bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in the distribution of CD34 epitopes on normal haemopoietic progenitor cells and leukaemic blast cells
Authors: Steen Rita , TjØnnfjord Geir E. , Gaudernack Gustav , Brinch Lorentz , Egeland Torstein
Published: November 2003

The CD34 molecule expressed on haemopoietic progenitor cells contains a large number of epitopes whose expression may be related to the maturation or function of the cells. Monoclonal antibodies specific for different epitopes have been reported to detect different numbers of CD34+ leukaemic blast cells. We wanted to confirm this observation and study whether parallel findings could be observed for normal haemopoietic progenitor cells. The cells were immunophenotyped by flow cytometry with a series of monoclonal antibodies reactive with different CD34 epitopes. Class III epitopes (resistant to enzymatic cleavage with neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica) showed a broader distribution on normal haemopoietic progenitor cells and leukaemic blast cells than class I epitopes (sensitive to cleavage with all three enzymes) and class II epitopes (sensitive to degradation with glycoprotease and chymopapain, and resistant to neuraminidase). The subpopulation of normal progenitor cells which exclusively expressed class III epitopes had flow cytometric characteristics compatible with mature myeloid progenitor cells, whereas class I, II and III epitopes were equally expressed on cells enriched for immature subsets. No discordant epitope expression could be observed for the more immature leukaemias (AML‐M0/1) and a higher percentage of the more mature leukaemic blast cells (AML‐M3 and AML‐M4/5) expressed class III epitopes compared to the percentage expressing class I and II epitopes.

These data indicate that CD34 class III epitopes are more broadly distributed on normal haemopoietic progenitor cells and leukaemic blast cells than class I and II epitopes, and that class I and II epitopes may be down‐regulated prior to class III epitopes during normal haemopoietic progenitor cell differentiation. These findings should be considered when selecting CD34 mabs for quantification and positive selection of haemopoietic progenitor cells for research and clinical purposes.
Oligonucleotide clonospecific probes directed against the junctional sequence of t(14;18): a new tool for the assessment of minimal residual disease in follicular lymphomas
Authors: Galoin Sandrine , Al Saati Talal , Schlaife r Daniel , Huynh Anne , Attal Michel , Delsol Georges
Published: November 2003

Follicular lymphomas are often associated with t(14;18) chromosomal translocation. The rearrangement site (bcl‐2/JH junctional region) between the two chromosomes is hypervariable regarding its size and DNA sequence, and is a potential specific marker for the neoplastic clone of each patient. We report the use of the polymerase chain reaction (PCR) technique for detecting and sequencing clonal bcl‐2/JH rearrangements in lymph nodes and/or bone marrow specimens from patients with follicular lymphoma. 53 patients at diagnosis (n = 40) or at relapse (n = 13) were studied. 25 of these 53 cases were found to have the t(14;18) translocation involving either the major breakpoint region (MBR) (n=21) or the minor cluster region (mcr) (n = 4). Since our PCR technique could detect the translocation in 1/106 cells we had to distinguish mal‐ignant cells from possible t(14;18)‐bearing non‐malignant cells which could be present during and after treatment. The bcl‐2/JH junctional regions were therefore sequenced in order to synthesize an anti‐junction oligonucleotide probe specific for each patient’s malignant clone (clonospecific probe). Using these clonospecific probes for hybridization it was possible to detect one malignant cell mixed with 106 normal cells. 28 patients with advanced stage (stage III and IV), had been enrolled for treatment with myeloablative chemoradiotherapy and autologous bone marrow transplantation (ABMT). In 12 of these patients bcl‐2/MBR translocation was found at diagnosis and used as a marker to detect the presence of residual lymphoma cells in serial bone marrow (BM) and peripheral blood (PB) samples. In three relapsed patients (with available tissue samples at diagnosis and relapse), clonospecific probes clearly demonstrated the same bcl‐2/JH junction, thus confirming that the relapse occurred from the same malignant clone, and which remained stable without any clonal evolution of its junctional region throughout the course of the disease. These results demonstrate the value of the t(14;18) clonospecific probes as a diagnostic tool in the detection of minimal residual disease and relapses in patients with follicular lymphoma.
ENDOTHELIAL CELL SURFACE ACTIN SERVES AS A BINDING SITE FOR PLASMINOGEN, TISSUE PLASMINOGEN ACTIVATOR AND LIPOPROTEIN(a)
Authors: Dudani Anil K. , Ganz Peter R.
Published: November 2003

One of the mechanisms by which endothelial cells (ECs) regulate fibrinolysis is through the regulated assembly of proteins such as plasminogen, tissue plasminogen activator (tPA) and urokinase (uPA) on their membrane surface. Receptors for many of these fibrinolytic factors have been isolated and characterized. A unique 45 kD plasminogen receptor present on ECs derived from vein vasculature has been identified and resolved into two plasminogen binding components. One component consists of the unique 45 kD plasminogen receptor (pI = 6.3) whereas the other component (pI = 5.1) is identified as the cytoskeletal protein, actin. Immunofluorescent studies of isolated ECs confirm the presence of actin on their extracellular surface. This observation is consistent with a number of other recent reports of actin externally localized on other cell types. In vitro studies using purified actin confirm that plasminogen binds to actin both saturably and with relatively high affinity. Competition studies with lysine indicated that the binding was largely kringle‐dependent, and when binding of tPA to actin was assessed, it also bound to actin with 70–80% of binding inhibited by lysine. Lipoprotein(a), which shows homology with plasminogen, also interacted with actin. Addition of plasminogen and low‐density lipoproteins inhibited Lp(a) binding to actin in a dose‐dependent fashion. Moreover, in competition with tPA, partial inhibition of plasminogen binding to actin was also observed. In experiments using anti‐actin antibodies added in excess to cultured ECs, binding of plasminogen was inhibited by 45%, tPA binding was inhibited by 46% and Lp(a) binding was reduced by 56%, confirming actin as a binding site for these various ligands whilst attesting to the presence of other EC receptors for these proteins. Collectively, the data presented are consistent with actin playing a major role in localizing binding not only of plasminogen, but also of tissue plasminogen activator and Lp(a) to the surface of human endothelial cells.
Soluble L‐selectin in the connective tissue diseases
Authors: Blann A. D. , Sanders P. A. , Herrick A. , Jayson M. I. V.
Published: November 2003

