bluuebunny/crossref_metadata_2025 huggingface dataset:
2‐Chlorodeoxyadenosine treatment after splenectomy in patients who have myelofibrosis with myeloid metaplasia
Authors: Tefferi Ayalew , Silverstein Murray N. , Li Chin‐Yang
Published: November 2003

2‐Chlorodeoxyadenosine (2‐CdA) is a purine nucleoside analogue with therapeutic activity in low‐grade lymphoproliferative disorders. In addition, 2‐CdA has a potent myelosuppressive effect, and it has been shown to be toxic to malignant myeloid cells both in vitro and in vivo. In this pilot study we treated nine patients who had advanced myelofibrosis with myeloid metaplasia (MMM) and progressive hepatomegaly or symptomatic thrombocytosis after therapeutic splenectomy. 2‐CdA was administered at 0.05–0.1 mg/kg/d for 7 d for one to five treatment cycles. A reduction in liver size associated with marked improvement in fatigue and control of thrombocytosis and leucocytosis was achieved in seven of the nine patients (78% response rate). In four of the seven responding patients the reduction in liver size was durable (4–28 months) and was associated with a decrease in serum alkaline phosphatase levels. However, no patient had improvement in anaemia, and two of the seven initially responding patients have since died of acute leukaemia or progressive disease. Improvement in bone marrow fibrosis was noted in two of five available post‐treatment marrow examinations. Toxicity was mainly myelosuppression, which was severe in two patients. 2‐CdA may be considered a palliative therapeutic agent after splenectomy in noncytopenic patients with MMM who have progressive hepatomegaly or extreme thrombocytosis.
Flow cytometric detection of cytomegalovirus antigen in peripheral blood cells after bone marrow transplantation
Authors: Honda Junichi , Okubo Yasumitsu , Imamura Yutaka , Kusaba Mikako , Saruwatari Naoko , Oizumi Kotaro
Published: November 2003

Cytomegalovirus (CMV) immediate‐early (IE) antigen within the nucleus of cells was detected by flow cytometry in peripheral blood obtained from five bone marrow transplant recipients. In patients with an unexplained fever, CMV antigens were detected in monocytes or lymphocytes. On the other hand, in patients with CMV pneumonia, CMV antigens were detected in polymorphonuclear leucocytes (PMNLs). We suggest that the detection of CMV antigen in monocytes or lymphocytes may be related to CMV activation or reactivation, and the positive results in PMNLs indicate that the patient has a CMV‐associated disease (CMV pneumonia). Our method may be useful in monitoring CMV activation or reactivation, and analysing the mechanisms of CMV infection.
Cladribine (2‐CDA) given as subcutaneous bolus injections is active in pretreated Waldenström's macroglobulinaemia
Authors: Betticher D. C. , Hsu Schmitz S.‐F. , Ratschiller D. , Von Rohr A. , Egger T. , Pugin P. , Stalder M. , Hess U. , Fey M. F. , Cerny T.
Published: November 2003

Clinical trials with intravenous cladribine infusions in pretreated patients with Waldenström's macroglobulinaemia have shown a response rate of 40%. Our pharmacokinetic studies revealed that the bioavailability of subcutaneous cladribine is complete but that the concentration–time profile is very different from intravenous administration. We designed this phase II multi‐institutional trial to study the activity and toxicity of cladribine given as s.c. bolus injections in patients with symptomatic Waldenström's macroglobulinaemia.

Between May 1993 and October 1995, 25 patients were accrued: male/female 18/7, median age 65 years (range 44–85). All except one patient had been pretreated with more than one regimen (median 2, range 0–10). 18 patients had progressed under previous therapy and six were in relapse. All patients received cladribine for a total dose of 0.5 mg/kg per cycle as s.c. bolus injections divided over 5 d at  4 week intervals, for a maximum of six cycles.

All 25 patients were evaluable for toxicity and response. A total of 67 cycles were administered (median 3 cycles, range 1–6). Overall response rate including disease stabilization which had been progressive under previous therapy was 68%. 10 patients (40%, 95% CI 21–61%) achieved a partial remission. Seven responders had been progressive under previous therapy. Maximum responses were reached no later than the third cycle. Median time to treatment failure and remission duration were 4.4 (range 0.5–33) and 8 months (5–29), respectively. Four patients (16%) suffered from infections W.H.O. grade  2 (pneumonia grade 2, Staphylococcus septicaemia grade 3, viral encephalitis and pneumonia, both grade 4 with complete resolution). No other severe adverse events were observed.

Cladribine given as s.c. 5 d bolus injections was found to be active in pretreated Waldenström's macroglobulinaemia and resulted in durable remissions.
Poor prognosis in non‐villous splenic marginal zone cell lymphoma is associated with p53 mutations
Authors: Baldini L. , Guffanti A. , Cro L. , Fracchiolla N. S. , Colombi M. , Motta M. , Maiolo A. T. , Neri A.
Published: November 2003

We have recently reported a series of 15 non‐villous splenic marginal zone lymphoma patients, six of whom showed p53 mutations (40%). This molecular alteration did not correlate with any particular clinico‐pathologic feature at diagnosis. After a median follow‐up of 56 months, four cases evolved into aggressive fatal non‐Hodgkin's lymphoma (NHL) and two had refractory progressive disease; interestingly, p53 mutations were demonstrated in five of these patients at diagnosis. As the patients with wild‐type p53 presented responsive or indolent disease, this genetic alteration may be an early marker of aggressive transformation or refractoriness. p53 evaluation at diagnosis could be advisable in this particular subset of NHL.
Non‐transferrin‐bound iron induced by myeloablative chemotherapy
Authors: Bradley S. J. , Gosriwitana I. , Srichairatanakool S. , Hider R. C. , Porter J. B.
Published: November 2003

