bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha‐interferon (α‐IFN) protects B‐chronic lymphocytic leukaemia cells from apoptotic cell death in vitro
Authors: Panayiotidis Panayiotis , Ganeshaguru Kanagasabai , Aafa Shireen , Jabbar B. , Hoffbrand A. Victor
Published: March 2008

B‐chronic lymphocytic leukaemia cells (CLL) are prone to apoptotic cell death when cultured in vitro. Apoptosis and loss of the bcl‐2 protein is prevented in CLL cells cultured in the presence of interleukin‐4. In this study we analysed the effects of α‐IFN on the DNA fragmentation, bcl‐2 protein levels and cell survival in purified B‐cells from 16 CLL patients. α‐IFN (103 U/ml) reduced the degree of spontaneous DNA fragmentation of CLL cells after a 30 h culture period (from a mean of 22·2% in control cultures to 10·5%, P<0·01). This inhibition was accompanied by preservation of bcl‐2 protein and an increased survival of CLL cells compared to control cultures. In parallel, α‐IFN inhibited hydrocortisone induced DNA fragmentation in CLL cells. The effects of α‐IFN on DNA synthesis of CLL cells were variable (in two patients a decrease and in seven an increase in 3H‐thymidine uptake) and did not correlate with the effect on DNA fragmentation. In conclusion, our data suggest that α‐IFN, like IL‐4 and γ‐IFN, inhibits apoptosis of CLL cells. These in vitro data indicate that the clinical responses of some CLL patients to α‐IFN cannot be explained by a direct cytotoxic effect of α‐IFN on circulating CLL cells. Alternatively, α‐IFN may inhibit the proliferation of the small fraction of clonogenic CLL progenitors, or interfere with cellular interactions necessary for the survival and growth of CLL cells.
Characterization of specific functional receptors for HuIFN‐α on a human megakaryocytic cell line (Dami): expression related to differentiation
Authors: Martyré Marie‐Claire , Wietzerbin Juana
Published: March 2008

Summary Interferon‐alpha (IFN‐α) treatment has been shown to be highly effective in inhibiting human megakaryocytopoiesis and controlling thrombocytosis in patients with myeloproliferative disorders. These observations suggest that IFN‐α might play some role in the biological feature of the megakaryocytic lineage and led us to investigate the presence of specific receptors for IFN‐α on human megakaryocytic cells, i.e. the Dami cell line, and to study the regulation of their expression. Our study demonstrates that [125I]‐recombinant human IFN‐α ([125I]rHu‐IFN‐α) binds to high‐affinity specific receptor on these cells. Scatchard analysis of binding data indicates the presence of homogeneous binding sites estimated in the range of 3000–5000. with an apparent equilibrium dissociation constant, Kd, of 1–2 × 109m. Also, [125I]rHuIFN‐α binding capacity decreased in Dami cells incubated with unlabelled rHuIFN‐α. This down‐regulation which was dose‐dependent appeared to result from a reduction of IFN‐α cell surface receptors and was observed at doses that elicited antiproliferative effects in Dami cells. Crosslinking of [125I]rHuIFN‐α to Dami membrane proteins using a bifunctional reagent yielded to a radioactive complex of 150,000 kD on SDS‐PAGE. Furthermore, in response to PMA, which induces the differentiation/maturation of the Dami cells as evaluated by surface marker and ploidy analysis, a 3‐fold increase of the number of specific membrane receptors for IFN‐α was observed, without any modification of either the affinity or the Mr value of the cross‐linked complex. Such an increase appeared to be restricted to IFN'‐a receptors; actually it was not observed in [125I]IFN‐y binding experiments. Transcript analysis indicated that down‐regulation and increased expression of the IFN‐α receptor after PMA treatment are post‐transcriptional events.
Endogenous haemopoietic growth factors in neutropenia and infection
Authors: Cebon Jonathan , Layton Judith E. , Maher Darryl , Morstyn George
Published: March 2008

Summary Haemopoietic growth factors (HGFs) are being administered to patients with neutropenic fever; however, little is known about the endogenous HGF response in these patients. Specific assays were used to study four HGFs, granulocyte (G‐) CSF, granulocyte‐macrophage (GM‐) CSF, macrophage (M‐) CSF and interleukin (IL‐) 6 levels in the blood of patients with neutropenic fever (46 episodes). For comparison, levels were also measured in three control populations: normals (20), afebrile neutropenic (14), and bacteraemic but not neutropenic patients (20).