Plasma levels of the leucocyte adhesion molecule L‐selectin were measured by ELISA in 41 patients with rheumatoid arthritis, 18 with ankylosing spondylitis, 18 with systemic sclerosis and 27 with vasculitis together with 42 age‐ and sex‐matched controls. Low levels of soluble L‐selectin were found in systemic sclerosis (797 ± 302 ng/ml, P < 0.05) and vasculitis (941 ± 329 ng/ml, P < 0.05) relative to controls (1244 ± 269 ng/ml). The exact reasons for low levels of soluble L‐selectin are unclear, but may reflect reduced shedding from leucocytes and/or strong binding to its cell membrane ligand(s). An approximate inverse relationship between soluble L‐selectin and disease severity may have clinical relevance.
Detection of microsatellite markers in leukaemia using DNA from archival bone marrow smears
Authors: Pabst Thomas , Schwaller Jürg , Tobler Andreas , Fey Martin F.
Published: November 2003

We describe a simple PCR method to detect highly polymorphic repetitive DNA markers (microsatellites) in leukaemia using DNA from fresh and archival bone marrow smears. Neither the period of storage nor staining of the slides affected microsatellite PCR. Comparison of slide DNA with constitutive DNA from buccal epithelium revealed loss of heterozygosity (LOH) with at least one marker in four of 11 leukaemias. Marrow smears offer an attractive archival source of DNA for the detection of microsatellite markers. Applications include detection of LOH or tracing the origin of cell populations, e.g. in bone marrow transplant recipients.
Fludarabine, as well as 2‐chlorodeoxyadenosine, can induce eosinophilia during treatment of lymphoid malignancies
Authors: LA¨rfars Gerd , Udén‐Blohmé Ann‐Mari , Samuelsson Jan
Published: November 2003

We treated 25 patients suffering from low‐grade lymphoid malignancies with nucleoside analogues. In 11 patients undergoing treatment with fludarabine, two patients with follicular lymphoma developed a marked absolute peripheral blood eosinophilia devoid of clinical symptoms which resolved without medical intervention. Of 15 patients treated with 2‐chlorodeoxyadenosine, one patient with B‐cell chronic lymphocytic leukaemia (CLL) experienced repeated episodes of eosinophilia that required steroid treatment. Eosinophilia in these three patients was not coupled to tumour lysis, and we found no correlation between the occurrence of eosinophilia and clinical response to treatment. Thus, for the first time, we can report eosinophilia as a side‐effect of fludarabine treatment, and these are also the first published cases of this complication occurring in patients treated with nucleoside analogues for follicular lymphoma and CLL.
Expression of the nerve growth factor receptor c‐TRK in human myeloid leukaemia cells
Authors: Kaebisch Andreas , Brokt Stefanie , Seay Ulrike , Lohmeyer Juergen , Jaeger Ulrich , Pralle Hans
Published: November 2003

Nerve growth factor (NGF) is of major importance for the survival, development and maintenance of peripheral sympathetic and central neuronal tissue. Most of the cellular effects are mediated by binding to their high‐affinity receptor c‐TRK, a transmembrane receptor tyrosine kinase. C‐TRK protein has been detected in neuronal tissue and also in mast cells, monocytes and some haemopoietic progenitor cells. Here we report c‐TRK gene expression in myeloid leukaemic cell lines (HEL, K562 and KG‐1) and for the first time in the primary leukaemic cells of 44% (n = 59) of patients with acute myelogenous leukaemia (AML). Moreover, in the human promyelocytic cell line HL‐60, c‐TRK expression was inducible by differentiation induction with tetradecanoylphorbol 13‐acetate (TPA). In c‐TRK gene‐expressing cells the transmembrane receptor tyrosine kinase was detectable by Western blotting and by in vitro kinase assay.  In the AML group, c‐TRK expression was not correlated to the FAB‐classified morphology or any other clinical parameter. In all cases tested we could not detect NGF mRNA by means of reverse transcriptase PCR, excluding an autocrine loop involving the TRK/NGF receptor–ligand system in leukaemogenesis. Our results show another example of possible communication between neuronal and haemopoietic tissue. However, we still lack positive evidence of a c‐TRK function in haemopoiesis.
A nonsense mutation in the GPIIb heavy chain (Ser 870 → stop) impairs platelet GPIIb–IIIa expression
Authors: VINCIGUERRA C. , KHELIF A. , ALEMANY M. , MORLE F. , GRENIER C. , UZAN G. , GULINO D. , DECHAVANNE M. , NEGRIER C.
Published: November 2003

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder, caused by a quantitative or qualitative defect of the GPIIb–IIIa integrin (αIIbβ3), which functions as the platelet fibrinogen receptor. We report a case of type I GT due to a homozygous mutation resulting in Ser 870 to stop codon substitution. This residue is located near the proteolytic cleavage site of proGPIIb. The mutation results in a GPIIb truncated of 138 amino acids, including transmembrane and intracytoplasmic domains. Cotransfection of an expression vector containing the mutant GPIIb and wild‐type GPIIIa showed that the mutant Ser 870 → stop GPIIb was able to associate to GPIIIa. However, this heterodimer failed to mature as shown by endoglycosidase‐H digestion and was therefore not expressed at the COS‐7 cell surface. This report is the first description of a homozygous nonsense mutation in the GPIIb gene and highlights the role of the GPIIb light chain.
Treatment of high‐risk myelodysplastic syndromes with lymphoblastoid alpha interferon
Authors: Petti M. C. , Latagliata R. , Avvisati G. , Aloe Spiriti M. A. , Montefusco E. , Spadea A. , Mandelli F.
Published: November 2003

