bluuebunny/crossref_metadata_2025 huggingface dataset:
Inducibility of lymphokine activated killer (LAK) cells in patients with acute myelogenous leukaemia in complete remission and its clinical relevance
Authors: Archimbaud Eric , Bailly Mireille , Doré Jean‐Françlois
Published: March 2008

Peripheral blood mononuclear cells (PBMC) from 42 patients with acute myelogenous leukaemia (AML) in complete remission (CR) and from normal donors were activated into LAK cells in the presence of 1000 U/ml of recombinant interleukin‐2 (rIL‐2). Cytotoxicity of LAK cells was assayed against K562, Daudi, and Raji cell lines, and autologous and/or allogeneic thawed leukaemic blasts. Fresh unactivated PBMC from normal donors and AML patients served as controls. Mean f standard deviation (SD) percentage lysis of the different targets by patient LAK cells were: K562 61 ± 20%. Daudi 62 ± 23%, Raji 48 ± 24%, autologous blast cells 12 ± 16% and allogeneic blast cells 13 ± 10%. Lysis of the different targets by LAK cells from normal donors was similar to that achieved with LAK cells from AML patients. Overall there was a good correlation between the lysis of the different targets. There was no significant difference between the percentage lysis of autologous and allogeneic thawed blast cells, although LAK cells from seven out of the 18 patients tested were unable to lyse autologous leukaemic cells. Activity of patient LAK cells did not correlate with the initial characteristics of the patient nor with the time spent in CR before harvesting PBMC for activation. At the time of analysis, 32 patients were in continuing CR and 10 had relapsed. Multivariant analysis for prognostic factors showed that patients whose LAK cells had more lytic activity on K 562 (P=0·005) and fresh blast cell (P=0·02) targets had significantly less risk of relapse than patients with little inducible LAK cell activity. These observations are in favour of clinical trials of r‐IL2 aimed at prolonging CR in AML.
Alpha interferon raises serum beta‐2‐microglobulin in patients with multiple myeloma
Authors: Tienhaara Anri , Remes Kari , Pelliniemi Tarja‐Terttu
Published: March 2008

Serum β2‐microglobulin (β2m) levels were followed in eight patients with multiple myeloma who were treated with leucocyte alpha interferon (αIFN) 6 × 106 IU daily for 1‐2 weeks before chemotherapy. Serial measurements of serum β2m were carried out regularly during the first week on αIFN. A rise in β2m was observed in all patients, with maximum increments ranging from 29% to 185% (mean 109%). The increase in β2m was most marked in patients with elevated pretreatment levels of serum β2m and creatinine. In six patients the peak value was reached between the third and the fifth day, after which the levels decreased. In two patients serum β2m remained elevated for 3 and 4 months, respectively. A marked increase in serum β2m can be induced by αIFN in myeloma, which should be taken into consideration when using β2m for the assessment of tumour response during αIFN therapy.
Differentiation between neutrophil‐bound antibodies and immune complexes
Authors: Klaassen R. J. L. , Goldschmeding R. , Vlekke A. B. J. , Rozendaal R. , Kr. von dem Borne A. E. G.
Published: March 2008

It is often unknown whether neutrophil‐bound immunoglobulins (NBIg) in patients with chronic diseases consist of bound immune complexes (IC) or of anti‐neutrophil antibodies. Until now, the detection of IC in serum by the C1q‐binding test has been used to define the nature of NBIg. Here we present studies on methods for characterization of NBIg using a bivalent IC and heat‐aggregated IgG as IC models and neutrophil alloantibodies as antibody models. Heat‐aggregated IgG was readily detected in the C1q‐binding test. However, the bivalent IC, which is capable of binding to both low‐affinity Fc receptors (FcRII and FcRIII) on neutrophils, was not detected in this test. This was due to failure of the bivalent IC to bind to C1q, as was demonstrated by studies with immobilized C1q. However, the bivalent IC as well as heat‐aggregated IgG, when bound to neutrophils from healthy donors, were not or only marginally eluted from the cell surface by lowering the pH, in contrast to a number of different cell‐bound antibodies. This led to the observation that eluates, prepared from sensitized cells, showed reactivity with donor neutrophils, whereas eluates, prepared from cells to which immune complexes had bound, did not. Thus, a negative result in the C1q‐binding test does not prove that a serum does not contain neutrophil‐binding IC, but the reactivity of an eluate can distinguish between anti‐neutrophil antibodies and bound IC.
Simple in vivo bioassay for erythropoietin
Authors: Kawamura Akinori , Imai Nobuo , Kawacuchi Tsutomu , Hayakawa Takao
Published: March 2008

A new method of in vivo bioassay for erythropoietin (EPO) is described. This method is based on the measurement of immature reticulocytes in EPO‐treated mice using an automatic microcell counter, and is simpler and more precise than the existing methods of polycythaemic mouse assay and starved rat assay.

Normal mice were injected subcutaneously for 3 successive days with EPO at the doses betweeen 0 and 9·6 IU/mouse. On the following day, 20 μl of peripheral blood from each EPO‐treated mouse was collected and haemolysed with a stromatolysing agent, Quicklyser. Residual particles derived from immature reticulocytes in the stromatolysed blood cells were counted using a microcell counter (Sysmex CC‐180A) and a cell monitor (Sysmex CM‐5). The number of the residual particles increased in a dose‐dependent manner in EPO‐treated mice. The mean of correlation coefficients of five log dose‐response lines was 0·924, and the mean of precision indices was 0·138. A good correlation was also observed between the residual particle counts and reticulocyte counts obtained from smears.
Lupus‐like anticoagulant properties of murine monoclonal antibodies to β2‐glycoprotein I
Authors: Arvieux J. , Pouzol P. , Roussel B. , Jacob M. C. , Colomb M. G.
Published: March 2008

