bluuebunny/crossref_metadata_2025 huggingface dataset:
NPM/ALK gene fusion transcripts identify a distinct subgroup of null type Ki‐1 positive anaplastic large cell lymphomas
Authors: Simonitsch Ingrid , Panzer‐Gruemayer Eva Renate , Ghali Daniel W. , Zoubek Andreas , Radaszkiewicz ThaddÄus , Gadner Helmut , Kovar Heinrich
Published: November 2003

The chromosomal aberration t(2;5) resulting in the juxtaposition of NPM and ALK genes is a well‐known feature of several Ki‐1+ anaplastic large cell lymphomas (ALCL) of the T‐cell type. However, conflicting results have been reported concerning the presence of this gene rearrangement in other ALCL and Hodgkin’s disease (HD), respectively. We performed NPM/ALK RT‐PCR on 14 cases of ALCL expressing distinct myelomonocytic markers, e.g. CD11c, CD13, CD14 or CD68, but neither T‐cell nor B‐cell associated antigens (null cell phenotype). The specific translocation was found exclusively in six childhood tumours previously diagnosed as malignant histiocytosis (MH), whereas all adult lymphomas (three ALCL without characteristics of MH, three secondary ALCL following HD) and two paediatric cases of secondary ALCL following HD did not show NPM/ALK gene fusion products. By Southern blotting, the status of T‐cell receptor (TCR) and immunoglobulin heavy chain genes (IgH) were investigated; two patients with initially diagnosed MH had the TCRδ‐chain gene rearranged (Dδ2–Dδ3 and Vδ1–Jδ1, respectively). IgH rearrangements were detected in only one patient with secondary ALCL.  Our data indicate a high association of previously diagnosed MH and NPM/ALK gene rearrangements. In one case, this specific translocation was demonstrated at an early stage of development; in another, a mature TCRδ‐chain gene rearrangement was detected. These data support the hypothesis of a lymphoid origin of this subgroup of Ki‐1 positive ALCL previously diagnosed as MH.
The impact of prophylactic treatment on children with severe haemophilia
Authors: Liesner R. J. , Khair K. , Hann I. M.
Published: November 2003

Twenty‐seven children with severe haemophilia receiving regular prophylactic factor concentrate were evaluated to examine the overall effectiveness of prophylaxis in modern haemophilia care. The median age at the start of prophylaxis was 6.2 years (range 1.3–15.9 years) and the cumulative length of follow‐up was 808 months (mean 30, range 7–76 months).

Nine patients required a central venous catheter for venous access (age range 1.3–5.2 years), eight boys could cannulate themselves and in 10 the parents performed regular venepuncture. The mean dose of concentrate given at the time of study was 31.8 U/kg three times weekly (range 12.5–52.6U/kg) or 4900 U/kg/year (range 1900–8200). None developed an inhibitor on prophylaxis, though four had previously had an antibody.

The median average annual number of bleeds in the 27 patients prior to prophylaxis was 14.7 (range 3.7–35.4). On prophylaxis this fell to 1.5 (range 0–12.5) (P < 0.001) and in the group as a whole the frequency of bleeds diminished in successive years on prophylaxis.

All 20 children with evidence of arthropathy improved on prophylaxis and eight had reversal of chronic damage such that their joints appeared normal at the time of study. There were reductions in the need for walking aids, in hospital admissions, and in numbers of school days lost for bleeding episodes. All families feel that prophylaxis has brought about an improvement in quality of life.
Leukaemic CD5+ B‐cell apoptosis: co‐incidence of cell death and DNA fragmentation with reduced bcl‐2 expression
Authors: Tangye Stuart G. , Raison Robert L.
Published: November 2003

Apoptosis and bcl‐2 expression were characterized in leukaemic B cells during in vitro culture. Prior to culture, >% of cells from each B‐CLL patient expressed high levels of bcl‐2. Despite this, all leukaemic B‐cell populations underwent apoptosis in vitro. Furthermore, bcl‐2 was down‐regulated such that the B cells displayed a bcl‐2high and/or bcl‐2low phenotype. However, the overall number of bcl‐2‐positive cells remained constant. We propose that although enhanced survival of leukaemic B cells in vivo is mediated by the sustained expression of bcl‐2, it is apparent that additional mechanisms are capable of overriding the protective effect of bcl‐2 when bcl‐2 is expressed at reduced levels.
Cytogenetic remission induced by interferon‐α in a myeloproliferative disorder with trisomy 8
Authors: Petrini Mario , Simi Paolo , Saglio Giuseppe
Published: November 2003

We report a case of an unusual myeloproliferative disorder with trisomy of chromosome 8 characterized by peripheral blood thrombocythaemia and pronounced bone marrow myelodysplastic features. Treatment with interferon‐α at intermediate dosages, together with a complete bone marrow and peripheral blood haematology remission, was able to induce a cytogenetic remission, shown by the complete disappearance at the conventional cytogenetic analysis of trisomy 8 abnormality characterizing the neoplastic clone at diagnosis. However, trysomy 8 was still detectable by FISH in a small proportion of cells, showing that profound suppression of, but not complete eradication of, the neoplastic clone had occurred. The ability of interferon‐α to induce a major cytogenetic remission in a +8, Philadelphia‐chromosome negative, myeloproliferative disorder has not previously been documented and may provide further indications for the use of this drug in these disorders.
The regulation of neutrophil phospholipase A2 by granulocyte‐macrophage colony‐stimulating factor and its role in priming superoxide production
Authors: Roberts Pamela J. , Williams Sion L. , Linch David C.
Published: November 2003