Seventeen patients with high‐risk myelodysplastic syndromes (HR‐MDS) received lymphoblastoid‐interferon alpha (Ly‐IFNα) for 3 months at escalating doses from 0.5 to 3 MU s.c. 3 times per week. Three patients stopped the treatment after 2 months because of cardiac failure (one patient) and cerebral haemorrhage (two patients); six had a partial response (PR) and continued Ly‐IFNα; six were resistant and stopped Ly‐IFNα; two evolved to acute myelogenous leukaemia (AML). Among the six partial responders, four achieved a complete response (CR) during subsequent Ly‐IFNα treatment (CR duration 3, 4+, 15+ and 29 months) and two did not achieve any further improvement (PR duration 3 and 9 months). Two resistant patients had an unexpected clinical improvement soon after Ly‐IFNα discontinuation and achieved a PR of 6+ and 14+ months respectively. No toxicity related to Ly‐IFNα treatment was observed and no reduction of dosage was needed. In conclusion: (1) Ly‐IFNα seems to be effective in some patients with HR‐MDS; (2) the best treatment duration seems to be of at least 6 months.
Circulating endothelial cell/leucocyte adhesion molecules in ischaemic heart disease
Authors: BLANN ANDREW D. , AMIRAL JEAN , MCCOLLUM CHARLES N.
Published: November 2003

Increased levels of the endothelial markers soluble E‐selectin (P = 0.011), soluble thrombomodulin (P = 0.0001) and von Willebrand factor (VWF, P < 0.0001) were found in 116 patients with ischaemic heart disease compared to an equal number of age‐ and sex‐matched asymptomatic controls. In a multivariate analysis of the markers versus the major risk factors for atherosclerosis, VWF correlated with total cholesterol (P = 0.002) and E‐selectin with sex (lower in women, P = 0.004) and triglycerides (P = 0.007). The data point to profound differences in the release mechanisms of these three endothelial cell products and suggests that further studies into the roles of these molecules in coronary artery disease are warranted.
Inhibition of the acute‐phase response in vivo by anti‐gp130 monoclonal antibodies
Authors: Harrison Paul , Downs Tamyra , Friese Paul , Wolf Roman , George James , Burstein Samuel
Published: November 2003

The acute‐phase response is believed to be an important systemic defence reaction to inflammation during infection, trauma, injury or neoplasia. Although the interleukin‐6 (IL‐6) family of cytokines appear to be the major regulators of the acute‐phase reaction, the exact biological significance of this process remains unknown. In this study, a panel of monoclonal antibodies (Mabs) was raised against the extracellular domain of human gp130 (the common signal transducing chain of the IL‐6 cytokine family) in order to inhibit the biological activity of IL‐6‐like cytokines in vivo. Mabs designated 4B11 and 2H4 were most effective in the inhibition of the in vitro acute‐phase response on hepatoma cells and prevented the IL‐6‐induced growth inhibition of A375 cells. Administration of the antibodies to dogs at a dosage of 8 mg/kg/d showed that 2H4 was a potent inhibitor of the IL‐6‐induced (40 μg/kg/d) acute‐phase response, abrogating IL‐6‐mediated increments in fibrinogen, C‐reactive protein and the platelet count. This antibody, the first described to abrogate the acute‐phase response in vivo, may not only permit development of a new anti‐inflammatory strategy, but provides an excellent tool for defining the function of acute‐phase proteins in inflammation and infection.
Reversible bone marrow depression by high‐dose piperacillin/tazobactam
Authors: RUIZ‐IRASTORZA GUILLERMO , BARREIRO GUILLERMO , AGUIRRE CIRIACO
Published: November 2003

Bone marrow toxicity, mainly neutropenia, has been described as an uncommon secondary effect of most beta‐lactams, and it is usually related to large cumulative doses. Although previously described for piperacillin, no cases of marrow depression caused by piperacillin/tazobactam have been reported to date. We report a case of reversible pancytopenia, with evidence of bone marrow depression, which occurred after a 17 d course of piperacillin/tazobactam. The drug was given to an underweight 18‐year‐old woman, at the usual dosage of 4/0.5 g three times a day. We stress the need for reducing the recommended dosage when treating underweight adult patients, and also of monitoring haematological parameters during prolonged treatments.
Thrombocytosis in patients with acute promyelocytic leukaemia during all‐trans retinoic acid treatment
Authors: Losada Rafael , Espinosa Edgardo , Hernãndez Carlos , Dorticos Elvira , Hernãndez Porfirio
Published: November 2003

In 26 acute promyelocytic leukaemia (APL) patients treated with all‐trans retinoic acid (ATRA), 23% had platelet counts between 459 and 800 ×109/l during treatment. These values, observed between days 28 and 45 of ATRA treatment, were transient and asymptomatic. We report two APL cases with platelet counts >1000 × 109/l during ATRA therapy who were treated with recombinant interferon alpha. In both cases ATRA doses were not modified, no complications secondary to thrombocytosis were seen, and they subsequently achieved complete remission. It is suggested that IL‐6 may play an important role in the pathogenesis of the thrombocytosis induced by ATRA. To our knowledge, this is the first report of thrombocytosis occurring during ATRA treatment.
Transplantation of peripheral blood progenitor cells from HLA‐identical sibling donors
Authors: Schmitz Norbert , Bacigalupo Andrea , Labopin Myriam , Majolino Ignazio , Laporte Jean‐Philippe , Brinch Lorentz , Cook Gordon , Deliliers Giorgio Lambertenghi , Lange Andrzej , Rozman Ciril , Garcia‐Conde Javier , Finke Jürgen , Domingo‐Albos Andrieu , Gratwohl Alois
Published: November 2003