The lipid‐binding inhibitor of coagulation, β2‐glycoprotein I (β2GPI), has been shown to form the antigen to which some autoantibodies against anionic phospholipids (aPL) are directed. Six murine monoclonal antibodies (MAbs) of the IgG1 isotype were raised against human β2GPI and could be subdivided into three groups on the basis of mutual competition experiments. MAbs 9G1 and 8C3 (group A) markedly inhibited the binding of immunoglobulins from aPL‐positive sera to β2GPI‐coated wells. Using a lipid‐based solid‐phase radioimmunoassay, the MAbs interacted with both anionic phospholipids and phosphatidylethanolamine, but not phosphatidylcholine, in a β2GPI‐dependent manner. A cross‐reaction between β2GPI from several (including bovine) species was seen with one of the MAbs (9G1). All six MAbs induced dose‐dependent prolongation of the DAPTT, DRVVT. KCT and TTI clotting times of human plasma, whereas 9G1 was the sole antibody to be inhibitory with plasma from bovine origin. Synergistic inhibitory effects were observed with MAbs used in pairs provided that they did not compete with each other for β2GPI binding. The anticoagulant activity of the MAbs was fully neutralized by the addition of freeze‐thawed platelets. The MAbs described here resemble lupus anticoagulants in several respects which makes them valuable to study the involvement of β2GPI in the autoimmune thrombotic pathophysiology.
Assessment of an EIA for measuring human serum erythropoietin as compared with RIA and an in‐vitro bioassay
Authors: Ma D. D. F. , Wei Ai‐Qun , Dowton L. A. , Lau K. S. , Wu Z.‐H. , Ueda M.
Published: March 2008

Summary A recently developed enzyme‐linked immuno‐sorbent assay (EIAZ, ELISA) using two murine monoclonal anti‐erythropoietin antibodies was compared with a radio‐immunoassay (RIA) and a commercial in‐vitro bioassay, EPOS, for measuring serum erythropoietin (Epo) in humans. Specificity and validity for Epo‐EIA and the other two assays were examined. The serum Epo in normal subjects was 18 ± 12 mU/ml (mean±SD, n= 80) for EIA compared with 22.5.18.5 mU/ml (n= 20) for RIA and 136.132 mU/ml (n= 14) for the bioassay. The serum Epo concentrations in normals and patients were highly comparable between EIA and RIA for Epo (P<0.01, r= 0.95). Epo concentrations by the EIA for normal female and male subjects were 20.5 ± 13 and 16.5 ± 10 mU/ml, respectively. Epo levels in patients with secondary polycythaemia or autoimmune haemolytic anaemia were significantly higher than normal subjects by the three methods. Epo levels in patients with chronic renal failure were within the normal range. By the EPOS bioassay, the Epo concentrations of normals and patients with renal failure were significantly higher than expected (136.132 and 447.273, respectively). Due to its inherent design, the EPOS bioassay possibly measures bone marrow proliferative activity in response to other serum growth regulators besides erythropoietin and was found to be unsuitable for clinical assessment of Epo. We concluded that the new EIA and RIA were similarly sensitive, reliable and accurate for measurement of serum Epo. The EIA method has the advantage of being less time consuming, more convenient and avoids the use of a radioisotope.
Unique genotypic features of infant acute lymphoblastic leukaemia at presentation and at relapse
Authors: Biondi Andrea , Rossi Vincenzo , Francia DI Celle Paola , Carbone Anna , Benvestito Serena , Busca Alessandro , Giudici Giovanni , Giachino Claudia , Basso Giuseppe , Foa Robin , Migone Nicola
Published: March 2008

Summary Acute lymphoblastic leukaemia (ALL) of infants aged less than 1 year represents a group of patients with peculiar biological features, poor response to therapy and unfavourable prognosis. In order better to characterize this type of leukaemia, we have investigated the immunoglobulin (Ig) and T‐cell receptor (TCR) genes configuration of 21 infants with ALL, and compared the genotypic features with the phenotypic and karyotypic data, as well as with the clinical outcome. All cases had a pre‐B phenotype; 12 (57%) of them were pre‐pre‐B ALL (CD10‐, CD 19 +). Six of the 16 cases evaluated (38%) displayed chromosomal abnormalities: five had the typical translocation t(4;11)(q21;23). Eleven cases presented with a white blood cell count greater than 100.109/1. The clinical course was unfavourable in 14 patients. The genotype of this group of ALL revealed several peculiarities. (1) Of the 21 cases, six (29%) displayed a multiple rearrangement pattern at the IgH locus. (2) In three cases (15%), the light chain genes were rearranged. (3) The TCR β and γ genes were rearranged in only one case (one case at the TCR β and one at the TCR γ locus). (4) The TCR chain was rearranged in eight cases (40%) and rarely deleted; the rearrangements observed were those most frequently observed in B cell‐precursor ALL. Two cases were evaluated both at presentation and at relapse. While the immunophenotype had remained unmodified, comparison of Ig heavy chain gene rearrangements revealed clonal variations in both cases. Taken together, these findings further underline the biological peculiarities of infant ALL compared to ALL which occurs in older children and in adults, and stress the need of differentiated and aggressive therapeutic approach for these patients.
Rapid identification by denaturing gradient gel electrophoresis of mutations in the γ‐globin gene promoters in non‐deletion type HPFH
Authors: Gottardi E. , Losekoot M. , Fodde R. , Saglio G. , Camaschella C. , Bernini L. F.
Published: March 2008

Summary We have outlined a fast, non‐radioactive strategy to identify point mutations in the 5’flanking region of the γ‐globin genes using denaturing gradient gel electrophoresis (DGGE) of amplified DMA. In a sample of previously characterized carriers of non deletion‐type hereditary persistence of fetal haemoglobin (HPFH) the different point mutations in both γ gene promoters could be easily identified by DGGE of a 327 bp fragment. A 4 bp deletion at position —225 to —222 of the Aγ globin gene was unexpectedly found in several samples and shown to represent a frequent polymorphism.