Previous studies have suggested that non‐transferrin‐bound plasma iron (NTBI) is present in patients undergoing cytotoxic chemotherapy, and that this may exacerbate untoward organ damage and increase the risk of bacterial infections following chemotherapy. However, the source of NTBI during myelosuppressive chemotherapy is controversial. In this study we have examined the kinetics of the appearance and disappearance of NTBI with chemotherapy. NTBI was present in only two out of 24 patients prior to chemotherapy but, following chemotherapy, was present in 19 patients, with peak NTBI levels at 5 d after onset of chemotherapy (mean 3.06 μm). Thereafter levels fell, but were still detectable in seven patients 14 d after the end of chemotherapy. The appearance of NTBI was associated with a sudden rise in transferrin saturation, but NTBI was detected on four occasions in the absence of full transferrin saturation. We have also established that daunorubicin cannot itself liberate NTBI from serum. There was no relationship between induced NTBI levels and serum iron or ferritin levels, previous or current blood transfusions, disease stage, or the type of chemotherapy given. The appearance of NTBI following chemotherapy was inverserely related to the fall in reticulocytes and serum transferrin receptor (TfR) levels, suggesting that NTBI formation is a consequence of suspension of erythropoietic activity.
No effect of a new second‐generation B‐domain‐deleted recombinant product on lymphocyte transformation in vitro: a study of plasma‐derived and recombinant products
Authors: Astermark Jan , Ekman Maj , Berntorp Erik
Published: November 2003

Immunomodulatory effects of various factor VIII and factor IX clotting factor concentrates (CFCs) and of albumin were evaluated by a sensitive assay measuring the incorporation of 3H‐thymidine in phytohaemagglutinin‐stimulated lymphocytes in the presence of monodansylthiacadaverine.

In contrast to previous findings by others, we found lymphocyte transformation to be inhibited by all plasma‐derived factor VIII concentrates at concentrations of 0.02, 0.2 and 2.0 IU/ml, including those purified by monoclonal antibodies (P < 0.05). Kryobulin TIM3 had the most pronounced effect. In addition, three plasma‐derived human albumin preparations exerted a similar inhibitory effect as the factor VIII concentrates, whereas the corresponding plasma‐derived factor IX concentrates only manifested minor immunomodulatory effects.

Of the recombinant preparations, only Recombinate exerted an inhibitory effect at 0.02 and 0.2 IU/ml, whereas both Kogenate and Recombinate decreased 3H‐thymidine incorporation at 2.0 IU/ml (P = 0.01). No immunomodulatory effect at all was observed with r‐VIII SQ, a new B‐domain‐deleted recombinant factor VIII preparation free from added albumin. The significance of this finding regarding immunological side‐effects including inhibitor development remains to be evaluated, but this second‐generation recombinant product opens up new and interesting perspectives yet to be explored.
Plasma levels and production of soluble stem cell factor by marrow stromal cells in patients with aplastic anaemia
Authors: Kojima Seiji , Matsuyama Takaharu , Kodera Yoshihisa
Published: November 2003

Defective marrow stroma or microenvironment have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). Stem cell factor (SCF), the ligand for the c‐kit receptor, is produced mainly by marrow stromal cells and seems to reflect the haemopoietic function of bone marrow stroma. We measured the plasma levels of soluble SCF in 87 patients with AA and investigated the production of soluble SCF by the marrow stromal cells of 46 patients with acquired AA. The mean plasma SCF concentrations in the AA patients and normal controls were not significantly different (1098 ± 398 pg/ml versus 1160 ± 316 pg/ml, respectively), and there was no significant correlation between the peripheral blood counts and the SCF concentrations. However, the mean SCF concentration in patients who received prednisolone ± anabolic steroids at the time of sampling was significantly lower than that in the patients who did not receive both agents. We did not find any correlation between the changes in SCF concentrations and the response to immunosuppressive therapy, although it did increase significantly after bone marrow transplantation. The ability of marrow stromal cells to release soluble SCF did not differ significantly between the patients with AA and normal controls. We conclude that soluble SCF production does not appear to be altered in patients with AA and that defective production of soluble SCF is unlikely to be the cause of AA in most patients.
Differences in specificity of heparin‐dependent antibodies developed in heparin‐induced thrombocytopenia and consequences on cross‐reactivity with danaparoid sodium
Authors: Pouplard Claire , Amiral Jean , Borg Jeanne‐Yvonne , Vissac Anne‐Marie , Delahousse Bénédicte , Gruel Yves
Published: November 2003

Heparin‐induced thrombocytopenia (HIT) is frequently associated with antibodies (Abs) to heparin–PF4 complexes (H‐PF4). In order to investigate whether there are variations in specificity of Abs, we studied 63 samples from patients with suspected HIT. Two groups of samples were separated after comparing their reactivity against H‐PF4 or recombinant PF4 (r‐PF4) using ELISA. In group Ab1 (n = 46), Abs only or mainly bound to H‐PF4 complexes and thus most of the epitopes recognized probably involved both heparin and PF4. In group Ab2 (n = 17), Abs exhibited similar reactivity to r‐PF4 and H‐PF4, and the antigens recognized were possibly neoepitopes mainly expressed by modified PF4 and by H‐PF4 complexes. Platelet activation tests were positive with 56 samples containing high titres of Abs to H‐PF4. Most samples (n = 59) contained IgG antibodies, often associated with IgA antibodies which were more frequently found in group Ab2, and/or IgM. With unfractionated heparin treatment, HIT was associated with Ab1 or Ab2 antibodies, whereas only Ab1 antibodies were detected after low‐molecular‐weight heparin (LMWH). Furthermore, cross‐reactivity with danaparoid sodium was present only in group Ab1 and mainly involved LMWH‐treated patients.
A rapid non‐invasive method for the detection of the haemochromatosis C282Y mutation
Authors: Merryweather‐Clarke Alison T. , Liu Yan‐Tat , Shearman Jeremy D. , Pointon Jennifer J. , Robson Kathryn J. H.
Published: November 2003