In febrile patients, levels of G‐CSF (median, range) (0·46, <0·10–142 ng/ml), IL‐6 (0·054, 0·05·24–3 ng/ml), and M‐CSF (18·5, 9·9–79·1 ng/ml) were elevated compared with afebrile subjects (<0·10, <0·0–1–6·2 ng/ml), (0·008, 0·002–0·024 ng/ml) and (6·45, < 5·0–31·3 ng/ml) respectively. GM‐CSF was not elevated (<0·02, <0·02–8·0 ng/ml) compared with afebrile subjects (0·021, < 0·02–0·20 ng/ ml).

Variables significantly associated (P < 0·05) with elevated cytokine levels were determined by multiple regression analyses. Factors associated with G‐CSF elevation were fever, neutropenia, pathogen type and raised bilirubin and creatinine. In contrast, neutropenia was not associated with IL‐6 elevation although there was an association between IL‐6 elevation and fever, Gram‐negative and fungal infections and raised creatinine and bilirubin. M‐CSF elevation was associated with fever, renal impairment and known pathogen. Elevated G‐CSF and IL‐6 levels normalized rapidly (hoursdays) with the resolution of infection, whereas M‐CSF concentrations remained elevated for up to 10 d. Cytokine levels remained elevated in septic neutropenic patients who did not recover.

In summary, G‐CSF, IL‐6 and M‐CSF levels were significantly elevated in sepsis. In contrast, GM‐CSF levels were not elevated. These studies should assist the development of therapeutic strategies using HGFs in the treatment of sepsis.
The acute lymphoblastic leukaemia cell line SEM with t(4;11) chromosomal rearrangement is biphenotypic and responsive to interleukin‐7
Authors: Greil Johann , Gramatzki Martin , Burger Renate , Marschalek Rolf , Peltner Markus , Trautmann Udo , Hansen‐Hagge Thomas E. , Bartram Claus R. , Fey Georg H. , Stehr Klemens , Beck Jörn
Published: March 2008

Summary A cell line, designated SEM, was established from the peripheral blood of a 5‐year‐old girl in relapse with acute lymphoblastic leukaemia (ALL). Both the lymphoblasts of the patient and the cells of the cell line SEM showed the t(4:11) chromosomal rearrangement. The analysis of the immunophenotype of the SEM cell line revealed the B‐cell differentiation antigens CD19, CD22 and CDw75 in the absence of CD20. CD24 and immunoglobulin expression. Besides B‐lineage antigens. SEM cells were positive for the myeloid antigens CD13, CD15, CD33 and CDw65. Immunogenotypic analysis of SEM cells showed a monoclonal rearrangement of immunoglobulin heavy‐chain (IgH), T‐cell receptor (TCR) γ and δ genes. Addition of interleukin (IL)‐7 promoted the growth of the patient's lymphoblasts in culture and enhanced the proliferation of SEM cells. The SEM cells also express messenger RNA (mRNA) for the IL‐7 receptor (IL‐7R), but no evidence for autocrine production of IL‐7 by the cell line was found. Addition of IL‐4, tumour necrosis factor (TNF)‐α, interferon (IFN)‐α, or IFN‐γ resulted in a profound inhibition of SEM growth. Thus, these cytokines may have important growth regulatory activities for biphenotypic leukaemic ALL cells.
Improved survival for acute lymphoblastic leukaemia in infancy: the experience of EORTC‐Childhood Leukaemia Cooperative Group
Authors: Ferster Alina , Bertrand Yves , Benoit Yves , Boilletot Annie , Behar Catherine , Margueritte Geneviève , Thyss Antoine , Robert Alain , Mazingue Françoise , Souillet Gérard , Philippe Noël , Solbu Gabriel , Suciu Stefan , Otten Jacques
Published: March 2008

Summary Out of 744 newly diagnosed ALL children under the age of 18 years treated according to the EORTC‐CLCG protocols 58831 and 58832, 28 (4%) were infants less than 1 year of age. An elevated risk factor, which takes into account the sizes of the liver and spleen and the number of circulating blasts, was present in 25 cases.