Experiments were performed to investigate the relative role of phospholipase A2(PLA2) in the activation and cytokine‐mediated priming of neutrophil superoxide production. PLA2 activity was measured with a radiometric assay which discriminates between PLA2 and the downstream enzyme, 5‐lipoxygenase. In cells that had not been primed by prior incubation with granulocyte‐macrophage collony stimulating factor (GM‐CSF), PLA2 and NADPH oxidase were differentially stimulated by the chemotactic peptide N‐formyl‐met‐leu‐phe (FMLP), calcium ionophore, or phorbol ester. In addition, inhibition of PLA2 by mepacrine (0–100 μmol/l) did not concomitantly inhibit FMLP‐stimulated superoxide production. These findings suggest that the activity of PLA2 and NADPH oxidase may be uncoupled in the unprimed cell. In cells preincubated with GM‐CSF, time‐ and dose‐dependent priming of FMLP‐stimulated PLA2 responses were observed and inhibition of PLA2 by mepacrine was accompanied by the inhibition of FMLP‐stimulated superoxide production down to the level of unprimed cells. The effect of mepacrine was not due to inhibition of FMLP receptor expression. These data suggest that a mepacrine‐sensitive PLA2 may have a role in the GM‐CSF mediated priming of superoxide production. Using ionophore‐stimulated PLA2 activity as a model, we showed that Bordatella pertussis toxin did not inhibit GM‐CSF mediated priming, demonstrating that a pertussis‐sensitive GTP‐binding protein does not mediate signal transduction from the GM‐CSF receptor to PLA2. The tyrosine kinase inhibitor, genestein, selectively inhibited GM‐CSF primed but not unprimed PLA2 activity, demonstrating that GM‐CSF‐mediated priming requires tyrosine kinase activity.
Bone marrow transplantation from partially HLA‐matched related donors in adults with leukaemia: The Experience at the University Hospital of Essen, Germany
Authors: Ottinger H. , Beelen D. , Sayer H. , Schaefer U. W. , Grosse‐Wilde H.
Published: November 2003

The Seattle group has demonstrated that bone marrow transplantation (BMT) using partially HLA‐mismatched related donors is feasible in principal. However, it was unclear whether these results can also be achieved at smaller‐sized BMT units. Therefore a matched pair analysis enrolling 52 BMTs from partially HLA‐mismatched relatives and 52 control BMTs from HLA‐identical siblings was performed at the University Hospital of Essen. Overall survival (OS) and incidence of graft‐versus‐host disease (GVHD) did not differ significantly after study and control BMTs (OS: 52% v 63% 1 year, 46% v 48% 5 years post transplant; acute GVHD: 37% v 35%, chronic GVHD: 67% v 55%). After study BMTs, however, therapy‐related mortality (P = 0.018) and incidence of graft failure (P = 0.002) were increased, whereas relapse rate was reduced (11% v 27%). Two or three mismatches in HVG direction implied the same risk of graft failure as one mismatch. Therefore, (i) OS after BMT from one HLA antigen mismatched relative and from HLA‐identical siblings does not differ significantly even when performed at a ‘smaller’ centre; (ii) two or even three HLA mismatches in host‐versus graft (HVG) direction might be acceptable in family BMT for leukaemia.
Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia
Authors: Vervoordeldonk Susan F. , Merle Pauline A. , Behrendt Henk , Steenbergen Eric J. , Van Leeuwen Eleonore F. , Van Den Berg Henk , Von Dem Borne Albert E. G. Kr. , Van Der Schoot C. Ellen , Slaper‐Cortenbach Ineke C. M.
Published: November 2003

In this study we describe a fast and sensitive method using three‐colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10+CD19+ cells and CD20−CD22+ cells in the CD34+ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10–28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM samples indicated that only a subpopulation of the CD34+CD10+CD19+ and CD34+CD20−CD22+ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three‐colour immunofluorescence assay is warranted.
Serum levels of GM‐CSF are elevated in patients with thrombocytopenia
Authors: Abboud Miguel , Laver Joseph , Xu Fushen , Weksler Babette , Bussel James
Published: November 2003

Serum levels of GM‐CSF, IL‐3 and IL‐6 were measured in patients with immune thrombocytopenia (ITP), non‐immune thrombocytopenia (NIT), autoimmune haemolytic anaemia (AIHA) and neutropenia. 8/10 children with ITP had elevated serum levels of GM‐CSF (mean 18.4 pg/ml) while thrombocytopenic, but only two had detectable levels (mean 4.5 pg/ml) after normalization of the platelet count. In patients with NIT a significant inverse correlation between platelet count and serum levels of GM‐CSF was observed. IL‐3 and IL‐6 levels were not significantly elevated in thrombocytopenic patients and only two of the nine patients with either AIHA or neutropenia had detectable levels of GM‐CSF. Thus, GM‐CSF may play a role in the response to severe thrombocytopenia.
IL‐12 is involved in the activation of CD3+ granular lymphocytes in patients with lymphoproliferative disease of granular lymphocytes
Authors: Zambello Renato , Trentin Livio , Cassatella Marco , Raimondi Roberto , Cerutti Andrea , Enthammer Christine , Facco Monica , Agostini Carlo , Semenzato Gianpietro
Published: November 2003