We describe the rapid single‐step detection, by mutagenically separated polymerase chain reaction (MS‐PCR), of the HLE C282Y mutation, for which > 90% of haemochromatosis patients in the U.K. are homozygous. In addition to using purified DNA as a template, whole blood and lysed buccal cell extracts from mouthwash samples can be used. Therefore sample collection may be non‐invasive and purification steps kept to a minimum.
New insights into the biology of Philadelphia‐chromosome‐positive acute lymphoblastic leukaemia using a combination of May‐Grünwald‐Giemsa staining and fluorescence in situ hybridization techniques at the single cell level
Authors: Haferlach Torsten , Winkemann Martin , Ramm‐Petersen Lotte , Meeder Marlies , Schoch Robert , Weber‐Matthiesen Klaus , Schlegelberger Brigitte , Schoch Claudia , Ludwig Wolf‐Dieter , Hiddemann Wolfgang , Löffler Helmut
Published: November 2003

It is sometimes difficult to discriminate chronic myeloid leukaemia (CML) in lymphatic blast crisis from Ph‐chromosome positive acute lymphoblastic leukaemia (ALL). Previous studies have suggested that ALL is restricted to the lymphatic lineage only, whereas CML involves all cell lineages.

In four cases of Ph‐positive ALL we combined the standard May‐Grünwald‐Giemsa staining with FISH at the single cell level and were able to demonstrate that  98% of lymphatic blasts carried the Philadelphia chromosome. Erythropoiesis was not involved when this technique was applied. Using immunological identification of single cells (FICTION), we detected the t(9;22) in 100% of CD19‐positive B lymphoblasts in all four cases, in some CD3‐positive T cells in two patients, and in  98% of CD34‐positive precursor cells. However, in two out of four patients the myeloid cell compartment was involved in the malignant transformation, unequivocally demonstrated not only by the combination of MGG and FISH but also by FICTION using the antibodies CD13 and CD33. The observation that both patients with myeloid cell lineage involvement had a myeloid co‐expression on their blasts and a better survival supports the concept of a separate, biologically determined subgroup in Ph‐positive ALL, leading to further investigations, and individually tailored treatment strategies.
HbC compound heterozygotes [HbC/Hb Riyadh and HbC/Hb N‐Baltimore] with opposing effects upon HbC crystallization
Authors: Hirsch Rhoda Elison , Witkowska H. Ewa , Shafer Frank , Lin Margaret J. , Balazs Tatiana C. , Bookchin Robert M. , Nagel Ronald L.
Published: November 2003

Compound heterozygotes of variant haemoglobins (Hbs) with HbC, with or without novel phenotypic changes, have provided insight into the molecular basis of the interacting haemoglobins and information concerning the role of specific residues in the crystallization of oxy HbC. A high phosphate buffer system has proved useful for studying the effects of variant haemoglobins (naturally co‐existing with HbC in the red cell) on the oxy HbC crystallization process and has led us to conclude that β87 and β73 are contact sites of the oxy HbC crystal. We now present investigations from two HbC compound heterozygotes which exhibit opposing effects upon HbC crystallization: HbC/Hb N‐Baltimore (β95 Lys → Glu) and HbC/Hb Riyadh (β120 Lys → Asn). The latter inhibits the in vitro crystallization of HbC, explaining the lack of erythrocyte abnormalities (with the exception of microcytosis) in the doubly heterozygous infant. In contrast, Hb N‐Baltimore accelerates the crystallization of HbC, contributing to multiple abnormalities in red cell morphology, albeit in the absence of morbidity. We conclude that (1) β120 and β95 are additional contact sites in the crystal, and (2) the HbC/Hb Riyadh haemoglobinopathy demonstrates that crystallization may not be required for the generation of the observed microcytosis and increased red cell density in HbC‐containing red cells.
Thrombopoietin measurement in thrombocytosis: dysregulation and lack of feedback inhibition in essential thrombocythaemia
Authors: Pitcher L. , Taylor K. , Nichol J. , Selsi D. , Rodwell R. , Marty J. , Taylor D. , Wright S. , Moore D. , Kelly C. , Rentoul A.
Published: November 2003

Essential thrombocythaemia (ET), a myeloproliferative disorder (MPD) manifested by excessive platelet production, lacks a specific diagnostic test to facilitate differentiation from other thrombocytoses. We studied thrombopoietin (TPO) levels in 41 patients with thrombocytosis: 25 ET patients, eight with other MPD, and eight with reactive thrombocytosis. Mean age and platelet counts for these groups were comparable. TPO levels for 96 healthy individuals provided a reference range for normal. The majority of ET patients (19/25 or 76%) had normal TPO levels. No patient with ET had a TPO level below 75 pg/ml, compared with 57% of healthy donors and 8/16 (50%) patients with other thrombocytoses (P < 0.05). TPO levels in ET are not appropriately down‐regulated, as occurs with cytokines relevant to other MPD. In thrombocytosis, a TPO level <75 pg/ml indicates that ET is unlikely.
Incidence of AML1/ETO fusion transcripts in patients entered into the MRC AML trials
Authors: Langabeer S. E. , Walker H. , Rogers J. R. , Burnett A. K. , Wheatley K. , Swirsky D. , Goldstone A. H. , Linch D. C. ,   on behalf of the MRC Adult Leukaemia Working Party
Published: November 2003

Acute myeloid leukaemia (AML) with the t(8;21)(q22;q22) is deemed to be a ‘good‐risk’ disease. 396 patients with AML at diagnosis were screened for the presence of t(8;21) and AML1/ETO fusion transcripts by cytogenetic and RT‐PCR techniques respectively. 32 cases of t(8;21) were detected, all of which were also PCR positive. A further 19 cases were detected at the molecular level, predominantly but not exclusively in M1 and M2 FAB types. Approximately 12% of all new cases of AML are estimated to have AML1/ETO fusion transcripts and it is suggested that molecular screening should be performed in all cases with the possible exception of the M3 FAB type.
Expression of AML1/MTG8 transcripts in clonogenic cells grown from bone marrow of patients in remission of acute myeloid leukaemia with t(8;21)
Authors: Saunders Melanie J. , Brereton Michelle L. , Adams Julie A. , Tobal Khalid , Liu Yin John A.
Published: November 2003