Transplantation of peripheral blood progenitor cells (PBPCs) has largely replaced autologous bone marrow transplantation. The same might occur in the allogeneic setting if the favourable initial experience with allogeneic PBPCT is confirmed. We analysed all primary transplants utilizing unmodified PBPC from HLA‐identical sibling donors reported to the European Group for Blood and Marrow Transplantation (EBMT) for 1994. 59 patients with a median age of 39 years received myeloablative therapy for acute myelogenous leukaemia (23 patients), acute lymphoblastic leukaemia (13), chronic myelogenous leukaemia (nine), lymphoma (seven), or other diagnoses (seven) mostly of advanced stages followed by transplantation of allogeneic PBPC.

Three patients died soon after grafting, the others showed prompt haemopoietic recovery with median times to recover an absolute neutrophil count (ANC) above 0.5 and 1.0×109/l of 15 (range 9–27) and 17 d (range 10–28) respectively. Time to platelet recovery above 20 or 50×109/l was 16 (range 9–76) and 18 d (range 12–100) respectively. 27 patients (46%) developed no or mild acute graft‐versus‐host disease (GVHD). The incidence of moderate (grade II) disease was 27%; 24% of the patients developed severe acute GVHD (grades III or IV). 55% of patients who were alive 90 d after transplantation developed chronic GVHD, the probability to develop extensive chronic GVHD was 32% (95% confidence interval 22–42) with a median follow‐up of 14 months. Overall and event‐free survival (EFS) at 1 year were 54% (CI 48–60) and 50% (CI 43–57), respectively, the relapse incidence was 23% (CI 17–29). EFS was 67% (CI 55–79) in patients transplanted for acute leukaemias in first complete remission, chronic myelogenous leukaemia in first chronic phase, or severe aplastic anaemia.

Transplantation of allogeneic PBPC resulted in prompt and durable engraftment. The incidence and severity of acute and chronic GVHD seemed comparable to that observed after allogeneic BMT. Overall and event‐free survival in this cohort of patients, most of whom suffered from advanced leukaemia or lymphoma, is encouraging, suggesting that the high numbers of T lymphocytes and/or natural killer cells contained in a typical PBPC collection product exert a vigorous graft‐versus‐leukaemia effect. Further evaluation of allogeneic PBPCT is highly desirable.
Pathogenesis of chronic Epstein‐Barr virus infection: detection of a virus strain with a high rate of lytic replication
Authors: Jä ger Michael , Prang Nadja , Mitterer Manfred , Larcher Clara , Huemer Hartwig P. , Reischl Udo , Wolf Hans , Schwarzmann Fritz
Published: November 2003

In rare cases Epstein‐Barr virus (EBV) leads to chronic active infection (CAEBV) which is characterized by persistant symptoms of infectious mononucleosis. Previously we described a case of persisting polyclonal B‐cell lymphocytosis (PPBL) that was associated with CAEBV. Using reverse transcription and polymerase chain reaction we showed that in late passages of a spontaneous cell line, SM, latent EB viral genes such as EBNA1, EBNA2, EBNA3A/3B/3C, LMP1 and LMP2A were active. The master gene of the lytic cycle, BZLF1, was silent. This indicated that there was no general defect in immortalization and establishing latency by this CAEBV isolate SM. We obtained virus from the standard immortalizing strain B95‐8 and the CAEBV strain SM from latently infected LCL, quantified the number of virus particles by competitive PCR and demonstrated that the impaired capacity to immortalize umbilical cord blood lymphocytes was a virus strain‐specific property, and was not due to an incapability to infect purified CD19+ B lymphocytes. Transcription of latency‐ and immortalization‐ associated genes such as EBNA1, EBNA2 and LMP2A was reduced, in contrast to a strongly enhanced activity of the master gene of the lytic cycle, BZLF1. A scenario for an antagonistic regulation of lytic and latent cycle genes is presented and a role for the pathogenesis of CAEBV is discussed.
Biochemical, histomorphometric and densitometric changes in patients with multiple myeloma: effects of glucocorticoid therapy and disease activity
Authors: Diamond T. , Levy S. , Day P. , Barbagallo S. , Manoharan A. , Kwan Y. K.
Published: November 2003

It is unknown whether bone changes which can occur in multiple myeloma (MM) are due to cytokine‐induced osteoclastic bone resorption from a clone of abnormal plasma cells or high‐dose glucocorticoid therapy.

We studied 25 MM patients treated for 1–12 years with combination chemotherapy, subdivided into two groups. Group 1 consisted of 12 patients with stage I and II myeloma and group 2 consisted of 13 patients with stage III MM. Their serum biochemistry, tetracycline‐labelled bone histomorphometry and bone densitometry were compared to age‐ and sex‐matched controls.

Patients with MM demonstrated increased indices of bone resorption (P<0.001 versus controls) and, to a lesser extent, increased indices of bone formation (P<0.01 versus controls). No patient had evidence of a mineralization defect.

Lumbar spine, femoral neck and total body bone mineral density measurements (BMD) were significantly lower in group 2 compared with group 1 (P<0.05). Following 12 months of therapy, lumbar spine BMD decreased by 6.6% (95% CI, 2.7% to −9.3%) and femoral neck BMD decreased by 9.5% (95% CI, −3.2% to −15.9%). In a stepwise regression analysis, cumulative prednisolone dosage (B Coef.=−0.39; P=0.03) and plasma cell infiltrate (B Coef.=−0.08; P=0.05) were the most important predictors of lumbar spine bone loss, whereas serum paraprotein (B Coef.=−0.35; P=0.02) and plasma cell infiltrate (B Coef.=−0.20; P=0.04) were the most important predictors of femoral neck bone loss.