Analysis of a 681 bp fragment specific for the 5’region of the Aγ gene, showed that this can be used to determine the haplotype of the chromosome under study.

This technique may be useful in the study of sequence variations associated with high Hb F expression in physiological and pathological conditions.
Monitoring of multiple myeloma and bone marrow fibrosis with aminoterminal propeptide of type III collagen (PIIINP)
Authors: Taube Taina , Franssila Kaarle , Risteli Leila , And Juha Risteli , Elomaa Inkeri
Published: March 2008

Summary Bone marrow fibrosis is known in myelomatosis and depends on the extent of plasma cell infiltration. The serum concentration of the aminoterminal propeptide of type III procollagen (PIIINP) has previously been reported to reflect fibrogenesis in the marrow in myelofibrosis. Here we followed 15 consecutive patients with newly diagnosed multiple myeloma with repeated PIIINP measurements during treatment with intermittent courses of melphalan and prednisolone. PIIINP was found to change with clinical behaviour of the disease, nonresponders and patients with recurrent disease having elevated values and the values in responders decreasing to the normal level or remaining there. Collagen fibres in plasma cell infiltrates of biopsies from bone marrow or skeletal tumours of these patients stained heavily with antibodies against PIIINP. Our results suggest that PIIINP works as a noninvasive indicator of bone marrow fibrogenesis. In multiple myeloma PIIINP is a sensitive, but not specific marker of disease course.
Massive abdominal lymphadenopathy in hairy cell leukaemia: a report of 12 cases
Authors: Mercieca J. , Matutes E. , Moskovic E. , Maclennan K. , Matthey F. , Costello C. , Behrens J. , Basu S. , Roath S. , Fairhead S. , Flandrin G. , Brizard A. , Catovsky D.
Published: March 2008

Summary. Lymphadenopathy is an uncommon finding in hairy cell leukaemia (HCL). We report 12 HCL patients in whom relapse was associated with massive abdominal lymphadenopathy. All but one had long‐standing HCL (range 3–25 years; median 10 years); in one it was discovered at presentation. Nine patients had been splenectomized and seven had previously been treated with 2'deoxycoformycin (DCF) and/or alpha‐interferon (αIFN): three had achieved complete remission and four a partial response. The computerized tomography (CT) scan appearances were similar in all cases with a primary lymph node mass centred around the coeliac axis and involving upper para‐aortic and retropan‐creatic regions. Histology and/or cytology confirmed nodal involvement by HCL in six patients. Large immature hairy cells were seen in both lymph nodes and bone marrow, suggesting a degree of transformation. Nine patients were treated with DCF: one had complete resolution, six responded with 50–90% reduction of the lymphadenopathy, one did not respond and one is still on treatment; α‐IFN was used concomitantly or sequentially in two of the responders. One responding patient died of sepsis after four injections of DCF. Three patients received either α‐ or β‐IFN alone with no response. One elderly patient was not treated. Abdominal lymphadenopathy could be part of the natural history of HCL and/or may represent a transformation analogous to that seen in other low‐grade tymphoproliferative disorders. Routine abdominal CT scanning should be part of the work up of all patients with HCL.
Marrow proliferation and the appearance of giant neutrophils in response to recombinant human granulocyte colony stimulating factor (rhG‐CSF)
Authors: Campbell L. J. , Maker D. W. , Tay D. L. M. , Boyd A. W. , Rockman S. , McGrath K. , Fox R. M. , Morstyn G.
Published: March 2008

During a study of recombinant human granulocyte colony stimulating factor (rhG‐CSF) administration, 15 patients received twice daily i.v. infusions and nine patients received daily s.c. infusions of rhG‐CSF for 5 d prior to cytotoxic therapy, and then a second course subsequent to melphalan administration. There was a striking dose‐related neutrophilia and the appearance in the blood of early myeloid cells that express the intercellular adhesion molecule CD54. In addition, giant neutrophils or macropolycytes were observed in the peripheral blood of all patients. These cells were evident on the display of the Technicon H*1 as a population of large peroxidase positive cells, and using Feulgen staining these cells were shown to be tetraploid. Bone marrow kinetics studies performed on Day 4 or 5 indicated an increase in the proportion of bone marrow cells in S phase, G2 and mitosis, reflecting a proliferative response of the marrow. Large myeloid precursors and occasional binucleate promyelocytes were seen in the bone marrows done on Days 14 and 18 but not on Day 5. These findings indicate that administered G‐CSF has both quantitative and qualitative effects on myeloid cells in vivo.
New insights into the regulation of megakaryocytopoiesis by haematopoietic and fibroblastic growth factors and transforming growth factor β1
Authors: Han Zhong C. , Bellucci Sylvia , Wan Hai Y. , Caen Jacques P.
Published: March 2008