We investigated the effects of IL‐12 on functional properties of CD3+CD8+ granular lymphocytes (GL) of patients with lymphoproliferative disease of granular lymphocytes (LDGL). To this aim, in 10 cases with a clonal CD3+ GL proliferation (nine cases with an associated TCR α/β receptor and one case with a TCR γ/δ receptor) we studied the proliferative and cytotoxic activities of resting and αCD3 monoclonal antibody (mAb) activated cells in the presence of rIL‐12 and anti‐IL‐12 blocking antibodies. Specific mRNA for IL‐12 p40 subunit was also investigated.  Our results showed that rIL‐12 increased the proliferation of αCD3 pre‐stimulated GL (2 to 6 times). Further, anti‐ IL‐12 antibodies partially inhibited αCD3‐induced cell growth, suggesting a role for this cytokine in the αCD3‐mediated GL activation. The addition of antibodies blocking the p55 and p75 chains of IL‐2 receptor (IL‐2R) did not inhibit the rIL‐12‐mediated cell proliferation, indicating that the activity of rIL‐12 is independent of IL‐2 in the in vivo expanded GL of patients under study. Concerning the cytotoxic activity, rIL‐12 increased the αCD3‐mediated NK activity against K‐562 target cells and αCD3 redirected cytotoxicity against P815 target cells. Molecular analysis pointed out that, following αCD3 stimulation, patients' GL increased the expression of specific mRNA for the p40 subunit of IL‐12 as compared to baseline conditions.  Our data indicate that IL‐12 is involved in the mechanisms of activation of clonal CD3+ GL in patients with LDGL; these features are consistent with the possibility that this discrete subset of GL might represent in vivo primed cytotoxic T lymphocytes.
Prothrombin fragment 1+2 and thrombin–antithrombin complex levels in patients with inherited APC resistance due to factor V Leiden mutation
Authors: SIMIONI P. , SCARANO L. , GAVASSO S. , SARDELLA C. , GIROLAMI B. , SCUDELLER A. , GIROLAMI A.
Published: November 2003

Inherited activated protein C (APC) resistance is a newly described pathological condition associated with familial thrombophilia. A recent report on a family with APC resistance showed increased levels of prothrombin fragment 1+2 (F1+2) in the affected individuals. No data concerning thrombin–antithrombin complex (TAT) levels in patients with inherited APC resistance are presently available.  The aim of this study was to assess the plasma levels of F1+2 and TAT in patients with inherited APC resistance due to factor V (F.V) Leiden mutation and to evaluate F1+2 and TAT levels in symptomatic and asymptomatic patients with the defect (‘carriers’) as compared to their family members having no evidence of F.V Leiden mutation (‘non‐carriers’).  One hundred and twenty‐nine individuals belonging to 30 families with inherited APC resistance due to F.V Leiden mutation were studied. F1+2 and TAT levels were determined using two commercially available ELISA kits and cut‐off values were defined as the higher limits of normal ranges obtained in healthy volunteers.  Out of the 129 family members investigated, 36 were non‐carriers, 85 were heterozygous and eight homozygous for F.V Leiden mutation. Thrombosis had occurred in 2/36 (6%) non‐carriers, in 36/85 (42.3%) heterozygous and in 5/8 (63%) homozygous. Median levels of F1+2 and TAT were above cut‐off values in carriers, whereas they were below in non‐carriers.  An overall percentage of 68.8% of carriers exhibited F1+2 levels above the cut‐off value as compared to 38.9% of non‐carriers. For TAT, an overall percentage of 63.4% of carriers presented with levels above the cut‐off compared with 28% of non‐carriers.  In conclusion, patients with inherited F.V Leiden mutation may exhibit increased levels of F1+2 and TAT. There are no differences in F1+2 and TAT median levels among symptomatic and asymptomatic carriers. The percentage of carriers of F.V Leiden with levels of F1+2 and TAT above cut‐off appears to be higher than that found in other clotting inhibitors defects and in this respect the defect might be considered different. However, these findings and the presence of a high percentage of non‐carriers presenting with increased F1+2 and TAT levels may suggest the possible coexistence in these families of other unknown defects predisposing to thrombosis.
Additional neoplasms and HCV infection in low‐grade lymphoma of MALT type
Authors: Luppi Mario , Longo Giuseppe , Ferrari Maria Grazia , Ferrara Leonardo , Marasca Roberto , Barozzi Patrizia , Morselli Monica , Emilia Giovanni , Torelli Giuseppe
Published: November 2003

Several chronic inflammatory conditions and genetic alterations are likely to be involved in the pathogenesis of low‐grade lymphoma of MALT type. In a well‐characterized series of 27 patients with low‐grade lymphoma of MALT type, we studied: (1) the incidence of other neoplasms, which might be indicative of genetic instability, apparently a characteristic of this disease; (2) the prevalence of serologic and molecular markers of HCV infection, which has been found in association with other lymphoproliferative disorders. Three patients had one or more additional cancers; a total of eight tumours, five of which occurred in the same patient, suggests the presence of some genetic instability in at least some cases of the disease. Rather unexpectedly, anti‐HCV antibodies and HCV RNA sequences were documented in 50% of the patients examined, without elevation of serum transaminases. Of interest, the two patients with parotid and conjunctival MALT lymphomas, respectively, with a previous history of Sjögren's syndrome, were HCV positive. We suggest, for the first time, that HCV may be considered, in addition to Helicobacter pylori, as another potential infectious co‐factor in the multistep pathogenesis of low‐grade lymphomas of MALT type.
Interferon‐alpha 2b (IFNα) for Early‐Phase Chronic Lymphocytic Leukaemia with High Risk for Disease Progression: Results of a Randomized Multicentre Study
Authors: Langenmayer Irmgard , Nerl Christoph , Knauf Wolfgang , Dempster Sarah , Hallek Michael , Adorf Dagmar , Dietzfelbinger Hermann , Busch Raymonde , Ziegler‐Heitbrock Hans Werner , Thiel Eckhard , Emmerich Bertold
Published: November 2003

