymphoma (FL) cells express CD20 and are associated in most cases with the t(14;18) chromosomal translocation. A multicentre study was undertaken between January 1997 and January 1998 to assess the complete response rate (CR) and overall response rate (RR) to rituximab, a chimaeric anti‐CD20 monoclonal antibody. Seventy patients with previously treated FL received rituximab (375 mg/m2/week ×4, by intravenous infusion). Restaging studies were performed 1 and 2 months after therapy. Molecular monitoring for the presence of cells harbouring the Bcl‐2/JH gene rearrangement in the peripheral blood (PB) and bone marrow (BM) was performed before and after treatment using a two‐step semi‐nested polymerase chain reaction (PCR) assay. The overall RR was 32/70 (46%), being highest in patients who had received only one previous treatment (12/15, 80%). However, only two patients achieved a CR. The median duration of response was 11 months. Thirteen of 21 evaluable ‘PCR‐positive’ patients (62%) became ‘PCR‐negative’ in PB and/or BM samples 1 month after rituximab, although this did not correlate with clinical response. Treatment was generally well tolerated, although one patient developed Stevens–Johnson syndrome. Rituximab was shown to be active in FL, and in some cases PB and/or BM became PCR negative. Studies in combination with cytotoxic chemotherapy to increase the CR rate are warranted.
A prospective randomized study to determine the optimal dose of intravenous vitamin K in reversal of over‐warfarinization
Authors: Hung A. , Singh S. , Tait R. C.
Published: March 2003

The optimal management of asymptomatic overanticoagulated patients remains unknown. We measured international normalized ratio (INR), activated partial thromboplastin time (APTT) and prothrombin fragment 1 + 2 (F1 + 2) over 7 d in 24 asymptomatic or mildly haemorrhagic patients on warfarin with prolonged INR of > 7·0 who were randomized to receive 0·5 mg, 1 mg or 2 mg intravenous vitamin K. Of six severely overanticoagulated patients (INR > 9·5 with APTT ratio > 2), five failed to achieve an INR ≤ 4·0 on day 1, irrespective of vitamin K dose given. In the remaining 18 cases, an optimal response (INR 2–4 at day 1) was observed in 67% of those receiving 0·5 mg vitamin K, but only in 33% of those receiving 1 or 2 mg, the majority of whom developed an INR < 2·0. Our results support an optimal dose of 0·5 mg i.v. vitamin K for most overanticoagulated patients, with possibly a repeat dose in the small group of severely overanticoagulated patients.
Paroxysmal cold haemoglobinuria in an adult with chicken pox
Authors: Papalia Mary Anne , Schwarer Anthony P.
Published: March 2003

Paroxysmal cold haemoglobinuria (PCH) is an autoimmune disorder characterized by intravascular haemolysis causing haemoglobinuria. It is due to a biphasic haemolysin known as the Donath–Landsteiner antibody, which binds specifically to the P antigen of red blood cells at low temperatures, leading to complement activation and red cell lysis at 37°C. PCH is a rare disease which predominantly affects the paediatric population, occurring mostly during viral infections. We report on what is possibly the first case of PCH in an adult to be precipitated by chicken pox infection.
Assay discrepancy in mild haemophilia A due to a factor VIII missense mutation (Asn694Ile) in a large Danish family
Authors: Schwaab R. , Oldenburg J. , Kemball‐Cook G. , Albert T. , Juhler C. , Hanfland P. , Ingerslev J.
Published: March 2003

Factor VIII gene analysis in a large consanguinous Danish family comprising 24 affected males and four homozygously affected females revealed an Asn694Ile mutation within the A2 domain. The factor VIII gene mutation led to a mild haemophilia A phenotype with factor VIII function displaying discordance between one‐stage clotting and chromogenic two‐stage assays. In one‐stage assays, values ranged from 0·05 to 0·30 IU/ml (males) and from 0·19 to 0·29 IU/ml (homozygous affected females), whereas the chromogenic two‐stage assay produced values of around only 50% of the one‐stage result [0·02–0·12 IU/ml (males); 0·06–0·10 IU/ml (females)]. The differences are suggested to be caused by the effect of the mutation on the active cleaved form of the factor (F)VIII protein. As the original amino acid (Asn) is conserved in all known FVIII A2 sequences, but not in ceruloplasmin, we suggest that Asn694 is involved in an A2‐specific functional role. Examination of a homology model of the A domains predicts that the Asn694Ile mutation (i) results in the loss of two potential hydrogen–bonding interactions and (ii) hampers the integration of the bulky side‐chain of Ile into the A2 domain core, probably causing an altered stability and/or folding of the protein. Interestingly, the disease in this Danish family was originally proposed to be von Willebrand–Jürgens disease. However, the current study rules out the co‐existence of either von Willebrand's disease or the presence of the Normandy variant of von Willebrand factor (type 2N).
The monoclonal antibody TER‐119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage
Authors: Kina Tatsuo , Ikuta Koichi , Takayama Eiji , Wada Katsuya , Majumdar Anis Sen , Weissman Irving L. , Katsura Yoshimoto
Published: March 2003