Effects of cytokines on murine megakaryocyte (MK) colony formation from either unfractionated marrow cells or purified early haematopoietic cells were studied. Recombinant interleukin‐3 (IL3), interleukin‐6 (IL6), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), erythropoietin (Epo) and acidic and basic fibroblast growth factor (aFGF and bFGF) each was able to stimulate MK colony growth although they varied somewhat in their potential. IL6 and FGFs, in addition to their effect on MK colony growth, increased the size of individual MK. The combination of IL3 with IL6 or FGF resulted in an additive action. Monoclonal anti‐IL6 antibody completely neutralized the activity of mouse IL6 and FGFs but had no effect on human IL6, mouse IL3 and GM‐CSF. When using purified lineage negative marrow cells, only IL3 and IL6 promoted MK colony formation. Transforming growth factor β1 (TGF‐β1) at 10–200 pg/ml selectively inhibited IL3‐induced MK colony formation, and at 0·2–0·5 ng/ml it still had no obvious effect on the activity of IL6 or GM‐CSF but caused an inhibition of FGF‐induced MK colony formation. These data suggest that differential mechanisms are involved in the regulation of megakaryocytopoiesis by IL3, IL6, FGFs and GM‐CSF, and that TGF‐β1 negatively regulates MK development mainly by interfering with the action of IL3.
Molecular analysis of clonality in plasma cell dyscrasias
Authors: Miglino Maurizio , Gaetani Gian Franco , Canepa Letizia , Meloni Tullio , Forteleoni Gavino , Ferraris Anna Maria
Published: March 2008

It has been suggested that multiple myeloma, generally considered a neoplastic disorder of mature plasma cells, may arise from a pluripotent haemopoietic stem cell. The possibility that circulating lymphocytes derive from the same neoplastic progenitor has been tested in a large number of studies in the past few years, as proof of the interest that this subject is raising among scientists, and also of its elusiveness. We studied a group of 29 patients with plasma cell dyscrasias in order to evaluate clonality of haemopoietic cell populations. The X‐linked markers hypoxantine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase (PGK) disclosed no monoclonal component in seven heterozygous women. Analysis of immunoglobulin gene rearrangement with four probes showed a germline configuration in samples from 25/29 patients. Only four bone marrow samples from subjects with aggressive disease had rearranged Cμ sequence; one had rearrangement of JH and Cμ.
Ber‐H2 (anti‐CD30)‐saporin immunotoxin: a new tool for the the treatment of Hodgkin's disease and CD30+ lymphoma: in vitro evaluation
Authors: Tazzari Pier Luigi , Bolognesi Andrea , Totero Daniela de , Falini Brunangelo , Lemoli Roberto M. , Soria Marco R. , Pileri Stefano , Gobbi Marco , Stein Harald , Flenghi Leonardo , Martelli Massimo F. , Stirpe Fiorenzo
Published: March 2008

An immunotoxin containing an anti‐CD30 monoclonal antibody (Ber‐H2) and saporin, a ribosome‐inactivating protein type 1, is described. It specifically inhibits protein synthesis by Hodgkin derived target cell lines with a very high efficiency (IC50 ranging from 5 × 10–12 M to 5.10∼14 M, assaporin), while irrelevant immunotoxins do not. Present results suggest that this immunotoxin could be used for in vivo therapy as well as for ex vivo bone marrow purging in Hodgkin's disease and CD304+ lymphomas.
Long‐term follow‐up of patients with hairy cell leukaemia treated with pentostatin: lymphocyte subpopulations and residual bone marrow infiltration
Authors: Thaler Josef , Grünewald Kurt , Gattringer Claus , Ho Anthony D. , Weyrer Katharina , Dietze Otto , Stauder Reinhard , Fluckinger Thomas , Lang Alois , Huber Heinz
Published: March 2008

Summary. Peripheral blood lymphocyte (PBL) subsets and bone marrow biopsies were analysed in six patients with hairy cell leukaemia (HCL) treated with 2′‐deoxycoformycin (pentostatin, DCF) according to a phase II trial of the EORTC Leukemia Cooperative Group. All patients responded to DCF with four complete and two partial remissions according to conventional criteria.

Within the PBL subsets, major changes concerned the CD4± T cells, which during DCF therapy were distinctly suppressed to nadir values of 0–038–0–18 (median 0–126) × 109/‐1. In five patients these cells returned to normal 3‐0–49–5 (median 14·5) months after the last DCF injection. CD8± cells were decreased to a lesser extent, and NK cell numbers improved during treatment.

Bone marrow immunohistology applying the MoAb B‐ly7 demonstrated residual hairy cells (HCs) in all of the six patients following DCF treatment with nadir HC numbers of 0·2–3·0% of bone marrow cells. Immunoglobulin gene rearrangement analysis of DNA obtained from these biopsies showed only germline bands, whereas rearranged bands had been present on the pretreatment specimens. Within the observation period of 15·5–54·0 (median 47·0) months after discontinuation of DCF therapy, immunohistology demonstrated a continuous increase in HC numbers in five of the six patients with clonal rearrangement detectable in bone marrow specimens from three of these patients at last follow‐up date. Although established on the basis of a small number of patients, these data suggest that DCF treatment as currently employed in HCL is unable to eradicate the malignant B cell clone.
Role in growth regulation of cytokines and cytokine receptors in acute lymphoblastic leukaemia expressing myeloid markers
Authors: Komada Yoshihiro , Zhou Yan‐Wen , Zhang Shao‐Li , Azuma Eiichi , Sakurai Minoru
Published: March 2008

Summary. The biological roles of cytokines and cytokine receptors were examined in acute lymphoblastic leukaemia cells expressing myeloid antigens (My+ ALL). Interleukin‐3 (IL‐3), granulocyte‐colony stimulating factor (G‐CSF), granulocyte‐macrophage‐CSF (GM‐CSF), and low molecular‐weight B‐cell growth factor (LW‐BCGF) could induce DNA synthesis in certain cases of My+ ALL. Whereas in My− ALL the stimulatory effects were shown only with LW‐BCGF. Acute myelogenous leukaemia (AML) cells were activated with multiple cytokines including interleukin‐1 (IL‐1), IL‐3, G‐CSF and GM‐CSF. Specific receptors for IL‐1 and IL‐3 were strongly expressed on both My+ and My− ALL cells. These receptors, however, were weakly detectable on AML cells. Additionally we studied the production of tumour necrosis factor‐α and IL‐1 by leukaemic blasts and found that distinct amounts of both cytokines were released from My+ ALL cells and AML cells, but not from My− ALL cells.