The efficacy of interferon‐alpha 2b (IFNα) to prolong progression‐free (PFS) and/or overall survival (OS) in early B‐CLL (Binet stage A) was examined in a risk‐adapted phase III study. 99 previously untreated B‐CLL patients were recruited. 44 patients with expected high risk for disease progression, defined by non‐nodular bone marrow infiltration and lymphocyte doubling time ≤12 months or serum thymidine kinase levels ≥5 U/l, were randomized to either receive IFNα (group 1, n = 21) or not (group 2, n = 23). 55 low‐risk patients were observed to evaluate this risk stratification (group 3). During a median observation time of 36 months, four patients in the IFNα group achieved a partial remission (PR), no patient had stable disease (SD), and 17 patients experienced progressive disease (PD). The four responders had less extensive disease at study entry and tended to exhibit a rise in serum IgG levels. In group 2, no PR, seven SD and 16 PD, whereas in group 3, no PR, 37 SD and 18 PD occurred. PFS in group 1 (6.7 months) was not different from group 2 (13.3 months, P = 0.22), but PFS of groups 1 and 2 differed from group 3 (37 months, P ≤ 0.001). OS was 44.9 months (group 1), 43.1 months (group 2) and 57.9 months (group 3). OS was not significantly different for group 1 v 2, but was significant between groups 1 and 3 (P = 0.023). The higher percentage of PD in group 2 compared to group 3 (70% v 29%) shows that the selected risk factors allow the definition of CLL stage A patients at risk for disease progression within about a year. In conclusion, our data indicate that IFNα does not prolong PFS or OS in stage A CLL patients with high risk for disease progression.
Cryosupernatant as replacement fluid for plasma exchange in thrombotic thrombocytopenic purpura
Authors: ROCK G. , SHUMAK KENNETH H. , SUTTON DAVID M. C. , BUSKARD NOEL A. , NAIR RAMA C. , and the Members of the Canadian Apheresis Group
Published: November 2003

The current established treatment of thrombotic thrombocytopenic purpura (TTP) is plasma exchange with fresh frozen plasma (FFP). With this treatment, there is a 49% response after seven exchanges and a 78% survival at 1 month. Although the exact cause of TTP is unknown, the presence of von Willebrand factor (VWF) multimers has been implicated in the disease. Accordingly, it has been suggested that cryosupernatant (plasma from which cryoprecipitate has been removed), which is relatively deficient in VWF multimers, might be an effective replacement fluid during plasma exchange.

Patients from six centres were treated by plasma exchange with cryosupernatant. 18 patients who had failed a first course (average 7.7 exchanges) of plasma exchange with FFP, received a further seven exchanges with cryosupernatant. Subsequently, 40 previously untreated patients were exchanged with cryosupernatant.

Of the 18 previously treated patients, 11 responded (defined as an increase in platelet count to >150 × 109/l and no neurological events) after seven exchanges and 15 (83%) of the patients were alive at 1 month. The response rate in the 40 previously untreated patients was 75% at the end of seven exchanges and 95% of the patients were alive at 1 month. These values are significantly different (P<0.05) from those reported in our earlier study and in other patients concurrently treated at the same centres with FFP when cryosupernatant was not available.

Some patients who have failed to respond to plasma exchange with FFP replacement will respond to further exchange with cryosupernatant. Cryosupernatant replacement may be more effective as first‐line treatment of TTP than FFP.
Monocyte‐mediated killing of human leukaemia is enhanced by administration of granulocyte‐macrophage colony stimulating factor following chemotherapy
Authors: Williams Marc A. , Newland Adrian C. , Kelsey Stephen M.
Published: November 2003

We studied the capacity of recombinant human granulocyte‐macrophage colony stimulating factor (rhGM‐CSF) to modulate monocyte anti‐leukaemic activity when administered to patients following myelosuppressive chemotherapy. The leukaemic cell lines K562, U937 and KG‐1 were used as models of human leukaemia as they exhibit differential sensitivity to cell‐mediated or TNF‐mediated cytotoxicity. Monocyte tumouricidal activity was augmented by rhGM‐CSF or lipopolysaccharide (LPS) alone in vitro against leukaemic blasts, whereas granulocyte‐colony stimulating factor (rhG‐CSF) was without effect. rhGM‐CSF and LPS exhibited an additive effect in stimulating the cytotoxic effect of monocytes against K562 blasts compared with either agent alone (P < 0.001). Both cell‐mediated and soluble TNF‐mediated killing of leukaemic blasts was augmented by rhGM‐CSF administration to patients following chemotherapy. This effect persisted for up to 4 weeks after cessation of GM‐CSF therapy. The administration of rhGM‐CSF significantly increased the anti‐leukaemic activity of monocytes against leukaemic targets that were resistant to secreted TNF, probably via a transmembrane TNF‐dependent mechanism. Therapy with rhG‐CSF exhibited a minimal effect. We conclude that administration of rhGM‐CSF, but not rhG‐CSF, augments the tumouricidal properties of the monocyte‐macrophage system, particularly during recovery from myelosuppressive chemotherapy. Moreover, the killing mechanism is direct and not mediated by an antibody‐dependent cellular cytotoxic (ADCC) mechanism. Killing of TNF‐resistant leukaemic cells in particular may be augmented via cell‐to‐cell contact.
Antibodies to CD44 enhance adhesion of normal CD34+ cells and acute myeloblastic but not lymphoblastic leukaemia cells to bone marrow stroma
Authors: Bendall Linda J , Kirkness Jason , Hutchinson Amy , Bianchi Alessandra , Makrynikola Vicki , Bradstock Kenneth F , Gottlieb David J
Published: November 2003