The antigen specificity of a rat monoclonal antibody TER‐119 was investigated. In adult mice, TER‐119 reacted with mature erythrocytes, 20–25% of bone marrow cells and 2–3% of spleen cells but not with thymocytes nor lymph node cells. In fetal haematopoietic tissues, 30–40% of d 10 yolk sac cells, 80–90% of d 14 fetal liver cells and 40–50% of newborn liver cells were reactive with TER‐119. TER‐119+ cells in adult bone marrow expressed significant levels of CD45 but not myeloid (Mac‐1, Gr‐1) or B‐cell (B220) markers. Morphological examination and haematopoietic colony‐forming assays for isolated TER‐119+ cells revealed that TER‐119 reacts with erythroid cells at differentiation stages from early proerythroblast to mature erythrocyte, but not with cells showing typical erythroid blast‐forming unit (BFU‐E) and erythroid colony‐forming unit (CFU‐E) activities. Erythroleukaemia cell lines do not express the TER‐119 antigen even after stimulation with dimethylsulphoxide. TER‐119 immunoprecipitated protein bands with molecular masses of 110 kDa, 60 kDa, 52 kDa and 32 kDa from erythrocyte membrane, whereas only a 52‐kDa band was detected by TER‐119 in Western blot analysis. Further molecular and cellular analyses indicated that the TER‐119 antigen is a molecule associated with cell‐surface glycophorin A but not with glycophorin A itself.
Quality of life in 244 recipients of allogeneic bone marrow transplantation
Authors: Chiodi S. , Spinelli S. , Ravera G. , Petti A. R. , Van Lint M. T. , Lamparelli T. , Gualandi F. , Occhini D. , Mordini N. , Berisso G. , Bregante S. , Frassoni F. , Bacigalupo A.
Published: March 2003

The number of long‐term survivors after allogeneic bone marrow transplantation (BMT) has been increasing over the past years, and quality of life (QOL) has become an important end‐point. We studied 244 patients undergoing an allogeneic BMT to identify factors and events influencing psychosocial outcome. Patients enrolled received the Psychosocial Adjustment to Illness Scale (PAIS) questionnaire assessing psychological and social adjustment to chronic illness or its sequelae. Eighty‐two per cent of patients had a haematological disease. The median age was 28 years at BMT, and the median follow‐up was 61 months. The median overall PAIS score for all patients was 56 (range 22–76): 25% (n = 61) of patients were considered to have a good QOL (≤ 25 percentile score); 44% (n = 108) of patients had an intermediate QOL (26–75 percentile score) and 31% (n = 75) had a poor QOL (> 75 percentile score). Factors associated with a poor QOL in multivariate analysis were: patients' age at BMT (> 25 years, P < 0·01); presence of long‐term sequelae (P < 0·01); chronic graft‐versus‐host disease (GVHD) (P < 0·05); and a short interval from BMT (< 5 years; P < 0·05). The QOL improved with time: 12% of patients reported a good QOL within 5 years compared with 38% after this time point and, conversely, 38% reported a poor QOL within 5 years compared with 24% after this time point (P < 0·0001). Older patients had significantly poorer QOL compared with younger patients (≤ 25 years; P = 0·01). Females had significantly poorer scores when compared with males in the sexual (P < 0·0001) and psychological domains (P = 0·001). The data suggest that (i) one‐third of patients undergoing allogeneic BMT report a poor QOL; (ii) factors associated with poor QOL are older age, presence of long‐term sequelae, chronic GVHD and short follow‐up; (iii) QOL is superior in long‐term survivors; and (iv) BMT affects different aspects of life in males and females. A longitudinal study is ongoing to prove the effect of time on quality of life.
Desmopressin: therapeutic limitations in children and adults with inherited coagulation disorders
Authors: Nolan B. , White B. , . Smith J , O'reily C. ,   C McMahon, Fitzpatrick B. , Smith O.P.
Published: March 2003