Patients in long‐term remission of acute myeloid leukaemia (AML) M2 with t(8;21) after chemotherapy, with or without bone marrow transplantation, are known to retain residual cells which express AML1/MTG8 transcripts in bone marrow, detectable by RT‐PCR. In order to determine whether these residual cells are clonogenic, we have grown remission bone marrow samples in standard semi‐solid culture and picked individual CFU‐GM and BFU‐E colonies which were then analysed for the expression of AML1/MTG8 transcripts using a rapid specific RT‐PCR technique. Nine patients were tested in remission, six between 1 and 83 months post chemotherapy, one 103 months post autologous bone marrow transplant and one 41 months post allogeneic bone marrow transplant. One of these patients also had quantitation of AML1/MTG8 transcripts on five occasions after recovery from each course of chemotherapy and at the end of treatment. There was a significant correlation between the percentage of positive colonies and the level of AML1/MTG8 transcripts. Between two and 80 CFU‐GM and between two and 21 BFU‐E colonies were analysed from each patient sample; 0–23% CFU‐GM and 0–17% BFU‐E colonies were found to express AML1/MTG8 transcripts suggesting that these residual cells are clonogenic in vitro and that the cell of origin is a multipotent myeloid progenitor.
The clinical outcome of 65 cases of mantle cell lymphoma initially treated with non‐intensive therapy by the British National Lymphoma Investigation Group
Authors: Vandenberghe E. , De Wolf‐Peeters C. , Vaughan Hudson G. , Vaughan Hudson B. , Pittaluga S. , Anderson L. , Linch D. C.
Published: November 2003

Mantle cell lymphoma (MCL) was first described as a distinct biological entity on the basis of its association with the t(11;14)(q13;q32) resulting in over‐expression of the cyclin D1 gene. Recognition of the morphological, immunophenotypic and clinical characteristics of MCL has enabled the accurate diagnosis of this entity and appreciation of its poor prognosis. Most published series of patients with MCL have used anthracycline‐containing regimens. In contrast the British National Lymphoma Investigation (BNLI) group have treated 65 patients with MCL with non‐intensive ‘low‐grade lymphoma’ therapy. The median overall survival of 57 months and progression‐free survival of 24 months compares favourably with the more intensively treated series. Although the disease was generally more aggressive than other low‐grade lymphomas, some patients were asymptomatic and had indolent disease. When compared to 1853 patients with non‐MCL low‐grade lymphomas entered on the BNLI database, patients were found on average to be older (P = 0.02), to have more extra‐nodal disease (P < 0.00001), and a higher proportion to have a raised ESR (P = 0.02) and a low serum albumin (P = 0.002). Multivariate analysis of significant prognostic markers in all BNLI low‐grade lymphomas failed to identify MCL as an independent prognostic factor.
Expression of interleukin‐5 by human bone marrow microvascular endothelial cells: implications for the regulation of eosinophilopoiesis in vivo
Authors: Möhle Robert , Salemi Parissa , Moore Malcolm A. S. , Rafii Shahin
Published: November 2003

We have shown that bone marrow microvascular endothelial cells (BMEC) support growth and differentiation of haemopoietic progenitors in vitro by elaboration of haemopoietic cytokines. Since generation of eosinophils can be observed in these coculture experiments, and BMEC do not produce interleukin (IL)‐3, we evaluated BMEC for expression of IL‐5, a specific growth factor for the eosinophilic lineage. Using RT‐PCR, IL‐5 mRNA was expressed by BMEC after stimulation (12 h) with lipopolysaccharide (LPS), IL‐1, IL‐2 and phorbol myristate acetate (PMA), but not by resting BMEC, after stimulation with TNF‐α or interferon (IFN)‐γ. Moreover, IFN‐γ suppressed expression of IL‐5 in response to LPS and IL‐2. The identity of the PCR products was confirmed by restriction enzyme digestion, which resulted in fragments of the predicted size. T lymphocytes were not present in the endothelial cultures as demonstrated by absence of CD2 mRNA. Using a sensitive (1 pg/ml) ELISA assay, IL‐5 was detected after 48 h incubation of BMEC with IL‐2 (4.1 pg/106 cells) or with a combination of LPS and IL‐2 (4.8 pg). However, the number of eosinophils generated after 4 weeks coculture of CD34+ haemopoietic cells with BMEC was not increased by addition of IL‐2. RT‐PCR revealed that BMEC in coculture with haemopoietic cells expressed IL‐5 even without addition of exogenous cytokines or stimulating agents. In conclusion, expression of IL‐5 by BMEC can be stimulated by cytokines (IL‐1, IL‐2), LPS, PMA, and coculture with proliferating haemopoietic cells. Thus, BMEC may support proliferation and differentiation of eosinophils in the bone marrow. IFN‐γ represents a cytokine with an inhibitory effect on IL‐5 expression by BMEC. In addition, eosinophilia in response to circulating IL‐2 or bacterial products (LPS) in vivo may be partially mediated by BMEC or vascular endothelium.
Anaemia of lung cancer is due to impaired erythroid marrow response to erythropoietin stimulation as well as relative inadequacy of erythropoietin production
Authors: Dowlati Afshin , R'Zik Samir , Fillet Georges , Beguin Yves
Published: November 2003