The profiles of cytokines and cytokine receptors expressed by My+ ALL showed both similaries and differences to those in My− ALL or AML. The proliferation of My+ ALL cells was dependent on multiple cytokines that would regulate growth and maturation in a lineage‐restricted fashion. These data suggested that My+ ALL cells might originate from uncommitted haematopoietic precursor cells coexpressing features of both lymphoid and myeloid lineages.
Subclinical autonomic dysfunction in patients with Hodgkin's disease and non‐Hodgkin's lymphoma
Authors: Turner Marc L. , Boland Oliver M. , Parker Alistair C. , Ewing David J.
Published: March 2008

Summary. A prospective study was carried out to determine the prevalence of autonomic dysfunction in patients with lymphoma, and to assess the effect on this of chemotherapy. Twenty consecutive patients presenting with Hodgkin's disease, high‐grade non‐Hodgkin's lymphoma, or low‐grade non‐Hodgkin's lymphoma were studied. All had advanced disease, requiring combination chemotherapy which included the use of vinca alkaloids. Clinical assessment and standard cardiovascular autonomic function tests were carried out prior to and following completion of chemotherapy. Although no patients had clinical evidence of autonomic neuropathy at presentation, 16 (80%) had abnormal cardiovascular autonomic function tests. There was no correlation with the presence or absence of mediastinal disease. There was significant improvement in autonomic scores with treatment despite the use of drugs of known neurological toxicity. Some patients showed residual abnormalities of autonomic function despite disease resolution. We suggest that subclinical autonomic dysfunction is common in patients with lymphoma, and probably represents a paraneoplastic syndrome—the pathogenesis and prevalence of which deserve further study. This phenomenon may predispose patients with lymphoma to develop gastrointestinal and genitourinary dysfunction, or postural hypotension, and should be considered during the evaluation of the neurotoxicity of chemotherapy regimens.
Lymphotoxin, tumour necrosis factor and interleukin‐6 gene transcripts are present in Hodgkin and Reed‐Sternberg cells of most Hodgkin's disease cases
Authors: Foss Hans‐Dieter , Herbst Hermann , Oelmann Elisabeth , Samol Jens , Grebe Maren , Blankenstein Thomas , Matthes Jesko , Qin Zhi‐Hai , Falini Brunangelo , Pileri Stefano , Diamantstein Tibor , Stein Harald
Published: March 2008

Summary. Tissue specimens from 26 cases of Hodgkin's disease (HD) and six HD‐derived cell lines were analysed for tumour necrosis factor (TNF), lymphotoxin (LT), and interleukin (IL)‐6 RNA transcripts by in situ hybridization, in some cases subsequent to immunohistology for CD30 antigen. LT and TNF transcripts were found in tumour cells of all cases; IL‐6 gene transcripts were detectable in 19/23 cases. Presence of RNA specific for these cytokines was not correlated with any of the following parameters: sex, symptoms and histotype, as well as immunophenotype and association of the tumour cells with Epstein‐Barr virus. Rather, the presence of LT, TNF and IL‐6 transcripts appeared to characterize Hodgkin and Reed‐Sternberg cells in general, supporting concepts which suggest that HD represents a malignancy of cytokine secreting activated cells, and that many of the features distinguishing HD from other malignant lymphomas may ultimately be due to expression of cytokines. LT and TNF RNA transcripts were also found in five HD‐derived cell lines, whereas supernatants of these cell lines contained high levels of LT, but low or undetectable levels of TNF activity. This suggests that, although not detectable at the level of RNA transcripts, differences between HD cases may exist on the level of cytokine gene transcript processing, translation and polypeptide secretion.
Myelodysplastic syndrome with eosinophilia in bone marrow
Authors: Matsushima Takafumi , Murakami Hirokazu , Sawamura Morio , Tamura Jun'ichi , Sakura Tohru , Matsumoto Morio , Hirabayashi Hisami , Miyawaki Shuichi , Simano Shunichi , Sato Sadao , Tsuchiya Jun
Published: March 2008

Summary. Clinical features were investigated in 114 patients with de novo myelodysplastic syndrome (MDS) diagnosed over the past 10 years, and eight cases (7%) were complicated with eosinophilia in the bone marrow. Two patients had refractory anaemia (RA), five had RA with excess of blasts (RAEB), and one had RAEB in transformation. Their bone marrow cells exhibited trilineage dysplasia and morphological abnormalities in eosinophils. Cytogenetics revealed major karyotype abnormalities (MAKA) in five patients. Survival durations were significantly shorter than those of other MDS patients. Our study suggests that marrow eosinophilia in MDS is strongly related to the coexistence of MAKA.
Direct sequence analysis of TCR Vδ2–Dδ3 rearrangements in common acute lymphoblastic leukaemia and application to detection of minimal residual disease
Authors: Langlands K. , Eden O. B. , Micallef‐Eynaud P. , Parker A. C. , Anthony R. S.
Published: March 2008