We conclude that MM is characterized by high bone turnover with osteoblast–osteoclast uncoupling. Both disease activity and high‐dose glucocorticoid therapy may be responsible for the ongoing bone loss seen with MM.
EPIDEMIOLOGY OF COAGULATION FACTORS, INHIBITORS AND ACTIVATION MARKERS: THE THIRD GLASGOW MONICA SURVEY I. ILLUSTRATIVE REFERENCE RANGES BY AGE, SEX AND HORMONE USE
Authors: Lowe G. D. O. , Rumley A. , Woodward M. , Morrison C. E. , Philippou H. , Lane D. A. , Tunstall‐Pedoe H.
Published: November 2003

Coagulation factor activity (fibrinogen, VII, VIII and IX), coagulation inhibitor activity (antithrombin, protein C, protein S), and coagulation activation markers (prothrombin fragment F1,  2; thrombin–antithrombin complexes) were measured in 747 men and 817 women aged 25–74 years, randomly sampled from the north Glasgow population in the Third MONICA Survey. Significant effects of age, sex, menopause and hormone use were observed and specific reference ranges are presented to illustrate these effects. Significant correlations were observed between several coagulation factors and inhibitors. Increased levels of factors VII, VIII and IX and decreased levels of protein C were associated with increased coagulation activation. In general, increases in coagulation factors with age were greater than increases in coagulation inhibitors, especially in men; this imbalance may favour increased coagulation activation and hence increased thrombotic risk with age.
PREVALENCE AND CLINICAL SIGNIFICANCE OF HEPATITIS G VIRUS INFECTION IN ADULT BETA‐THALASSAEMIA MAJOR PATIENTS
Authors: Sampietro Maurizio , Corbetta Noemi , Cerino Mario , Fabiani Paola , Ticozzi Anna , Orlandi Anna , Lunghi Giovanna , Fargion Silvia , Fiorelli Gemino , Cappellini Maria Domenica
Published: November 2003

The risk of polytransfused patients for hepatitis C virus (HCV) infection is likely to extend to another recently identified member of the Flaviviridae, hepatitis G virus (HGV). We investigated the prevalence of HGV in 40 adult Italian patients with transfusion‐dependent thalassaemia and evaluated the clinical significance of HGV infection. HGV‐RNA was detected in 9/40 patients (22.5%). HGV infection was significantly associated with HCV viraemia (P =0.0012), with all patients positive for HGV being also viraemic for HCV. Overall, the clinical picture of patients with HCV/HGV co‐infection was not different from that of patients with isolated HCV. However, patients co‐infected with both viruses had lower values of alanine‐transferase (P =0035) and a lower titre of HCV viraemia (P =0042) in the absence of other evident factors which could influence the clinical expression of HCV infection. In conclusion, HGV is highly prevalent among Italian polytransfused patients. No evidence of a clinically significant pathogenic role for HGV in liver disease could be found in these patients. In a subset of cases a possible interference of HGV with HCV infection was observed.
In‐vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with β‐thalassaemia major
Authors: Ruf Andreas , Pick Marjorie , Deutsch Varda , Patscheke Heinrich , Goldfarb Ada , Rachmilewitz Eliezer A. , Guillin Marie C. , Eldor Amiram
Published: November 2003

Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with β‐thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p‐selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual‐colour flow cytometry. A significantly (P < 0.01) higher fraction of FITC‐annexin V‐labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation (P < 0.001) was found in TM patients between the number of RBC‐bound annexin V molecules and the fraction of CD62p (p‐selectin) or CD63‐positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelarate thrombin generation in vivo which, in turn, triggers platelet activation.
Molecular characterization of G6PD deficiency in Southern Italy: heterogeneity, correlation genotype–phenotype and description of a new variant (G6PD Neapolis)
Authors: Alfinito Fiorella , Cimmino Amelia , Ferraro Filomena , Vittoria Cubellis Maria , Vitagliano Luigi , Francese Matteo , Zagari Adriana , Rotoli Bruno , Filosa Stefania , Martini Giuseppe
Published: November 2003

We report on the molecular basis of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in Southern Italy (Campania region). Thirty‐one unrelated G6PD‐ deficient males were analysed at DNA level for the presence of G6PD gene mutations. Nine different G6PD variants were identified, eight of which have already been described (Mediterranean, Seattle, two different A−, Santamaria, Cassano, Union and Cosenza). G6PD Mediterranean, Santamaria, A− and Union were associated with haemolytic episodes. G6PD Seattle, which is polymorphic in several populations, Cassano and Cosenza appeared to be asymptomatic. A new variant (G6PD Neapolis) is reported here. The 467Pro→Arg substitution reponsible for G6PD Neapolis is discussed in the light of the current 3D model of human G6PD and in comparison with other natural mutations which occur in the proximity of residue 467.
Spleen sizing by ultrasound in polycythaemia and thrombocythaemia: comparison with SPECT
Authors: Messinezy M. , MacDonald L. M. , Nunan T. O. , Westwood N. B. , Chinn S. , Pearson T. C.
Published: November 2003

Detection of non‐palpable early splenic enlargement may aid diagnosis of primary polycythaemia (PP) and primary thrombocythaemia (PT). In this study linear spleen sizing by ultrasound has been compared with spleen volume estimation by single photon emission computerized tomography (SPECT) in 26 patients. Spleen length by ultrasound correlated well with SPECT volume estimation.