Many studies have been done in order to elucidate the pathogenesis of the anaemia of chronic disorders accompanying cancer, with conflicting results. This is probably due to the heterogeneity of the patient population selected for these studies (many patients treated by chemotherapy). To avoid this pitfall, in this study a very homogenous group of chemotherapy and radiotherapy‐naive patients with lung cancer were selected. Serum erythropoietin and soluble transferrin receptor measurements suggested that the anaemia of non‐treated lung cancer is mainly due to an impaired erythroid marrow response to erythropoietin stimulation. However, a relative inadequacy of erythropoietin production may also contribute.
Simultaneous SIL‐TAL1 RT‐PCR detection of all tald deletions and identification of novel tald variants
Authors: Delabesse Eric , Bernard Muriel , Landman‐Parker Judith , Davi Frederic , Leboeuf Daniel , Varet Bruno , Valensi Françoise , Macintyre Elizabeth A.
Published: November 2003

Site‐specific deletions of the 5′ part of the TAL1 gene (tal d ) are among the most frequent non‐random genetic abnormalities in T‐cell acute lymphoblastic leukaemia (T‐ALL). They are usually detected by PCR from DNA with several primer pairs or by Southern blot analysis. Since tald lead to expression of a SIL‐TAL1 fusion transcript, irrespective of the genomic breakpoint, we have used a single monoplex RT‐PCR reaction to screen 55 T‐ALL patients at diagnosis. SIL‐TAL1 transcripts were demonstrated in 12 (22%) cases, including 7/27 (26%) children <15 years of age, 2/8 (25%) adolescents and 2/17 (12%) adults aged >20 years. SIL‐TAL1 RT‐PCR was preferrable to tal d DNA PCR since it allowed the simultaneous detection of tal d1, tal d2 and two previously undescribed tal d variants. SIL‐TAL1 RT‐PCR screening should therefore increase the detection rate of tal d by approximately 15–20%, with an at least comparable sensitivity to tal d genomic PCR, and represents a more practical and economic alternative to multiple DNA PCRs or Southern blotting when incorporated into molecular screening for multiple transcripts at diagnosis.
Colony counting is a major source of variation in CFU‐GM results between centres
Authors: Armstrong Lumley Matthew , Burgess Robert , Billingham Lucinda Jane , McDonald Dorothy Faith , Milligan Donald William
Published: November 2003

The results for colony forming unit granulocyte‐macrophage (CFU‐GM) assays vary substantially between centres. It is possible that colony counting is largely responsible for this discrepancy. In order to examine this exclusively from the many factors that make up the CFU‐GM assay, we performed a colony counting exercise involving 11 laboratories. Two‐way analysis of variance showed a highly significant difference (P =0 .0001) in the counts obtained from the centres. One centre was found to score consistently high and two others scored consistently low numbers of colonies. This suggests that identification of colonies is a major source of variation between centres.
Comparison of fluorescent consensus IgH PCR and allele‐specific oligonucleotide probing in the detection of minimal residual disease in childhood ALL
Authors: Owen R. G. , Goulden N. J. , Oakhill A. , Shiach C. , Evans P. A. , Potter M. N. , Morgan G. J.
Published: November 2003

The sensitivity of detection of residual disease by two IgH PCR strategies, fluorescent framework 3 (Ffr3) and allele‐specific oligonucleotide probing (ASOP), was compared in 57 ‘remission’ BM samples obtained from 19 children with B‐lineage acute lymphoblastic leukaemia (ALL). Oligonucleotide probing was more sensitive than FFr3 PCR in 10/16 cases, achieving a sensitivity of 0.01% or greater in 15/16 cases. Comparable sensitivities were obtained in the six remaining cases; the FFr3 PCR achieving a sensitivity of 0.1% or greater in 14/16 cases. 39/57 ‘remission’ BM samples analysed showed no evidence of MRD by either technique although 18 were positive by ASOP and 14 positive by FFr3 PCR. The level of disease was estimated to be 0.01% or less in the four false negative samples.
Re‐expression of functional P‐selectin molecules on the endothelial cell surface by repeated stimulation with thrombin
Authors: Kameda Hideto , Morita Ikuo , Handa Makoto , Kaburaki Junichi , Yoshida Tadashi , Mimori Tsuneyo , Murota Sei‐Itsu , Ikeda Yasuo
Published: November 2003

P‐selectin (GMP‐140, PADGEM, CD62P) is a cell adhesion receptor which is believed to play an important role in inflammatory diseases by supporting leucocyte rolling. P‐selectin is located on the granule membrane of Weibel‐Palade bodies in resting endothelial cells and is expressed on the cell surface during cellular activation with various stimulators such as thrombin. Thereafter, P‐selectin is internalized and sorted to the Golgi region and Weibel‐Palade bodies again. However, whether P‐selectin is re‐expressed upon subsequent cellular stimulation has, to date, been unclear.  To address this question, we measured the cellular content and surface expression of P‐selectin, using indirect immunofluorescence and confocal laser cytometry. Surface expression of P‐selectin reached a maximum < 2 min after thrombin stimulation and declined to basal levels after 180 min. Rechallenge with thrombin induced rapid surface re‐expression of P‐selectin, which was independent of de novo protein synthesis, since cycloheximide did not inhibit re‐expression. Moreover, re‐expressed P‐selectin supported the adherence of HL60 promyelocytic cells.  These results clearly demonstrated that functional P‐selectin molecule was recycled after repeated stimulation with thrombin, raising the possibility that P‐selectin is involved in chronic inflammation.
Concordant expression of class II and class III CD34 epitopes on haemopoietic cells in leukapheresis and cord blood samples with CD45/CD34 dual staining
Authors: Rudin Claudius E. , Garland John M. , Joyner Miles V.
Published: November 2003