Summary. T cell receptor δ chain (TCRδ) gene rearrangements were studied by Southern blot analysis in 36 patients with common acute lymphoblastic leukaemia, including 14 adults and 22 children. The majority of patients (68%) had either a rearrangement or deletion of one or more TCRδ genes. The most frequent rearrangement involved a partial recombination of Vδ2 to Dδ3 (55%). Dδ2–Dδ3 rearrangements were present in five patients (14%). To investigate the TCRδ rearrangement as a tumour marker in minimal residual disease studies, presentation samples from 18 patients were amplified by PCR and directly sequenced. Although the size of the Vδ2–Dδ3 junction varied by only 40 bp, sequence analysis showed extensive diversity. This was derived from four factors: deletion of the 5’end of Dδ3 gene (15/18) and 3’end of Vδ2 gene (16/18); the presence of Dδ2 sequences (6/18); insertion of N nucleotides (15/18); association of P nucleotides with intact Vδ2 and Dδ3 genes (5/18). N nucleotides were the major feature, contributing to 75% of the junction. Dδ1 sequences were not involved. Twenty base oligonucleotide probes, constructed from the junctional sequences, were capable of detecting residual tumour cells at the 10−4 sensitivity level. Cross hybridization studies confirmed the probes to be clone specific. Longitudinal studies on patients undergoing treatment were capable of detecting tumour in remission samples.
Markers of procoagulant imbalance in patients with localized melanomas and autoimmune disorders
Authors: Tripodi Armando , Mannucci Pier Mannuccio , Chantarangkul Veena , Bottasso Biancamaria , Arbini Arnaldo A. , Bella Silvia Della , Scorza Raffaella
Published: March 2008

Summary. Hypercoagulability can be defined as a condition of procoagulant imbalance due to heightened enzymatic activation of coagulation zymogens, but with no laboratory evidence of fibrin deposition nor clinical signs of thrombosis. The imbalance can be detected by measuring the plasma levels of prothrombin fragment 1 + 2 (F1 + 2), fibrinopeptide A (FPA) and thrombin–antithrombin III (TAT) complexes. The aims of this study were to establish the frequency of existence and biochemical pattern of hypercoagulability in patients with cancer and autoimmune disorders, clinical conditions associated with an increased risk of thrombosis, and to ascertain the most sensitive method for its diagnosis. In approximately one‐fourth of the patients hypercoagulability was identified by finding high levels of FPA F1 + 2 or TAT unaccompanied by signs of fibrin deposition (expressed by normal levels of D‐dimer). In a smaller proportion of patients (approximately 10%), the concomitant presence of high levels of D‐dimer indicated that the activation of the coagulation cascade had gone beyond the stage of heightened enzymatic activity to the point of cross‐linked fibrin deposition. Of the markers used to detect hypercoagulability, FPA seems to be the most sensitive, being significantly increased in all clinical conditions studied.
The generation of thrombin in vivo induces the selective loss of high molecular weight multimers of von Willebrand factor and the reversible sequestration of platelets
Authors: Toh Cheng Hock , Hoogendoorn Hugh , Giles Alan R.
Published: March 2008

Summary Various levels of thrombin generation were induced by the infusion of a combination of factor Xa (F.Xa) and phosphatidylcholine/phosphatidylserine (PCPS) vesicles into normal dogs and non‐human primates. In the dog, an immediate loss of von Willebrand factor antigen (vWF:Ag) with a progressive recovery to normal levels by 45 min was observed. Multimeric assay demonstrated a selective loss of high molecular weight multimers (HMWM) with subsequent replacement. At low doses, in non‐human primates (chimpanzees), identical changes to those seen in the dog were observed and this was associated with an equivalent loss of ristocetin co‐factor activity (vWF:RCoF). At high dose B reversal of the wWF response occurred with levels increasing to twice that of baseline values by 2 min and multimeric analysis demonstrated the presence of abnormally large multimers and increased vWF:RCoF specific activity, suggesting that the response at each dosage reflected a net balance of consumption over release. This was supported by in vitro simulation where increasing thrombin generation was associated with a selective loss of HMWM without replacement. In both species, an immediate fall in platelet count occurred and this was directly correlated with the amount of thrombin generated. Full recovery occurred within 45 min and isotopic labelling studies demonstrated that platelet sequestration rather than consumption was occurring. These studies demonstrate that thrombin generation in vivo is associated with a selective loss of the multimeric forms of vWF known to interact with platelets and this may provide an in vivo model to characterize the physiology/pathophysiology of this primary event in haemostasis.
Long‐standing remission after 25‐OH D3 treatment in a case of chronic myelomonocytic leukaemia
Authors: Mellibovsky Leonardo , Díez Adolfo , Aubia Jaume , Nogues Xavier , Pérez‐Vila Encarna , Serrano Sergi , Recker Robert R.
Published: March 2008

Summary This report describes a case of chronic myelomonocytic leukaemia (CMML) in whom a complete remission was achieved and sustained 15 months after treatment with 25‐OH vitamin D3. No side‐effects were observed. Although vitamin D3 has been used in the treatment of myelodysplastic syndromes, to our knowledge this is the first case of longstanding remission in a patient with CMML. This ‘differentiation therapy’ might be considered as a possible alternative in the management of this disease.
Nodular lymphocyte predominance Hodgkin's disease featuring blood atypical polyclonal B‐cell lymphocytosis
Authors: Mariette Xaviek , Tsapis Andréas , Oksenhendler Eric , Daniel Marie‐Thérèse , D'Agay Marie‐Françoised , Berger Roland , Brouet Jean‐Claude
Published: March 2008

Summary The nodular lymphocyte predominance form of Hodgkin's disease (NLPHD) is considered as a B cell derived lymphoproliferative disease. A patient with NLPHD presented with an absolute increase in blood B cells with cytological features of mantle zone cells: these B cells were polyclonal, did not exhibit bcl‐2 gene rearrangement. and some of them displayed non‐clonal chromosomal aberrations. EB virus genome was not detected by Southern analysis. Thus, this study, taking advantage of the presence of an unusual population of blood atypical B cells, confirms data obtained on lymph nodes where, however, malignant cells may be scarce, indicating that NLPHD is a polyclonal B cell lymphoproliferative disease of mantle zone origin.
Granulocyte‐colony‐stimulating factor induces increased serum levels of soluble interleukin 2 receptors preceding engraftment in autologous bone marrow transplantation
Authors: Dreger Peter , Grelle Karen , Eckstein Volker , Suttorp Meinolf , Müller‐Ruchholtz Wolfgang , Löffler Helmut , Schmitz Norbert
Published: March 2008