The role of CD44 in the adhesion of haemopoietic cells to bone marrow stromal layers has not been clearly defined in humans, although its importance in the murine system has been well documented. We have demonstrated that the CD44 antibody, NIH44‐1, enhances the adhesion of haemopoietic cells to bone marrow stroma. Normal human CD34+ haemopoietic progenitors and blasts from patients with acute myeloblastic, but not lymphoblastic, leukaemia responded to NIH44‐1. All CD44 antibodies tested which bound the same epitope as NIH44‐1 also augmented haemopoietic cell adhesion to bone marrow adherent layers; however, antibodies which bound to other CD44 epitopes showed mixed responses. Augmented adhesion was independent of cell metabolism, suggesting that antibody binding resulted in direct activation of the CD44 molecule. However, hyaluronic acid was not the ligand for induced adhesion, nor could we show a role for other CD44 ligands including fibronectin, laminin, collagen or chondroitin sulphate proteoglycan. Similarly, none of the 22 CD44 antibodies tested inhibited the stimulatory effect of the NIH44‐1. Expression of CD44 was not sufficient to determine NIH44‐1 responsiveness since cell lines and leukaemic cells which failed to respond to NIH44‐1 expressed high levels of CD44. Neither CD44 isoforms nor glycosylation patterns could be identified as predictive of response. CD44 antibodies enhanced binding of normal and leukaemic haemopoietic progenitors to bone marrow fibroblasts via an unidentified stromal ligand.
Severe acquired haemophilia A treated with recombinant factor VIIa
Authors: Shafi T. , Jeha M. T. , Black L. , Douri M. Al
Published: November 2003

Acquired haemophilia can be associated with various conditions including pregnancy, autoimmune diseases and lymphoproliferative disorders, though often no underlying cause is found. It often presents with a rapid onset of muscle bleeding and involves the IgG antibody. It may be treated with human or porcine factor VIII (FVIII), prothrombin complex concentrates, factor IX (FIX) complex concentrates, factor VIIa (FVIIa) or by immunosuppression. We report a case of acquired haemophilia in a 40‐year‐old woman diagnosed following laparotomy. She was treated unsuccessfully using human FVIII and cryoprecipitate, porcine FVIII and FIX complex concentrate, before being treated with recombinant FVIIa (NovoSeven, Novo Nordisk). On treatment with recombinant FVIIa, bleeding stopped rapidly with no side‐effects and the abdominal haematoma was evacuated with minimal post‐operative bleeding.
Cellular effects of hydroxyurea in Hb SC disease
Authors: Steinberg Martin H. , Nagel Ronald L. , Brugnara Carlo
Published: November 2003

Hb S polymerizes in high‐density Hb SC disease erythrocytes. We hypothesized that hydroxyurea — perhaps independent of increasing Hb F — might reduce the density of these cells. Six Hb SC disease patients were given 1000 mg of hydroxyurea daily and blood counts, cell volumes, and cell density were followed for 12 months. There was an increase in MCV and reticulocyte MCV. A fall in absolute reticulocyte counts, high‐staining (‘stress’) reticulocytes, serum bilirubin, and an increase in the ratio of mature red cell haemoglobin to reticulocyte haemoglobin, coupled with a rise in the haematocrit inferred a decrease in haemolysis and ‘stress erythropoiesis’. An improvement in cell hydration was suggested by a reduction in reticulocyte mean cell haemoglobin concentration (CHCMr), red cells and reticulocytes with CHCM > 38, and decreased dense cell counts estimated by phthalate ester and Percoll‐Larex gradients. After 4–5 months treatment, Hb F rose in a few patients but the overall difference in pre‐ and post‐treatment Hb F was not significant. A decline in cell density should diminish the polymerization tendency of Hb S in Hb SC disease.
Pulsed high‐dose dexamethasone in chronic autoimmune haemolytic anaemia of warm type
Authors: Meyer Oliver , Stahl Dorothea , Beckhove Philipp , Huhn Dieter , Salama Abdulgabar
Published: November 2003

The management of patients with autoimmune haemolytic anaemia of warm type (AIHA) is often problematic. Recently, pulsed high‐dose dexamethasone (HDD) has been shown to be effective in the treatment of autoimmune thrombocytopenic purpura (AITP).

In this study we treated seven patients with AIHA with HDD. The regimen recommended for treatment of refractory AITP (40 mg dexamethasone for 4 d at the beginning of each 28 d cycle) was employed in almost all cases. Prior to dexamethasone administration, haemolysis was decompensated in all seven patients. HDD was well tolerated and led to an improvement of haemolysis in all cases.
Aberrant FHIT transcripts in acute myeloid leukaemia
Authors: Lin Pai‐Mei , Liu Ta‐Chih , Chang Jan‐Gowth , Chen Tyen‐Po , Lin Sheng‐Fung
Published: November 2003