Ultrasound spleen length was also measured in 60 normal control subjects where the upper limit of the 95% reference range was 11.6 cm. Changes in spleen length with both age and body weight were substantial and overshadowed the imperfect reproducibility of this method. Therefore, interpretation of an individual's measured spleen length should be in relation to that predicted for adults of the same age and weight, particularly at the extremes of the younger, heavier patients and also the older, lighter patients.

Ultrasound spleen lengths of different patient groups (21 PP, 26 PT, 17 idiopathic erythrocytosis, 12 secondary polycythaemia, nine apparent polycythaemia) were compared both using the measured overall reference range and the differences from the values predicted for their age and weight. The comparison showed that almost all patients with PP whose spleens were not palpable had spleen lengths greater than the upper limit for the normal control group, but separation from the other patient groups was incomplete.

Detection of non‐palpable splenomegaly by ultrasound length should remain a ‘minor’ criterion amongst the ‘proposed modified diagnostic criteria’ of PP.
Microsatellite instability during the progression of acute myelocytic leukaemia
Authors: Tasaka Taizo , Lee Stephen , Spira Susanne , Takeuchi Seisho , Nagai Masami , Takahara Jiro , Koeffler H. Phillip
Published: November 2003

We studied microsatellite instability (MSI) at the onset and during progression of 17 individuals with acute myelocytic leukaemia (AML). These included two cases of M0, eight with M1 and seven with M4, according to the FAB classification. The DNA from diagnostic, remission and relapsed stages of their disease was analysed at 69 loci. Two MSI were found in the diagnostic and remission phase paired samples (12%), and eight MSI were identified in six of the relapsed phase samples (35%). These results indicate that mismatch repair errors such as MSI are unimportant at the onset of AML, but might have importance during the progression of the disease.
Further evidence for the importance of an androgen response element in the factor IX promoter
Authors: MORGAN GARETH E. , ROWLEY GABRIELLA , GREEN PETER M. , CHISHOLM MORAG , GIANNELLI FRANCESCO , BROWNLEE GEORGE G.
Published: November 2003

Previous work involving the characterizing of a factor IX promoter mutation (−26 G → C) of a 21‐year‐old patient with severe haemophilia B suggested that an androgen response element (ARE) was present in the wild‐type factor IX promoter but was disrupted in this patient. However, other theories not involving this ARE have been suggested for the mechanism of recovery in the more typical (Leyden) promoter patients, so that the ARE hypothesis requires further evidence if it is to be accepted. We now present a case history and functional data on another 48‐year‐old severe haemophilia B patient (UK232) with a different (G → A) mutation at the same position, −26. This mutation impairs transactivation of the minimal factor IX promoter region (−220 → +43) by HNF4 in transient transfection experiments in HepG2 and HeLa cells. It disrupts binding of both androgen receptor (AR) and HNF4 to oligonucleotides spanning this region (−40 → −9) in competition gel mobility shift assays. It impairs AR/testosterone transactivation of these oligonucleotides (−40 → −9) when tetramerized upstream of a CAT reporter gene in cotransfection assays in HeLa cells. And, finally, no clinical recovery has occurred since puberty. These results strengthen the evidence for the importance of nucleotide −26, both for the normal transcription of the gene in response to HNF4 and for the proposed Leyden recovery mechanism in response to AR and testosterone acting directly through the factor IX ARE.
The effect of intravenous iron on the reticulocyte response to recombinant human erythropoietin
Authors: Major Attila , Mathez‐Loic Fabien , Rohling Roman , Gautschi Kurt , Brugnara Carlo
Published: November 2003

We studied the effect of intravenous (i.v.) adminisration of 200 mg of iron sucrose following an i.v. bolus injection of recombinant human erythropoietin (r‐HuEPO; 300 U/kg body weight) in seven subjects and compared it with seven subjects treated with r‐HuEPO alone. Reticulocytes, serum erythropoietin (EPO) and ferritin levels were studied at baseline and daily for the following 8 d. Use of i.v. iron abolished the marked reduction in serum ferritin observed with r‐HuEPO administration. Although the total number of reticulocytes was not affected by i.v. iron administration, the reticulocyte Hb content and retHb (a measure in g/l of the Hb contained in all reticulocytes) were increased in the i.v. iron/r‐HuEPO group compared with the group who received r‐HuEPO alone. Therefore i.v. iron significantly potentiates the haemopoietic response to r‐HuEPO in normal subjects.
Epstein‐Barr virus associated B‐cell lymphoma after autologous bone marrow transplantation for T‐cell acute lymphoblastic leukaemia
Authors: Briz Montserrat , Forés Rafael , Regidor Carmen , Busto Maria‐José , Ramón y Cajal Santiago , Cabrera Rafael , Díez Jose‐Luis , Sanjuán Isabel , Fernández Manuel‐Nicolás
Published: November 2003

Epstein‐Barr virus associated lymphoproliferative disease after autologous bone marrow transplantation (ABMT) has rarely been reported. We report a case of B‐cell lymphoma following ABMT for T‐acute lymphoblastic leukaemia; bone marrow was purged in vitro with monoclonal antibodies to remove T cells. Immunoglobulin and T‐cell receptor gene rearrangement studies were used to demonstrate clonality and to show that this patient developed a second neoplasm after ABMT. EBV proteins and genome (type A) were present in post‐transplantation lymphoma, suggesting a causative role in its development.
Assessment of intracellular TAP‐1 and TAP‐2 in conjunction with surface MHC class I in plasma cells from patients with multiple myeloma
Authors: Crucian Brian E. , Moscinski Lynn C. , Androlewicz Matthew , Ballester Oscar F. , Widen Raymond H. , Yu Hua
Published: November 2003