Summary The serum levels of soluble interleukin 2 receptors (sIL‐2R) were determined in 19 patients who received highdose chemotherapy and an autologous or syngeneic bone marrow transplant (BMT) for treatment of Hodgkin's disease (n= 18) or non‐Hodgkin's lymphoma (n= 1). Twelve patients received granulocyte colony‐stimulating factor (G‐CSF) from day 0 or day +1 after autologous BMT until the white blood cell count had been stable for 9 d above 1 × 109/1, the remaining seven patients did not receive growth factors, In all G‐CSF‐treated patients the sIL‐2R levels increased steadily in the early post‐transplant course, even in the absence of infection. This increase was statistically significant 2‐4 d prior to the appearance of leucocytes in the peripheral blood (median 340 pm versus median 256 pm immediately after BMT, P<0.025) and peaked with the appearance of first peripheral blood leucocytes (median 536 pm, P<0.001). Cessation of G‐CSF administration resulted in a decline of sIL‐2R levels. In contrast, five of seven patients without G‐CSF treatment did not exhibit an sIL‐2R increase before or at the time of engraftment. Infection was associated with a rise of sIL‐2R levels. A correlation between sIL‐2R levels and total leucocyte count, lymphocyte count, or CD25 + lymphocyte count was not evident.

These data suggest that after autologous BMT G‐CSF induces increased sIL‐2R levels, which occur independent of lymphocyte activation. This may be compatible with involvement of immature bone marrow cells in G‐CSF‐induced sIL‐2R release.
Severe poikilocytosis associated with a de novoα28 Arg→Cys mutation in spectrin
Authors: Lorenzo F. , del Giudice E. Miraglia , Alloisio N. , Morle L. , Forissier A. , Perrotta S. , Sciarratta G. , Iolascon A. , Delaunay J.
Published: March 2008

Summary Severe poikilocytosis was observed in an Italian child. The mutation responsible was a de novoα28 Arg→Cys substitution (CGT→TGT) in spectrin, a mutation known to cause hereditary elliptocytosis or hereditary pyropoikilocytosis. In this particular case the severity of the manifestations were accounted for by the occurrence, in trans to the α28 mutation, of the αv/41 polymorphism. The latter has been shown previously to be associated with structural abnormalities at the αIV‐αV domain junction and with a low expression level. The pronounced alteration of the dimer self association process was also explained by the location of the α28 mutation. This mutation occurs in helix 3 of repeating segment α1, e.g. precisely in the head‐to‐head contact between the spectrin α and β chains. The present phenotype was compared to that yielded by another α28 mutation (Arg→His) also combined, in trans, with the αv/41 polymorphism. The pictures were very much alike, stressing the functional importance of residue α28. The de novo character of the present mutation strengthens the view that codon α28 is a ‘hot spot’ for mutations.
Assessment of graft status following allogeneic bone marrow transplantation for haematological disorders in children using locus‐specific minisatellite probes
Authors: Katz Fay , Hann Ian , Kinsey Sally , Ball Sarah , Morgan Gareth , Chessells Judith
Published: March 2008

We have examined peripheral blood or bone marrow DNA following allogeneic bone marrow transplantation (BMT) in children suffering from a variety of haematological disorders. Using either locus‐specific minisatellite probes separately or in combination with a Y specific probe for sex‐mismatched transplants, complete haematological chimaerism, autologous reconstitution, or mixed chimaeric states have been defined immediately post‐BMT. We have also been able to identify emerging autologous cells or relapse prior to morphological diagnosis.

Forty‐two children, mean age 6.4 years (range 9 months to 15 years), received an allogeneic BMT for: acute lymphoblastic leukaemia (ALL), n= 17; acute myeloid leukaemia (AML), n= 5; biphenotypic leukaemia, n= 1; myelodysplastic syndrome (MDS), n= 5; chronic granulocytic leukaemia (CGL), n= 1; severe aplastic anaemia (SAA), n= 7; familial erythrophagocytic lymphohistiocytosis (FEL), n= 2; beta thalassaemia major (beta thal), n= 1; and juvenile chronic myeloid leukaemia (JCML), n= 3. Immediately post‐transplant, 78% had achieved complete haematological chimaerism, 12% had failed to engraft (DNA analysis confirmed autologous reconstitution) and 10% had mixed chimaerism. SAA was the underlying disease in two of the chimaeric cases, the third case having had a matched unrelated donor (MUD) BMT for MDS. In 3/4 cases which subsequently relapsed, DNA analysis showed re‐emergence of autologous cells (indicative of relapse), prior to their morphological identification. We conclude that DNA analysis using minisatellite probes to assess graft status provides a useful contribution to patient management following allogeneic BMT.
Endogenous lymphokine activated killer cell activity and cytogenetic response in chronic myelogenous leukaemia treated with α‐interferon
Authors: Meseri A. , Delwail V. , Brizard A. , Lecron J. C. , Pelletier D. , Guilhot F. , Tanzer J. , de Laforest P. Goube
Published: March 2008