Desmopressin (DDAVP), a synthetic analogue of vasopressin has been successfully used in the treatment of type I von Willebrand's disease (VWD), mild factor VIII (FVIII) deficiency and intrinsic platelet function defects (PFDs) for almost three decades. However, there is limited published data documenting its efficacy and the reliability of circulating plasma FVIII:C as a surrogate marker of response to therapy in VWD. We report the haemostatic response to DDAVP in 133 consecutive patients (91 type I VWD, 20 mild FVIII deficiency and 22 PFDs). Minimal therapeutic response to DDAVP (0·3 µg/kg) was defined by normalization 30 min post‐ infusion of bleeding time for PFDs, factor VIII:C (FVIII:C) for mild haemophilia A, and von Willebrand factor antigen (VWF:Ag), von Willebrand factor functional activity (VWF:Ac) and FVIII:C for VWD. Nine out of 91 (10%) VWD patients failed to achieve minimal therapeutic response to DDAVP; plasma FVIII:C levels were an unreliable surrogate marker of DDAVP response as 6 out of 9 (67%) of these patients had normal post‐infusion FVIII:C levels. Five out of the 20 (25%) patients with mild FVIII deficiency and 5 out of 22 (23%) patients with PFDs failed to achieve a minimal therapeutic response to DDAVP. DDAVP is an effective therapy in the majority of patients with type I VWD, PFDs and mild FVIII deficiency. The significant failure rate associated with this therapy supports the recent recommendations that response should be assessed in all patients at the time of diagnosis. FVIII:C is an unreliable guide of response to DDAVP in patients with VWD and therefore VWF:Ag and VWF:Ac should also be assessed. Failure to demonstrate the response of VWF:Ag, VWF:Ac and FVIII:C to DDAVP in patients with VWD is likely to increase the risk of haemorrhagic complications in patients with bleeding episodes or who are undergoing surgery.
Familial chronic lymphocytic leukaemia: a survey and review of published studies
Authors: Yuille M. R. , Matutes E. , Marossy A. , Hilditch B. , Catovsky D. , Houlston R. S.
Published: March 2003

B‐cell chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia. To gain insight into the role of inherited factors in the disease, we have conducted a survey of the family histories of 268 CLL patients and have reviewed published familial cases and epidemiological studies. The results of our survey and published studies strongly support the hypothesis that a subset of the disease can be ascribed to a genetic predisposition. The most likely genetic model for inherited predisposition appears to be dominantly acting genes with pleiotropic effects because in many families CLL appears to be associated with other lymphoproliferative disorders.
T‐cell expansions in patients with multiple myeloma have a phenotype of cytotoxic T cells
Authors: Raitakari Maria , Brown Ross D. , Sze Daniel , Yuen Edna , Barrow Lisa , Nelson Margaret , Pope Belinda , Esdale Warren , Gibson John , Joshua Douglas E.
Published: March 2003

The presence of T‐cell clones in peripheral blood has been previously shown to be associated with a survival advantage in patients with multiple myeloma and suggests that the expanded T‐cell populations may be involved in an anti‐tumour response. We studied the T‐cell receptor (TCR) repertoire of 38 patients with myeloma to identify and characterize the expanded T‐cell populations by flow cytometry. T‐cell expansions were found in 79% of the patients. The expansions occurred randomly among the 21 variable regions of the TCR β chain (Vβ) studied, representing 62% of the V‐β repertoire, and were stable during an 18‐month follow‐up. The phenotype of the expanded V‐β populations was predominantly CD8+, CD57+, CD28− and perforin+, which differed significantly from the other non‐expanded Vβ populations. The expression of the apoptosis markers Fas (CD95) and bcl‐2 were similar between the expanded and non‐expanded Vβ populations. In conclusion, expanded T‐cell populations were frequent in patients with myeloma, they remained unchanged during follow‐up and had phenotypic characteristics of cytotoxic T cells. These data add further support to the concept that the T‐cell expansions may have an immunoregulatory role in myeloma.
Modelling haemopoietic stem cell division by analysis of mutant red cells
Authors: Vickers Mark , Brown Gary C. , Cologne John B. , Kyoizumi Seishi
Published: March 2003