Recently the FHIT gene (fragile histidine triad gene) has been identified at chromosome 3p14.2 and a high frequency of abnormalities in this gene has been demonstrated in various cancers. To determine the role of the FHIT gene in leukaemia, bone marrow or peripheral blood from 62 acute myeloid leukaemia patients and five haemopoietic cell lines (HL60, U937, Raji, KG‐1, K562) were analysed by reverse transcription of the FHIT mRNA followed by PCR amplification and sequencing of the products. To detect the deletion of the FHIT gene, 17 cases were evaluated using microsatellite polymorphism analysis. In this study, 17/62 (27%) AML patients expressed aberrant transcripts which lack two or more exons of the FHIT gene, and all the cell lines exhibited the aberrant FHIT transcripts. No cases exhibited a loss of the FHIT alleles. Our data indicated that the FHIT gene may play a role in myeloid carcinogenesis and may be indicated in the late progression of the disease.
Comparative genomic hybridization is a powerful tool, complementary to cytogenetics, to identify chromosomal abnormalities in childhood acute lymphoblastic leukaemia
Authors: Paszek‐Vigier Magda , Talmant Pascaline , Méchinaud Françoise , Garand Richard , Harousseau Jean‐Luc , Bataille Régis , Avet‐Loiseau Hervé
Published: November 2003

Cytogenetics has a strong prognostic value in childhood acute lymphoblastic leukaemia (ALL), but results are often incomplete because of the poor chromosome morphology. To improve this analysis, we tested comparative genomic hybridization (CGH) for the detection of chromosomal imbalances. 72 children were retrospectively analysed using CGH. Only 53% of the patients had been fully banded by standard methods. With CGH, 36 patients retained a normal chromosomal profile and 36 had unbalanced abnormalities. No amplification was detected. Fluorescence in situ hybridization (FISH) with centromeric and unique sequence probes was used in those cases with discrepancies or unsuccessful karyotype to validate CGH results. CGH enabled clear identification of unbalanced chromosomal abnormalities, even in some cases which had a normal karyotype. In view of the strong prognostic value of hyperdiploidy in childhood ALL, CGH appears to be a powerful technique, complementary to conventional cytogenetics.
Fibrinogen Kaiserslautern (γ 380 Lys to Asn): a new glycosylated fibrinogen variant with delayed polymerization
Authors: Ridgway Hayley J. , Brennan Stephen O. , Loreth Ralph M. , George Peter M.
Published: November 2003

An adult woman diagnosed with cerebral thrombosis following a caesarean section was found to have severely prolonged thrombin and reptilase times. Five other family members also had prolonged, but variable, thrombin and reptilase times. Analysis of purified fibrinogen on reducing SDS‐PAGE revealed an additional band, in all family members, which migrated immediately below the normal Bβ band. Western blotting indicated that this band was a gamma chain and endoglycosidase‐F digestion established that it contained an additional oligosaccharide side chain. Partial acid hydrolysis localized the new oligosaccharide to the C‐terminus of the gamma chain. Amplification of this region by PCR and subsequent DNA sequencing demonstrated a single base substitution altering the normal 380 Lys (AAG) codon to Asn (AAT), producing a new Asn‐Lys‐Thr glycosylation site. The propositus and one other family member were homozygous for this mutation but the remaining four family members were heterozygous. The polymerization of purified fibrin monomers from the propositus was grossly abnormal; however, the polymerization curve was almost normalized by the removal of terminal sialic acid residues. This suggests that the polymerization defect was primarily caused by additional negatively charged sialic acid residues present on the new oligosaccharide. Further analysis of the D domain of purified fibrinogen established that calcium binding to the high affinity site remained unaffected by the bulky carbohydrate side chain or negatively charged sialic acid residues.
Bcl‐6 gene hypermutations in diffuse large B‐cell lymphoma of primary gastric origin
Authors: Liang Raymond , Chan W. P. , Kwong Y. L. , Chan A. C. L. , Xu W. S. , Au W. Y. , Srivastava G. , Ho Faith C. S.
Published: November 2003

The presence of the bcl‐6 gene hypermutation was studied in 40 Hong Kong Chinese patients with diffuse large B‐cell lymphoma. The primary sites of involvement were nodal in 18 cases and gastric in 22. Hypermutations at the E1.11 segment of bcl‐6 gene were detectable in 16/22 (73%) primary gastric and 4/18 (22%) primary nodal lymphoma (P < 0.01). Three of the 22 cases of primary gastric but none of the nodal lymphoma had mutations at E1.12. The proportion of hypermutated cases in the group with bcl‐6 gene rearrangement was similar to that of the germ‐line group (70% v 75%). High frequency of bcl‐6 gene hypermutations was found in diffuse large B‐cell lymphoma of the stomach and they were independent of rearrangement of the gene as detected by Southern analysis. Unlike gene rearrangement, hypermutations of the bcl‐6 gene did not appear to carry any prognostic significance clinically.
Genetic analysis of 8;21 chromosomal translocation without AML1 gene involvement in MDS‐AML
Authors: Kawano Seiji , Miyanishi Setsuko , Shimizu Kimiko , Tanaka Kimio , Okumura Atsuko , Ohki Misao , Kamada Nanao , Ohno Yoh‐ichiro
Published: November 2003