We used flow cytometry with CD45/CD34 dual antibody labelling to investigate the relative expression of class II and class III CD34 epitopes on haemopoietic progenitor cells (HPC) in 22 leukapheresis and cord blood samples. There was a close correlation between class II (QBEnd‐10) and class III (clone 581) CD34+ cells (R2 = 0.975, ratio of class III to class II CD34+ count 1.24 ±0.04). The linear relationship between class II and III CD34 epitopes on HPC suggests that the choice of antibody class is not a source of random variation in the quality assurance of CD34+ cell enumeration.
Molecular basis of the D variant phenotypes DNU and DII allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein
Authors: Avent Neil D. , Jones Jeffrey W. , Liu Wendy , Scott Marion L. , Voak Douglas , Flegel Willy A. , Wagner Franz F. , Green Charles
Published: November 2003

The discovery of Rh partial D variant red cells by discrepant reactions with different monoclonal anti‐D has demonstrated the range of Rh D epitopes that have arisen due to alterations in Rh D protein structure. There are two current classification systems, one which uses a nine epitope model (epD1–epD9) whereas a more recent model proposes 30 different epitopes. We describe here the molecular basis of two D variants which lack epD4 and epD9 namely the DNU and DII phenotypes. These would have both been originally classified as DII phenotype individuals, but we have revealed subtle differences in the serological profile of these erythrocytes. Such a differential reactivity and determination of the molecular bases of these phenotypes allows us to predict critical amino acids for epD3, epD4 and epD9 expression. The DNU phenotype arises from a single point mutation in the RHD gene resulting in a single amino acid change (Gly353Arg). Sequence analysis of exon 7 of the RHD gene derived from the DII propositus indicates that there is a single point mutation in this exon resulting in a single amino acid change (Ala354Asp). It is likely that this point mutation gives rise to the DII phenotype. Both mutations result in the change to Rh D‐specific residues. Our results indicate that the following amino acids are crucial for epD3a (Asp350), epD3b (Asp350+Gly353), epD4a (Gly353 + Ala354), epD4b (Ala354), epD9a (Asp350 + Gly353 + Ala354) and epD9b (Asp350 + Ala354) expression. All of these amino acids reside on the predicted sixth external domain of the Rh D protein, so it is possible that epD3, 4 and 9 are continuous epitopes.
Circulating plasma cells in multiple myeloma: characterization and correlation with disease stage
Authors: Rawstron Andrew C. , Owen Roger G. , Davies Faith E. , Johnson Roderick J. , Jones Richard A. , Richards Stephen J. , Evans Paul A. , Child J. Anthony , Smith Graeme M. , Jack Andrew S. , Morgan Gareth J.
Published: November 2003

The aim of this study was to develop a flow cytometric test to quantitate low levels of circulating myeloma plasma cells, and to determine the relationship of these cells with disease stage. Cells were characterized using five‐parameter flow cytometric analysis with a panel of antibodies, and results were evaluated by comparison with fluorescent consensus‐primer IgH‐PCR.

Bone marrow myeloma plasma cells, defined by high CD38 and Syndecan‐1 expression, did not express CD10, 23, 30, 34 or 45RO, and demonstrated weak expression of CD37 and CD45. 65% of patients had CD19−56+ plasma cells, 30% CD19−56low, and 5% CD19+56+, and these two antigens discriminated myeloma from normal plasma cells, which were all CD19+56low.

Peripheral blood myeloma plasma cells had the same composite phenotype, but expressed significantly lower levels of CD56 and Syndecan‐1, and were detected in 75% (38/51) of patients at presentation, 92% (11/12) of patients in relapse, and 40% (4/10) of stem cell harvests. Circulating plasma cells were not detectable in patients in CR (n=9) or normals (n=10), at a sensitivity of up to 1 in 10 000 cells. There was good correlation between the flow cytometric test and IgH‐PCR results: myeloma plasma cells were detectable by flow cytometry in all PCR positive samples, and samples with no detectable myeloma plasma cells were PCR negative. Absolute numbers decreased in patients responding to treatment, remained elevated in patients with refractory disease, and increased in patients undergoing relapse. We conclude that flow cytometry can provide an effective aternative to IgH‐PCR that will allow quantitative assessment of low levels of residual disease.
The engagement of CD4 surface antigen in the HEL haemopoietic cell line up‐regulates the transforming growth factor‐β1 (TGF‐β1) promoter activity
Authors: Gibellini Davide , Celeghini Claudio , Panaya Roberto , Secchiero Paola , Capitani Silvano , La Placa Michele , Zauli Giorgio
Published: November 2003

In order to investigate the effect of CD4 engagement on the transforming growth factor β1 (TGF‐β1) promoter activity in haemopoietic progenitors, HEL cells were transiently transfected with a plasmid vector containing −453/+11 nucleotides of the TGF‐β1 promoter fused with the bacterial chloramphenicol acetyltransferase (CAT) gene and then treated with various agonists. Both cross‐linked CD4 mAb and envelope gp120 were able to significantly up‐regulate CAT activity with respect to the levels of activation observed in HEL cells treated with cross‐linked CD8 mAb or p24. By using deletion mutants of the TGFβ1 promoter, we found that the minimal DNA sequence still responsive to cross‐linked CD4 mAb or gp120 was located between nucleotides −453/−323 of the TGF‐β1 promoter, which contains two activating protein 1 (AP1) binding sites. In electromobility shift assays (EMSA) we could demonstrate that CD4 engagement of HEL cells induced a significant increase of AP1 binding activity at the nuclear level. Furthermore, the steady‐state mRNA of endogenous TGF‐β1 showed a small but reproducible increase when HEL cells were treated with cross‐linked CD4 mAb or gp120. Altogether, these findings suggest that the engagement of CD4 in HEL cells modulates TGF‐β1 expression, acting predominantly at the transcriptional level.
Identification of ‘short‐lived’ and ‘long‐lived’ patients at presentation of idiopathic myelofibrosis
Authors: CERVANTES Francisco , PEREIRA Arturo , ESTEVE Jordi , RAFEL Montserrat , COBO Francesc , ROZMAN Ciril , MONTSERRAT Emilio
Published: November 2003