Most patients had non‐common ALL. Among 15 patients studied by cytogenetics, nine present chromosonal abnormalities, six of them having a t(4:11) translocation.

Complete remission was achieved in 86% of cases. One patient died in complete remission of therapy‐related infection. The overall EFS is 43%. It is not statistically different in very young infants as compared to infants older than 6 months. Except for patients with AUL or with t(4:11) translocation, a continuous complete remission rate above 50% can be achieved with a median follow‐up of 4 years.

The results obtained in infant ALL with EORTC‐CLCG protocols are currently better than those obtained with some other protocols, but remains inferior when compared to the ones obtained in older children. Thus, further improvements are needed and should be evaluated in large cooperative trials.
Collagenase activity and collagenase‐inhibitory factors of serum and bone marrow fibroblast‐conditioned medium in chronic myelogenous leukaemia
Authors: Yonekura Shuji , Nagao Tadami
Published: March 2008

Summary Chronic myelogenous leukaemia (CML) is frequently accompanied by progressive myelofibrosis that is probably largely due to increased collagen production by bone marrow fibroblasts or decreased collagen degradation by collagenase. We investigated the activity of collagenase and collagenase‐inhibitory factors (CIF) in serum and bone marrow fibroblast‐conditioned medium obtained from patients with CML and healthy volunteers. The activity of both collagenase and CIF was significantly higher in the CML patients than in the normal volunteers. Our previous study showed that bone marrow fibroblasts from CML patients produce significantly more collagen than fibroblasts from normal subjects. Therefore, it appears that the in vitro production of collagenase and CIF by bone marrow fibroblasts is related to the degree of in vivo collagen production, and that both the increased collagenase and CIF activities are reflections of collagen overproduction in CML.
Fibrin degradation product D‐dimer induces the synthesis and release of biologically active IL‐1β, IL‐6 and plasminogen activator inhibitors from monocytes in vitro
Authors: Robson S. C. , Shephard E. G. , Kirsch R. E.
Published: March 2008

Summary Disseminated intravascular coagulation, characterized by circulating fibrin(ogen) degradation products (FDP), is associated with both acute and chronic inflammatory conditions. Since the association of FDP with monocytes could influence the release of cytokines and other regulatory proteins with significant clinical ramifications, we have studied cytokine synthesis and release following the interaction of D‐dimer (DD), a terminal degradation product of fibrin, with human monocytes in vitro.

Adherent peripheral blood monocytes were incubated with purified DD for 24 and 48 h and secreted or cell‐associated IL‐1β and IL‐6 antigen levels and activity determined. DD (50 μg/ml) boosted the secretion of IL‐1β antigen from median control levels of 659 pg/ml to 2704 pg/ml and that of IL‐6 antigen from 806 pg/ml to > 3000 pg/ml at 48 h (P<0.05). Similar increases in extracellular biologically active IL‐1 and IL‐6 were observed. Although DD increased cell associated IL‐1 β antigen levels from median values of 188 to 1600 pg/106 cells and IL‐6 antigen from 660 to 2215 pg/106 cells (P < 0.05). cell‐associated IL‐1 functional activity decreased from control levels of 98 inhibitor units ml to 65 units/ml for cells exposed to DD. Secreted plasminogen activator inhibitor (PAI) bioactivity and PAI type 2 antigen levels were significantly increased following exposure of monocytes to DD. This may explain the decreased cell associated IL‐1 activity observed in our study as PAI are known to inhibit biologically active membrane bound IL‐1.