The capacity of α‐interferon (α‐IFN) to induce lymphokine activated killer (LAK) cytotoxicity in the absence of interleukin‐2 (IL2) has prompted us to test whether or not its ability to reduce dramatically the number of Ph1 + clones in chronic myelogenous leukaemia (CML) patients is in part mediated through the generation of natural killer (NK) or LAK activity. The latter were tested using NK‐sensitive (K562) and NK‐resistant (Raji) cell lines in a target‐cell colony‐growth inhibition assay. Effector cells (E) were patient blood mononuclear cells (MC) without in vitro activation prior to their coculture with targets (T). Out of 16 patients tested so far, three failed to undergo cytogenetic remission under α‐IFN therapy. No NK nor LAK cells could be detected in the MC from two of them while the other displayed NK activity within upper normal limits. 13 patients underwent complete (eight) or partial (five) cytogenetic remission together with significantly high NK and/or LAK activity as compared to normal controls. These observations could favour the hypothesis of an indirect effect of α‐IFN on leukaemic cells, mediated by cells involved in immune surveillance.
Haemopoietic CD34+ progenitor cells are not infected by HIV‐1 in vivo but show impaired clonogenesis
Authors: Luca Andrea De , Teofili Luciana , Antinori Andrea , Iovino Michela Stefania , Mencarini Paola , Visconti Elena , Tamburrini Enrica , Leone Giuseppe , Ortona Luigi
Published: March 2008

Summary. We evaluated the role of CD34 + bone marrow progenitor cells in vivo, in the pathogenesis of AIDS‐related haematological abnormalities. The clonogenic activity of CD34+ cells from seven patients with HIV‐1 infection, without bone marrow involving opportunistic infections or neoplasms, was assessed in semisolid cultures. The number of CFU‐GM was significantiy reduced as compared to the controls (P=0.017). independently from myelotoxic therapy, while the number of BFU‐E was not. The presence of retroviral sequences in CFU‐GM colonies from four patients and in the total population of CD34 + cells from six patients with advanced stage HIV infection was investigated using the polymerase chain reaction. The presence of HIV‐1 sequences was also searched for in a purified suspension of CD34 + cells after 3 weeks liquid culture. All these cells were always HIV‐1 negative, while viral sequences were always detected in bone marrow mononuclear cells from these and other patients. The number of HIV‐1 DNA copies decreased with increasing enrichment. At most 1:10000 CD34+ cells are infected in vivo. Other mechanisms than direct viral infection of progenitor cells must account for the defective haemopoiesis in HIV‐1 infected patients.
An IVS‐I‐117 (G→A) acceptor splice site mutation in the α 1‐globin gene is a nondeletional α‐thalassaemia‐2 determinant in an Indian population
Authors: Clürük M. A. , Baysal E. , Gupta R. B. , Sharma S. , Huisman T. H. J.
Published: March 2008

Summary. In 1991 we reported the identification of two deletional α‐thalassaemia‐2 determinants (— 3.7 kb and — 4.2 kb) and one nondeletional α‐thalassaemia‐2 determinant (Hb Koya Dora α2 codon 142, TAA→TAA) in a tribal population in Central India (Gupta et al, 1991). Evidence was obtained at that time for the possible presence of an additional nondeletional α‐thalassaemia‐2 because of low levels of Hb S (<28%) in some Hb S heterozygotes with a simple α‐thalassaemia‐2 heterozygosity (—α/αα). This abnormality has now been identified as a G→A mutation at IVS‐I‐117 of the α1‐globin gene (acceptor splice site) which makes this gene nonfunctional. Its frequency was established at ˜6% which raises the total frequency of α‐thalassaemia determinants in this population to ˜60%. Subjects with a deletional α‐thalassaemia‐2 and the newly discovered α1 acceptor splice junction mutation in trans appear to have an α chain deficiency similar to that of an α‐thalassaemia‐2 homozygote (—α/—α). An additional change (C→G) at the Cap —4 site was observed in six α1‐ and one α2‐globin genes: this polymorphism is not associated with a decrease in α chain synthesis and is not linked to the IVS‐I‐117 (G→A) mutation.
Abnormal factor VIII Hiroshima: defect in crucial proteolytic cleavage by thrombin at Arg1689 detected by a novel ELISA
Authors: Kamisue Seiki , Shima Midori , Nishimura Takuya , Tanaka Ichiro , Nakai Hiroaki , Morichika Shogo , Takata Noboru , Kuramoto Atsushi , Yoshioka Akira
Published: March 2008

We have established an ELISA for detecting thrombin cleavage of the FVIII light chain at Arg1689. The method used a coating alloantibody which recognized amino acid residues 2248–2312 in the C2 domain, together with a second monoclonal antibody, NMC‐VIII/10, which recognized residues 1675–1684 in the amino‐terminal region of the light chain. FVIII antigen (FVIII:Ag) was measured after treatment of plasma with various concentrations of thrombin. The FVIII:Ag of normal plasma was reduced in a dose‐dependent manner by the thrombin, falling to 28% in the presence of 100 U/ml enzyme. The concentration of thrombin that achieved 50% reduction (IC50) was approximately 1·0 U/ml. The plasma of four haemophilia A positive (A+) and two haemophilia A reduced (AR) patients were analysed. The IC50 of all patients was more than 1·0 U/ml, indicating that thrombin cleavage of the FVIII light chain was defective. One haemophilia A+ plasma did not respond to thrombin in this ELISA system. The patient (TI) was a haemophiliac with FVIII coagulant activity of 0·04 U/ml and FVIII:Ag of 1·78 U/ml. In addition, immunoblotting of the purified FVIII from TI showed that thrombin cleavage of the 80 kilodalton (kD) light chain was impaired. The patient's DNA was amplified using the polymerase chain reaction with a set of synthetic oligonucleotide primers spanning amino acid residues 1646–1714. Sequence analysis of the amplified DNA fragments revealed a cytosine to thymine transition, converting an arginine 1689 to cysteine. This abnormal FVIII was designated as FVIII Hiroshima. Our ELISA system is a simple and useful method of evaluating the proteolytic cleavage by thrombin at Arg1689.