To contribute to a better knowledge of the prognosis of idiopathic myelofibrosis (IM), the prognostic value of the presenting features in 106 patients diagnosed with IM at a single institution during a 21‐year period was retrospectively analysed. Median survival was 59.4 months (95% CI 40.7–75.4). Using univariate analysis, age < 64 years, constitutional symptoms (fever, night sweats, weight loss), Hb < 10 g/dl, circulating blasts (≫1%), and serum LDH > 3 times upper normal level were associated with a significantly shorter survival; male sex, platelet count <100×109/l, blood percentage of immature granulocytes (excluding blasts), low cholesterol levels and advanced marrow histological stage had borderline significance. Using multivariate study, only age > 64 years, constitutional symptoms, Hb < 10 g/dl, and circulating blasts retained their prognostic relevance. The latter three variables confirmed their predictive value in patients above and below the series median age, and were able to identify two groups of patients: a low‐risk group of 67 patients with none or one bad prognostic factor, in whom IM had an indolent course (median survival 98.8 months, 95% CI 68.7–127.6), and a high‐risk group, including 39 patients with two or three factors, with a more aggressive disease (median survival 20.6 months, 95% CI 10–28.2). Finally, the application of two recently proposed scoring systems (in which three prognostic groups are considered) was unable to separate intermediate‐ from high‐risk patients.
Angina pectoris occurring during granulocyte colony‐stimulating factor‐combined preparatory regimen for autologous peripheral blood stem cell transplantation in a patient with acute myelogenous leukaemia
Authors: Fukumoto Yumiko , Miyamoto Toshihiro , Okamura Takashi , Gondo Hisashi , Iwasaki Hiromi , Horiuchi Takahiko , Yoshizawa Shigeru , INABA SOICHI , Harada Mine , Niho Yoshiyuki
Published: November 2003

We describe a patient with acute myelogenous leukaemia who developed angina pectoris during pre‐transplant conditioning for autologous peripheral blood stem cell transplantation (PBSCT); the conditioning regimen consisted of cytotoxic drugs in combination with granulocyte colony‐stimulating factor (G‐CSF). Neutrophilia and hypercoagulability were observed at the time of angina pectoris. Recurrence of angina pectoris was not seen after nitrate and aspirin therapy. Exercise stress testing performed after PBSCT suggested the presence of myocardial ischaemia. Therefore cases at risk of vascular events should be carefully managed with prophylactic treatment during G‐CSF administration.
Allogeneic peripheral blood progenitor cell (PBPC) transplantation in children with haematological malignancies
Authors: Diaz M. A. , Alegre A. , Villa M. , Benito A. , Bernardo M. R. , Lopez‐Botet M. , Madero L.
Published: November 2003

We report on five children with haematological malignancies who underwent allogeneic peripheral blood progenitor cell (PBPC) transplantation. PBPC were harvested from HLA‐identical sibling donors after G‐CSF (10 μg/kg/d s.c.) mobilization. Aphereses were carried out on day 5 after G‐CSF using a Cobe Spectra blood cell separator. All PBPC allografts were cryopreserved before transplantation. The median of CD34+ cells and CD3+ cells infused were 14.1×106/kg recipient body weight (range 4.92–22.3) and 2.40×108/kg recipient body weight (range 0.54–4.82), respectively. Engraftment occurred in all cases. The median time to a neutrophil count >0.5×109/l and a platelet count >20×109/l were 15 and 14 d, respectively. The incidence of severe acute graft‐versus‐host disease was 20%. These data suggest that allogeneic PBPC transplantation might be an alternative to bone marrow transplantation in children.
Novel band 3 variants (bands 3 Foggia, Napoli I and Napoli II) associated with hereditary spherocytosis and band 3 deficiency: status of the D38A polymorphism within the EPB3 locus
Authors: MIRAGLIA del GIUDICE Emanuele , VALLIER AgnÉs , MAILLET Philippe , PERROTTA Silverio , CUTILLO Stefano , IOLASCON Achille , TANNER Michael J. A. , DELAUNAY Jean , ALLOISIO Nicole
Published: November 2003

We report three novel variants of band 3 associated with hereditary spherocytosis: band 3 Foggia (311delC; ACCCAC → ACCAC), band 3 Napoli I (447insT; TCT → TTCT) and band 3 Napoli II (I783N; ATC → AAC). The first two mutations resulted in premature termination of translation, making one haploid set of band 3 mRNA unavailable. Since it affected a highly conserved position at the terminal end of transmembrane domain 11, the third mutation prevented one haploid set of band 3 from becoming incorporated or stabilized into the membrane. These three mutations resulted in a reduction of the band 3 level in the red cell membrane (by 20–25%) and were dominantly transmitted. The D38A substitution (GAC → GCC) is a low frequency change of band 3. In one compound heterozygote D38A/Napoli II, a markedly aggravated picture required early splenectomy. In contrast, the D38A change was not associated with deterioration in another compound heterozygote, carrying in trans, the previously recorded R760W mutation (CGG → TGG). In the aggravated case, SSCP analysis did not exhibit any additional change in the two EPB3 alleles. Nor did it show any alteration in the exons of the two ANK1 alleles, and the aggravating factor remained elusive. The D38A alteration should be regarded as an innocuous polymorphism.
Fas/APO‐1 (CD95)‐mediated cytotoxicity is responsible for the apoptotic cell death of leukaemic cells induced by interleukin‐2‐activated T cells
Authors: Komada Yoshihiro , Zhou Yan‐Wen , Zhang Xao‐Li , Chen Tong‐Xin , Tanaka Shigeki , Azuma Eiichi , Sakurai Minoru
Published: November 2003