We have investigated a case of acute myelocytic leukaemia derived from myelodysplastic syndrome (MDS‐AML) with an 8;21 translocation. In this case the AML1/MTG8 (ETO) fusion transcript was not detected by reverse transcriptase–polymerase chain reaction (RT‐PCR), and the rearrangement of the AML1 gene locus was not detected by Southern blot nor pulse field gel electrophoresis (PFGE) analyses using specific probes for the AML1 gene. Fluorescence in‐situ hybridization (FISH) study using cosmid probes for 21q22 revealed that the breakpoint of 21q22 was telomeric to the AML1 gene locus and centromeric from D21S259, 351, 3421 loci. This is the first report concerning the t(8;21)(q22;q22) carrying AMLs (de novo AML, MDS‐AML and therapy‐related AML) to show that the breakpoint at 21q22 is located outside the AML1 gene locus. It is also noteworthy that the cell‐surface antigen expression pattern of the bone marrow (BM) blasts was changed from CD7+CD2+CD13+CD33+CD19−CD11b+CD14+CD36+ to CD7− CD2− CD13+ CD19+ CD11b− CD14− CD33+ CD34+ CD36−CD56+ during leukaemic progression, and the pattern in leukaemic phase was similar to the characteristic phenotype of de novo AML cases with t(8;21), when the AML1/MTG8 fusion transcripts are always detected by RT‐PCR.
Malignant histiocytosis‐like B‐cell lymphoma, a distinct pathologic variant of intravascular lymphomatosis: a report of five cases and review of the literature
Authors: Murase Takuhei , Nakamura Shigeo , Tashiro Kazuhiro , Suchi Taizan , Hiraga Junji , Hayasaki Naoyuki , Kimura Masayuki , Murakami Masamoto , Mizoguchi Yoshikazu , Suzuki Takashi , Saito Hidehiko
Published: November 2003

Malignant histiocytosis (MH)‐like B‐cell lymphoma (BCL) is a neoplastic proliferation of large B cells clinically characterized by fever, hepatosplenomegaly, haemophagocytosis and abnormal laboratory data, without lymphadenopathy or skin lesions. Interestingly, most cases have been reported in Asian patients, and it is unclear whether MH‐like BCL is biologically distinct from conventional large B‐cell lymphomas. We report five Japanese patients with MH‐like BCL. Biopsied specimens of bone marrow, liver and/or spleen showed infiltration of neoplastic B cells accompanied by haemophagocytosing histiocytes. Lymphoma cells were positive for CD19, CD20 and HLA‐DR surface antigens, and negative for CD5 and CD10. In four cases elevated serum levels of interleukin (IL)‐6 and the soluble IL‐2 receptor isoform were noted, but not IL‐1β, IL‐2 or tumour necrosis factor‐α. Autopsies of two cases were pathologically diagnosed as intravascular lymphomatosis (IVL). Based on these observations, the current and nine previous cases reported as MH‐like BCL in Japan were re‐evaluated. They appear to form a peculiar variant of IVL, characterized by bone marrow involvement at presentation, haemophagocytic syndrome, and a rapidly aggressive clinical course, but rarely neurological complications or skin lesions. This variant may merit separate consideration because of the problems posed in the initial diagnosis and therapeutic approaches.
Autologous stem cell transplantation: evaluation of erythropoietic reconstitution by highly fluorescent reticulocyte counts, erythropoietin, soluble transferrin receptors, ferritin, TIBC and iron dosages
Authors: TESTA UGO , RUTELLA SERGIO , MARTUCCI ROBERT , SCAMBIA GIOVANNI , D'ONOFRIO GIUSEPPE , PIERELLI LUCA , SICA SIMONA , LUIGI PIER , PANICI BENEDETTI , MENICHELLA GIACOMO , FOTI ELVIRA , MASTROBERARDINO G. , MANCUSO SALVATORE , LEONE GIUSEPPE , PESCHLE CESARE
Published: November 2003

The plasma concentrations of erythropoietin (Ep), soluble transferrin receptors (sTfRs), iron, total iron binding capacity (TIBC) and ferritin were monitored in five leukaemia patients undergoing autologous bone marrow stem cell transplantation (BMSCT) and in 10 lymphoma and 21 ovarian cancer patients undergoing autologous peripheral blood SCT (PBSCT); 9/21 ovarian cancer patients received recombinant human G‐CSF and Ep and six recombinant human GM‐CSF and Ep following SCT. All parameters were evaluated in relation to the kinetics of erythroid reconstitution as evaluated by haemoglobin (Hb) and reticulocyte levels [including the fraction of immature reticulocytes, also called highly fluorescent reticulocytes (HFR)].

Leukaemia patients undergoing BMSCT showed only a delayed (occurring at days 35–50 after SCT) and partial RBC, neutrophil and platelet recovery, whereas all patients undergoing PBSCT exhibited a rapid (occurring at days 10–15 after SCT) and sustained haemopoietic recovery. The various levels of erythroid rescue observed among these patients markedly influenced the kinetics of the different parameters investigated: (i) in leukaemia BMSCT patients sTfRs declined following SCT and remained at low levels thereafter, whereas Ep, iron, TIBC and ferritin showed a progressive and significant increase; (ii) in the different groups of patients undergoing PBSCT: (a) sTfR levels first declined following SCT and then returned to pre‐therapy values at days 12–16, this response preceded erythropoietic recovery; (b) Ep, total iron, TIBC and ferritin showed an initial increase in the first days following SCT and then returned to pre‐therapy values.

Altogether, these observations indicate that: (i) both sTfR levels and reticulocyte counts are predictive parameters of erythropoietic recovery; (ii) coordinated changes of biochemical parameters underlying iron metabolism (iron, TIBC and ferritin) accompany erythroid rescue following SCT.
Hepatitis GB virus C genome in the serum of aplastic anaemia patients receiving frequent blood transfusions
Authors: MORIYAMA KOSEI , OKAMURA TAKASHI , NAKANO SHUJI
Published: November 2003