Data describing the number of human red cells mutated at the glycophorin A locus, measured flow cytometrically, are reported for 752 adults and 49 neonates. The variance increases with age more rapidly than the approximately linear increase in mean. It is postulated that this discrepancy is explained by the known property of asymmetric stem cell division, so that the division of a single mutant stem cell may result in zero, one or two progeny stem cells. A mathematical analysis allows description of this process with three parameters: stem cell number, mean division rate and mutation rate per division. The values of these parameters can not be deduced from the data presented here. However, estimates of either stem cell number or mutation rate from other sources enable deduction of the two other parameters. The mean number of divisions per stem cell per lifetime was estimated to be about 70. This analysis therefore implies that the rate at which blood cell telomeres shorten with age acts as a direct measure of stem cell turnover. Furthermore, it is argued that this low figure implies that mutations occurring during early life, including organogenesis, are relatively important in initiating stem cell‐derived malignancy. Finally, the number of human stem cell divisions per lifetime is similar to shorter‐lived mammals, suggesting this number is important in the ageing process.
Natural killer‐type receptors for HLA class I antigens are clonally expressed in lymphoproliferative disorders of natural killer and T‐cell type
Authors: Hoffmann T. , De Libero G. , Colonna M. , Wodnar‐Filipowicz A. , Passweg J. , Favre G. , Gratwohl A. , Tichelli A.
Published: March 2003

In recent years, natural killer (NK) cells, as well as subpopulations of T cells, have been found to express diverse NK receptors (NKRs) for HLA class I molecules. We have characterized NKR phenotypes in lymphoproliferative disorders of NK or T‐cell type. Peripheral blood of patients with lymphoproliferative disorders (n = 9) was analysed by multiparametric immunofluorescence flow cytometry with eight different antibodies against NKRs. Abnormal neoplastic cell populations from different types of NK or T‐cell lymphoproliferative disorders lacked diversity in their NKR repertoires, i.e. all or none of the abnormal cells expressed individual NKRs and this expression occurred at single levels of intensity. This pattern of expression was specific for lymphoproliferative disorders as these resticted NKR repertoires were not found either in healthy donors (n = 9) or in patients with viral or autoimmune disease (n = 5). We conclude that NKRs are clonally expressed in lymphoproliferative disorders of NK or T‐cell origin. NKR repertoires may represent a novel tool in diagnosing clonal disorders of NK and T‐cell type.
Lymphocyte function antigen 1 (LFA‐1) mediates early tumour necrosis factor α‐induced leucocyte adhesion in venules
Authors: Thorlacius H. , Vollmar B. , Guo Y. , Mak T. W. , Pfreundschuh M. M. , Menger M. D. , Schmits R.
Published: March 2003

A co‐ordinated expression of specific adhesion molecules regulates leucocyte–endothelium interactions in the microcirculation. In the present study, we used intravital microscopy of the cremaster muscle in CD11a gene‐targeted mice to examine the role of lymphocyte function antigen 1 (LFA‐1, CD11a/CD18) in tumour necrosis factor α (TNF‐α)‐induced leucocyte rolling and firm adhesion in venules. We found that 30 min after TNF‐α administration leucocyte rolling was unchanged compared with phosphate‐buffered saline (PBS)‐treated mice and similar in LFA‐1‐deficient and wild‐type animals. In contrast, firm leucocyte adhesion in venules increased by almost 10‐fold following 30 min of TNF‐α challenge in LFA‐1‐expressing animals, whereas no increase was observed in LFA‐1‐deficient mice. Four hours after intrascrotal administration of TNF‐α, venular leucocyte adhesion was found to be markedly increased, but similar in extent to LFA‐1‐deficient and wild‐type mice. Histological examination of haematoxylin–eosin‐stained tissue sections revealed that approximately 90% of the leucocytes in the TNF‐α‐stimulated venules in both wild‐type and LFA‐1‐deficient mice were polymorphonuclear. Taken together, our functional in vivo data demonstrate that LFA‐1 is an important adhesion molecule in early TNF‐α‐induced venular leucocyte adhesion.
The 4G/4G genotype at nucleotide position −675 in the promotor region of the plasminogen activator inhibitor 1 (PAI‐1) gene is less frequent in young patients with minor stroke than in controls
Authors: Endler G. , Lalouschek W. , Exner M. , Mitterbauer G. , Häring D. , Mannhalter C.
Published: March 2003