Immunotherapy involving cytotoxic T lymphocytes (CTLs) is an attractive alternative for treatment of various malignancies, including multiple myeloma. For tumour cells to be recognized and killed by CTLs they must express cell surface major histocompatibility complex (MHC) class I molecules and the transporter associated with antigen processing (TAP). However, loss of MHC class I and the TAP protein are common among several types of solid tumours. This study assessed the expression of TAP protein (by intracellular flow cytometry) and cell surface MHC class I molecules in three human myeloma cell lines as well as the plasma cell population (CD38+ bright) in bone marrow specimens from 13 multiple myeloma patients. In all of the patients, 100% of the plasma cell population expressed both the TAP subunits and cell surface MHC class I molecules, but at varying intensities. Both TAP and MHC class I were also expressed in the three myeloma lines. Additionally, the function of the antigen transport machinery was evaluated by a peptide transporter assay in the three myeloma lines. TAP transporter activity was readily detectable in two out of three myeloma lines, whereas the diminished activity in the third cell line was completely restored by co‐culturing with recombinant interferon‐γ (rIFN‐γ).
Thrombopoietin alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum‐free culture
Authors: Yoshida Makoto , Tsuji Kohichiro , Ebihara Yasuhiro , Muraoka Kenji , Tanaka Ryuhei , Miyazaki Hiroshi , Nakahata Tatsutoshi
Published: November 2003

We examined the effects of recombinant human thrombopoietin (TPO, c‐Mpl ligand) on the proliferation and differentiation of human haemopoietic progenitors other than megakaryocytic progenitors using serum‐free cultures. TPO alone supported the generation of not only megakaryocytic (MK) but also blast cell (blast) colonies from cord blood CD34+ cells. Delayed addition of a cytokine cocktail (cytokines; interleukin (IL)‐3, IL‐6, stem cell factor, erythropoietin, granulocyte‐macrophage colony‐stimulating factor, and TPO) to cultures with TPO alone on day 7 induced various colonies including granulocyte‐macrophage (GM) colonies, erythroid bursts (E), granulocyte‐erythrocyte‐macrophage‐megakaryocyte (GEMM) colonies. Replating experiments of blast colonies supported by TPO alone for culture with cytokines revealed that approximately 60% of the blast colonies contained various haemopoietic progenitors. Single cell cultures of clone‐sorted CD34+ cells indicated that TPO supported the early proliferation and/or survival of both primitive and committed haemopoietic progenitors. In serum‐free suspension cultures, TPO alone significantly stimulated the production of progenitors for MK, GM, E and GEMM colonies as well as long‐term culture‐initiating cells. These effects were completely abrogated by anti‐TPO antibody. These results suggest that TPO is an important cytokine in the early proliferation of human primitive as well as committed haemopoietic progenitors, and in the ex vivo manipulation of human haemopoietic progenitors.
A novel form of hereditary sideroblastic anaemia with macrocytosis
Authors: Tuckfield A. , Ratnaike S. , Hussein S. , Metz J.
Published: November 2003

We describe a pedigree with maternally inherited sideroblastic anaemia in which the red cells are dimorphic with a raised MCV. To our knowledge, this form of hereditary sideroblastic anaemia (HSA) has not been reported previously. 16 members of the family were investigated, revealing eight affected members. Two further family members were not tested but were presumed affected on the histories available. The proband, born in 1967, presented during pregnancy with a macrocytic anaemia (Hb 7.0 g/dl, MCV 106 fl) and a dimorphic red cell picture. Post partum, a bone marrow biopsy showed hypercellularity, mild dyserythropoiesis and ring sideroblasts. Cytogenetics were normal. Other causes of macrocytosis were excluded. Six other family members (three female, three male) have similar findings. There is no evidence of paternal transmission. An additional female relative who presented in 1992 with refractory anaemia with excess blasts in transformation and a dimorphic blood film, died from progression to AML. Affected members show a raised metal‐free red cell protoporphyrin level suggestive of a defect at the level of Fe2+ incorporation into protoporphyrin. We propose that this form of HSA is due to a mitochondrial mutation. A search for deletions or point mutations in the mitochondrial DNA is currently underway.
Short‐ and long‐term follow‐up of thyroid dysfunction after allogeneic bone marrow transplantation without the use of preparative total body irradiation
Authors: TOUBERT Marie‐Elisabeth , SOCIÉ Gérard , GLUCKMAN Eliane , ARACTINGI Sélim , ESPÉROU Hélène , DEVERGIE Agnès , RIBAUD Patricia , PARQUET Nathalie , SCHLAGETER Marie‐Hélène , BERESSI Jean‐Paul , RAIN Jean‐Didier , VEXIAU Patrick
Published: November 2003

We studied the incidence and potential prognostic value of thyroid abnormalities after allogeneic bone marrow transplantation (BMT) without total body irradiation (TBI) conditioning. 77 consecutive patients who received a chemotherapy‐alone‐based conditioning regimen pretransplant were included. Free serum thyroxine (FT4), free serum triiodothyronine (FT3) and serum thyrotropin (TSH) levels were assayed before and 3 and 14 months after BMT. Patients were classified in three categories: normal thyroid profile if FT3 and FT4 were within the normal range and TSH was normal or low, peripheral thyroid insufficiency (PTI) if TSH was >4 mIU/l, or an ‘euthyroid sick syndrome’ (ETS) if FT3 and/or FT4 were low and TSH was normal or low. The incidence of thyroid dysfunction at 3 months was 57%, and 29% at 14 months. This was mostly due to the occurrence of ETS which was more frequent at 3 months (48%, 29/61) than at 14 months (19%, 9/48). Furthermore, at 3 months, survival was significantly lower in the ETS group (34.5%) than in the euthyroid group (96.2%), or in the PTI group (83.3%) (P  <  0.0001). PTI was observed even in the absence of TBI in 11 patients (14%) and was equally distributed at 3 months (n  =  6) and 14 months (n  =  5).