Apoptotic cell death is induced by the cross‐linking of Fas/APO‐1 receptor (CD95) in acute myelogenous leukaemia (AML) cells. Since CD95 ligand (CD95L) is expressed on interleukin‐2 (IL‐2)‐activated T cells, we investigated the involvement of CD95‐CD95L pathway in T cell‐mediated cytotoxicity against AML cells. Activated CD8+ T cells could efficiently kill a parental CD95‐sensitive AML cell line, MML‐1 and, to a lesser extent, a CD95‐resistant clone, MML‐1R. Neither MML‐1 nor MML‐1R cells were killed by activated CD4+ T cells. The blocking monoclonal antibody (MoAb) against CD95, ZB4, caused a significant inhibition of T‐cell‐mediated cytotoxicity against MML‐1 cells, but not against MML‐1R cells. Interestingly, MML‐1 cells underwent the classic nuclear morphologic changes and DNA fragmentation characteristic of apoptosis when cultured with activated T cells. Enumeration of apoptotic and necrotic nuclei showed that both apoptosis and necrosis were induced in MML‐1 cells, whereas necrosis was exclusively observed in MML‐1R cells. Apoptosis of MML‐1 cells was completely blocked in the presence of ZB4 MoAb. Similarly, blocking by ZB4 MoAb significantly inhibited T‐cell‐mediated lysis of fresh AML cells in a CD95‐sensitive group, but not in a CD95‐refractory group. In addition CD8+ T cells expressed CD95L mRNA more abundantly than CD4+ T cells upon activation by IL‐2. These findings suggest that T‐cell‐mediated cytotoxicity against AML cells requires participation of CD95‐CD95L pathway for cytotoxic signal transduction leading to target apoptosis.
Treatment with recombinant human erythropoietin (rHuEpo) in a patient with paroxysmal nocturnal haemoglobinuria: evaluation of membrane proteins CD55 and CD59 with cytofluorometric assay
Authors: Astori Cesare , Bonfichi Maurizio , Pagnucco Guido , Bernasconi Paolo , Lazzarino Mario , Orlandi Ester , Bernasconi Carlo
Published: November 2003

We describe a 28‐year‐old man with paroxysmal nocturnal haemoglobinuria (PNH) and a high transfusion requirement. Prior to and during therapy with recombinant human erythropoietin (rHuEpo), we evaluated the levels of ‘decay‐accelerating‐factor’, CD55, and ‘membrane‐inhibitor‐of‐reactive‐lysis’, CD59, as markers of the disease, whilst CD58, a marker present on leucocytes, was utilized to monitor normal haemopoietic activity.

The patient became transfusion independent 1 month after beginning rHuEpo and remains well. The analysis of CD55, CD59 and CD58 suggests that the efficacy of rHuEpo was due to a selective rHuEpo action on normal erythroid clones.
β‐Spectrin Campinas: a novel shortened β‐chain variant associated with skipping of exon 30 and hereditary elliptocytosis
Authors: Bassères Daniela S. , Pranke Patricia H. L. , Sales Tereza S. I. , Costa Fernando F. , Saad Sara T. O.
Published: November 2003

β‐Spectrin Campinas is a novel spectrin variant associated with a shortened β‐chain in a kindred with hereditary elliptocytosis (HE). The propositus and her mother exhibited increased amounts of spectrin dimers and an increase in the αI 74 kD fragment from the α‐chain after partial tryptic digestion of spectrin. The shortened β‐chain appeared as an additional band of approximately 200 kD on SDS‐PAGE. In order to delineate the molecular defect of this abnormality at the gene level, reticulocyte mRNA was transcribed into cDNA and the last four exons of the β‐spectrin gene were amplified. Agarose gel of the amplification product of the propositus revealed the expected band of 487 bp as well as a shortened band of approximately 300 bp (size determined on gel). This shortened cDNA amplification product was cloned and nucleotide sequencing revealed the absence of the entire exon 30. In order to determine the underlying mutation responsible for this abnormal splicing, a genomic DNA fragment containing exons 30 and 31 was amplified and nucleotide sequencing revealed a G → A substitution at the 5′ donor splice site consensus sequence of intron 30 (nt + 1 IVS30). The skip splicing observed in this study results in a frameshift, creating a new stop codon and causing a deletion of 129 aminoacids at the very COOH‐terminus of the protein, thus impairing spectrin dimers self‐association. We classified this HE as spherocytic HE because the propositus presented a few spherocytes in addition to many elliptocytes in the blood smear, whereas her mother, who was splenectomized, showed many schizocytes, poikilocytes and spherocytes.
Mechanism of action of antithymocyte globulin in the treatment of aplastic anaemia: in vitro evidence for the presence of immunosuppressive mechanism
Authors: Teramura Masanao , Kobayashi Shoko , Iwabe Koji , Yoshinaga Kentaro , Mizoguchi Hideaki
Published: November 2003

Antithymocyte globulin (ATG) is one of the effective drugs used in the treatment of aplastic anaemia (AA). Although it has been speculated that the mechanism of action of ATG is mediated by its immunosuppressive effect on lymphocytes which might have an inhibitory effect on haemopoietic stem and progenitor cells, no definite evidence of the presence of such a mechanism has been demonstrated. In this study we investigated whether such a mechanism is truly operating in ATG therapy for AA. In five patients who responded to ATG, bone marrow cells were obtained after haematological recovery and CD34‐positive cells were separated by immunobeads. Autologous CD34‐positive cells were mixed with autologous peripheral CD4‐ or CD8‐positive cells obtained before ATG therapy and after haematological recovery, liquid‐cultured for 12 h, and then cultured in methylcellulose for 14 d in the presence of haemopoietic growth factors. In all five cases studied, only the CD8 cells obtained before ATG therapy suppressed the colony forming unit‐granulocyte‐macrophage (CFU‐GM)‐ and burst forming unit‐erythroid (BFU‐E)‐derived colony formation. This result is definite evidence that one of the mechanisms of action of ATG in AA is an inhibitory effect on CD8‐positive cells which have suppressive activity for the growth of haemopoietic progenitor cells.