Genetic risk factors play an important role in the aetiology of vascular diseases. The insertion/deletion polymorphism (4G/5G) in the promotor region of the plasminogen activator inhibitor 1 (PAI‐1) gene has been associated with an increased risk of myocardial infarction. We investigated 136 patients with minor stroke (MS) and transient ischaemic attack (TIA) and found a prevalence of 0·32 for the 4G/4G genotype in patients compared with 0·42 in 115 age‐matched healthy controls. The 4G/4G genotype was significantly less frequent among 61 patients symptomatic before the age of 60 years (prevalence 0·20) than in 75 patients symptomatic after 60 years of age (prevalence 0·42; odds ratio). Our results indicate that the 4G/4G genotype is not a risk factor for MS or TIA and may even be protective in young patients.
Mapping of leukaemia‐associated breakpoints in chromosome band 3q21 using a newly established PAC contig
Authors: Wieser Rotraud , Volz Armin , Schnittger Susanne , Jäger Ulrich , Grüner Helga , Meran Johannes G. , Wimmer Katharina , Ziegler Andreas , Fonatsch Christa
Published: March 2003

Chromosome aberrations affecting band 3q21 are associated with a particularly poor prognosis in patients with acute myeloid leukaemia. To facilitate the molecular characterization of such rearrangements, we established a PAC contig covering the relevant genomic region. Using these PACs as probes in fluorescence in situ hybridization (FISH) experiments, we showed that a number of 3q21 breakpoints in patient samples map to a previously defined ‘breakpoint cluster region’. Others, however, are located at varying distances centromeric of it. These results have important implications in the search for genes affected by 3q21 rearrangements.
Sight‐threatening varicella zoster virus infection after fludarabine treatment
Authors: Chee Y. L. , Culligan D. J. , Olson J. A. , Molyneaux P. , Kurtz J. B. , Watson H. G.
Published: March 2003

Varicella zoster virus (VZV) infection involving the posterior segment of the eye after fludarabine treatment has not previously been described. Two patients, who had completed fludarabine treatment 3 and 18 months previously, presented with visual loss that had been preceded by a recent history of cutaneous zoster. The use of the polymerase chain reaction (PCR) for VZV DNA from ocular specimens allowed rapid confirmation of clinical diagnosis and treatment with a good outcome in one patient. With the increasing use of fludarabine and other purine analogues, an awareness of such complications is important because of their potentially sight‐threatening consequences.
High cyclin‐dependent kinase inhibitors in Bcl‐2 and Bcl‐xL‐expressing CD34+‐proliferating haematopoietic progenitors
Authors: Marone Maria , Pierelli Luca , Mozzetti Simona , Masciullo Valeria , Bonanno Giuseppina , Morosetti Roberta , Rutella Sergio , Battaglia Alessandra , Rumi Carlo , Mancuso Salvatore , Leone Giuseppe , Giordano Antonio , Scambia Giovanni
Published: March 2003

We have previously described the isolation of primitive, slow‐proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5‐6‐carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE). CFDA‐SEbright (primitive) and CFDA‐SEdim (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA‐SEbright/CD34+/slow‐proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA‐1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA‐SEbright (bright) cells, whereas they increased in CFDA‐SEdim (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C‐myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl‐xL, which followed the trend we have previously described for Bcl‐2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin‐dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.
Monitoring of residual disease and guided donor leucocyte infusion after allogeneic bone marrow transplantation by chimaerism analysis with short tandem repeats
Authors: De Weger Roel A. , Tilanus Marcel G. J. , Scheidel Karen C. , Van Den Tweel Jan G. , Verdonck Leo F.
Published: March 2003

In this study, we analysed the chimaeric status of peripheral blood leucocytes (PBLs) in recipients of allogeneic bone marrow transplantation (BMT) with the use of short tandem repeat (STR) microsatellite markers for monitoring the efficacy of BMT and donor leucocyte infusions (DLIs). A set of four STR markers was used with a highly discrimative capacity between individuals. STRs were detected by polymerase chain reaction (PCR) and were analysed by gene scanning (STR‐GS). Between June 1990 and December 1998, 52 patients treated with BMT for chronic myeloid leukaemia (CML) were analysed. Seventeen patients relapsed after BMT and two patients never achieved remission after BMT. Fourteen of the 17 patients achieved a complete donor chimaerism after BMT, as detected by the presence of only donor STR‐GS fragments, and in three cases a weak recipient STR‐GS signal remained persistently detectable after BMT. A reappearance or increase of recipient STR‐GS signals was indicative of relapse, which was mostly detected by STR‐GS several months before relapse was diagnosed clinically. Nineteen patients were treated with DLI for reappearance of CML after BMT which resulted in complete remission in 17 patients, concordant with the disappearance of recipient STR‐GS signals. More importantly, DLI treatment could be guided based upon the STR‐GS data, which prevented unnecessary extra DLI courses that could cause toxicity. This study indicates that STR‐GS is an effective and reliable method for monitoring BMT recipients.