In conclusion, thyroid dysfunction is not a rare complication even without pretransplant TBI conditioning regimen. Hypothyroidism prevalence was 10%, and ETS, which was more frequently observed, displayed a dismal predictive value at 3 months.
Successful second bone marrow transplant for Fanconi's anaemia following escalation of conditioning
Authors: O'Donnell J. , Roberts I. , De La Fuente J. , Daly P. , New H. , Dokal I.
Published: November 2003

Allogeneic bone marrow transplantation represents the treatment of choice for severe bone marrow failure in patients with Fanconi's anaemia (FA). In view of the increased sensitivity to alkylating agents documented in this condition, much attention has focused on reducing the conditioning chemotherapy. We present a 13‐year‐old girl in whom sibling allogeneic BMT after conditioning with low‐ dose cyclophosphamide only resulted in graft rejection. However, a second transplant using the same donor proved successful following a more intensive conditioning regimen. This case demonstrates the phenotypic variability of FA, and highlights the need for tailoring the conditioning regimen for a given patient.
Protein C and protein S in homozygous sickle cell disease: does hepatic dysfunction contribute to low levels?
Authors: Wright J. G. , Malia R. , Cooper P. , Thomas P. , Preston F. E. , Serjeant G. R.
Published: November 2003

The aim of this study was to confirm reports of low protein C (PC) and S (PS) concentrations in steady‐state patients with homozygous sickle cell (SS) disease when compared to a racially matched normal haemoglobin (AA) control group and to examine the mechanisms of this reduction with respect to hepatic function, coagulation activation and haematological indices.

In 36 SS patients and 35 AA race‐matched controls PC (functional and immunoreactive), PS (free and total) were measured. C4B binding protein (C4B) was assessed by immunoelectrophoresis and D‐dimer by ELISA. Hepatic function was assessed by prothrombin (PT) time (49 SS, 64 AA), factor V (34 SS, 36 AA) and factor VII concentrations (28 SS, 29 AA). Proteins induced in vitamin K absence or antagonism (PIVKA) were sought in 12 SS's. The relationship between PC, PS and total bilirubin, haemoglobin (Hb) F and reticulocyte count was also assessed.

PC, PS and C4B were lower in SS disease. SS patients had longer PT times, and lower factor V and VII concentrations in comparison to AA controls. PC (functional and immunoreactive) and free PS correlated with PT. Within SS genotype PT correlated negatively with factor V and factor VII. Factor V and VII were positively correlated. PIVKAs were not detected. There was no correlation between PC, PS and D‐dimer, haemolytic rate or Hb F concentration.

Prolongation of PT time, low factor V and VII suggest that hepatic dysfunction, rather than coagulation activation or haemolytic rate, accounts for the reduced concentrations of PC and PS in steady‐state SS disease. The absence of PIVKAs suggests a hepatocellular problem.
t(8;20)(q22;p13): a novel variant translocation of t(8;21) in acute myeloblastic leukaemia
Authors: Xue Yongquan , Yu Fei , Xin Yongna , Lu Daru , Zou Zhenghui , Guo Yu , Xie Xing
Published: November 2003

A novel variant translocation t(8;20)(q22;p13) detected by karyotype analysis of bone marrow cells using R‐ and G‐banding techniques, is reported in a case of M2‐acute myeloid leukaemia (AML). The leukaemic cells were indistinguishable morphologically from that of M2‐AML with t(8;21)translocation. RT‐PCR revealed no AML1/ETO fusion transcript, but the wild‐type ETO‐3′ was expressed in the bone marrow cells suggesting that t(8;20) is a true simple variant translocation of t(8;21), and that a fusion gene consisting of ETO and an unidentified gene located in band 20p13 may exist in our case. Further study is required to clarify the entity of the assumed fusion gene.
Transplantation of HLA‐mismatched CD34+ selected cells in patients with advanced malignancies: severe immunodeficiency and related complications
Authors: Bacigalupo A. , Mordini N. , Pitto A. , Piaggio G. , Podestà M. , Benvenuto F. , Van Lint M. T. , Valbonesi M. , Lercari G. , Carlier P. , Lamparelli T. , Gualandi F. , Occhini D. , Bregante S. , Figari O. , Soracco M. , Vassallo F. , De Stefano G.
Published: November 2003

This trial was designed to test the use of CD34+ selected haemopoietic stem cells (HSC) in HLA‐mismatched donor–recipient pairs, following intensive conditioning with thiotepa, antilymphocyte globulin (ALG), cyclophosphamide and single‐dose total‐body irradiation (sTBI). 10 patients aged 16–50 with advanced malignancies and a two‐ or three‐antigen mismatched family donor entered this study. Donor marrow and G‐CSF primed peripheral blood cells were processed separately on CD34 columns (Ceprate). The median number of infused CD34+ cells were 5.66 × 106/kg, with 0.55 × 106/kg CD3+ cells. Nine patients received cyclosporin for graft‐versus‐host disease (GvHD) prophylaxis. Median neutrophil counts on day 21 were 2 × 109/l with a median platelet count of 60 × 109/l, but CD4 counts remained extremely depressed throughout the study. Acute GvHD was scored as grade 0–I in two patients, as grade II in seven, and grade III in one. Eight patients died at a median interval of 72 d from HSCT (range 20–144) due to cytomegalovirus (CMV) associated interstitial pneumonitis (IP) (n = 5), renal failure (n = 1), GvHD (n = 1) and Aspergillus meningitis (n = 1). Two patients are alive 365–495 d post transplant, one in remission and one in relapse.

This study suggests that large numbers of positively selected mismatched HSC can rapidly engraft after intensive conditioning regimen: however, profound post‐transplant immunodeficiency leads to a high risk of lethal infectious complications.