GB virus C (GBV‐C) RNA was detected in five of 18 patients with aplastic anaemia who had received blood transfusions, whereas it was not detected in eight patients who had not received any transfusions. Antibody against hepatitis C virus (anti‐HCV) was detected in nine patients in the transfusion group, compared with one of eight who had not received any transfusions. Therefore, the route of transmission of both GBV‐C and HCV in these patients appeared to have been multiple blood transfusion. Since all of the GBV‐C RNA‐positive patients harboured anti‐HCV, GBV‐C seems to frequently superinfect with HCV. Neither GBV‐C nor HCV is likely to have been a causative agent of the anaemia in the cases examined.
Ultrastructural analysis of the distribution of the vitronectin receptor (αvβ3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the α‐granule membrane
Authors: POUJOL CHRISTEL , NURDEN ALAN T. , NURDEN PAQUITA
Published: November 2003

The vitronectin receptor (VnR or αvβ3) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the β3 subunit in common: GP IIb–IIIa complexes (or αIIbβ3) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the αv subunit (LM142, AMF7, CLB‐706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti‐mouse IgG and a monoclonal anti‐biotin antibody provided staining enhancement. Results showed that although present on the surface, αvβ3 was mostly detected in internal membrane systems including those of α‐granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of αvβ3 was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb–IIIa, although intracellular staining was present in EM and α‐granules were again labelled.
Intrathecal release of pro- and anti-inflammatory cytokines during stroke
Authors: TARKOWSKI E , ROSENGREN L , BLOMSTRAND C , WIKKELSÖ C , JENSEN C , EKHOLM S , TARKOWSKI A
Published: November 2003

A growing body of evidence points out the potential role of inflammatory mechanisms in the pathophysiology of ischaemic brain damage. We have recently demonstrated that stroke patients display an intrathecal production of proinflammatory cytokines, such as IL-1β and IL-6 already within the first 24 h after the beginning of symptoms (Tarkowski et al., 1995). The aim of the present study was to investigate patterns of local inflammatory responses as a consequence of acute stroke. Thirty stroke patients were studied prospectively on days 0–3, 7–9, 21–26 and after day 90 with clinical evaluations, radiological assessments and analysis of cerebrospinal fluid (CSF) cytokine levels. In addition, 15 healthy control CSF samples were used. Significantly increased CSF levels of IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-10 were observed early during the stroke with a peak on day 2 for the proinflammatory cytokines IL-8 and GM-CSF, and on day 3 for the immunoregulatory cytokine IL-10. Patients with a brain infarct predominantly located in the white matter showed significantly higher levels of IL-8 in CSF than patients with an infarct mainly located in the grey matter. Also, high levels of intrathecal tumour necrosis factor-alpha (TNF-α) were associated with the presence of white matter disease. Our study demonstrates an intrathecal production of proinflammatory and immunoregulatory cytokines in patients with stroke, supporting the notion of localized immune response to the acute brain lesion. A better understanding of the inflammatory response in stroke may lead to new treatment strategies.
Modulation of hu/mu severe combined immunodeficient (SCID) mouse arthritis by local application of human recombinant IL-1β, IL-2 and IL-6
Authors: Kuhn H , Ermann J , Sack U
Published: November 2003

The contribution of interleukins produced by most inflammatory cells to chronic arthritis is not well understood. Therefore, we investigated the influence of several human recombinant interleukins (IL-1β, IL-2 and IL-6) on joint swelling, on the inflammatory process, and on serological parameters in a novel animal model of arthritis, the human/murine SCID arthritis. In this model an arthritis is induced by implanting human synovial tissue from patients with rheumatoid arthritis (RA) into the knee joint of mice with SCID. These mice tolerate the xenogeneic implant and develop a mixed human/murine pannus tissue. The interleukins were injected daily for 7 or 14 days after implantation. IL-1β led to a significant increase in joint swelling. It intensified the inflammatory process accompanied by enhanced migration of murine inflammatory cells into the knee joint. The production of human IL-6 in the transplanted tissue was stimulated through the application of IL-1β, and the serum level of human IL-6 was thus significantly higher than in controls. We could not observe a significant influence of IL-1β on the production of human IgG or IgM by the implant. The application of human IL-2 had a weak effect similar to that of IL-1β, but without statistical significance. Although IL-6 is a good marker for inflammation in RA, the application of recombined human IL-6 had no influence on the inflammatory process in this model.
IL-5 but not interferon-gamma (IFN-γ) inhibits eosinophil apoptosis by up-regulation of bcl-2 expression
Authors: Ochiai K , Kagami M , Matsumura R , Tomioka H
Published: November 2003

In order to determine regulatory mechanisms of eosinophil apoptosis, we examined the effect of recombinant IL-5 and interferon-gamma (IFN-γ) on eosinophil apoptosis and bcl-2 expression. rhIL-5 (2.5 ng/ml) significantly inhibited eosinophil apoptosis in 96 h in vitro culture compared with medium only-cultured eosinophils (89.4 ± 3.6% versus 31.3 ± 12.2% (mean ± s.d.); n = 7, P < 0.05). Further, rhIL-5 significantly increased bcl-2 protein and mRNA expression on cultured eosinophils. A phosphorothioate antisense oligonucleotide targeted at the ATG translation initiation codon of bcl-2 (10−5 m) could significantly block the supportive effect of rhIL-5 (0.25 ng/ml) for eosinophil survival compared with sense cDNA of bcl-2 on 96 h culture (inhibition rate 28.01 ± 4.56% versus 0.07 ± 1.73%; n = 4, P < 0.05). In contrast, rhIFN-γ (100 U/ml) significantly inhibited eosinophil apoptosis on 96 h in vitro culture (72.7 ± 10.5%; n = 7, P < 0.05), but did not significantly up-regulate bcl-2 protein and mRNA. These results indicate that IL-5 has inhibitory effects on eosinophil apoptosis by regulation of bcl-2 expression.