Our finding that DD enhances monocyte release of biologically active cytokines suggests the presence of positive feedback pathways for fibrinogen synthesis by hepatocytes. Furthermore, the association of monocytes with DD may potentiate localized coagulation processes by subsequent alterations in pericellular proteolysis.
The molecular basis of β thalassaemia in Punjabi and Maharashtran Indians includes a multilocus aetiology involving triplicated α‐globin loci
Authors: Garewal Gurjevan , Fearon Clair W. , Warren Tina C. , Marwaha Neelam , Marwaha Ram K. , Mahadik Chitra , Kazazian Haig H.
Published: March 2008

Summary We have analysed 201 β‐thalassaemia (β‐thal) genes from natives of the Punjab (156) and Maharashtra states of India and found the causative mutation in 200 of them. The most common β‐globin gene mutations differed significantly between these two groups and between these groups and Indian immigrants in the U.S.A. and the U.K. In the Punjabi Indians the IVS‐1, nt 1 (G–T) mutation accounted for nearly one‐quarter of β‐thal genes, whereas it was 5% or less in the other groups. Likewise, the cap+ 1 mutation was much more prevalent in the Punjabis, whereas the nonsense codon 15 allele had a higher frequency in the Maharashtrans of the Bombay region. The common IVS‐1, nt5 allele had a frequency of 60% of β‐thal genes in the Maharastrans, 35% in North American immigrants, and only 23% in the Punjabis. Two‐thirds of all β‐thal genes in Punjab were found in the merchant caste (Khatri‐Arora), whereas the menial caste (Shudra) was highly represented among those with β‐thal genes in Maharashtra. Two novel β‐globin alleles were each found once; a frameshift codon 55 (+ A) in Maharashtrans and a frameshift codons 47–48 (+ ATCT) in Punjabis.

Of three Punjabi patients with β‐thal intermedia in whom only a single severe β‐globin gene mutation was found, two had six α‐globin genes (homozygosity for a triplicated α‐globin locus) instead of the normal α‐globin gene number of four. Thus, these two individuals had a multilocus aetiology of β‐thal and their parents have the unusual recurrence risk of 1 in 8 for conceiving a third with β‐thal intermedia. Since 15% of 126 α‐globin clusters studied in Punjabis contained either single (10%) or triplicated (5%) α‐globin genes, the α‐globin gene number is a frequent modifier of the phenotype of β‐thal in this ethnic group.
Serum macrophage colony‐stimulating factor (M‐CSF) levels correlate with clinical response to interferon‐alpha in patients with early‐stage B‐CLL
Authors: Jewell Andrew P. , Yong Kwee L. , Worman Colin P. , Tsakona Chris P. , Giles Frank J. , Goldstone Anthony H.
Published: March 2008

Summary Interferon‐alpha (IFN‐α) reduces peripheral lymphocyte counts in B‐CLL (CLL). In eight patients with stage 0 CLL on IFN‐α therapy, peripheral lymphocyte counts fell to 61·7 19·5% of baseline at week 2 (P<0·01), while serum M‐CSF levels rose from 455 183 U/ml to 686 110 U/ml (P<0·05). Neopterin levels also showed a significant rise. M‐CSF levels were correlated with clinical response in these patients. Increased production of M‐CSF and the activation of mononuclear phagocytes may be involved in clinical responses to IFN‐α in early‐stage CLL.
In vitro growth of human fetal CD34+ cells in the presence of various combinations of recombinant cytokines under serum‐free culture conditions
Authors: Zauli Giorgio , Vitale Marco , Visani Giuseppe , Marchisio Marco , Milani Daniela , Capitani Silvano
Published: March 2008

Summary. CD34+ cells were purified from midtrimester human fetal blood and adult bone marrow samples and seeded in serum‐free fibrin‐clot cultures in order to evaluate the number and the responsiveness to recombinant cytokines of pluripotent (CFU‐GEMM), erythroid (BFU‐E), megakaryocyte (BFU‐meg and CFU‐meg) and granulocyte/macrophage (CFU‐GM) haemopoietic progenitor cells.

The number of the different haemopoietic progenitors/1 × 103 CD34+ cells, except CFU‐meg, was significantly higher in fetal blood than in adult bone marrow in cultures stimulated by any combination of cytokines including interleukin‐3 (IL‐3), granulocyte/macrophage colony stimulating factor (GM‐CSF) or stem cell factor (SCF) plus erythropoietin (Epo). Nevertheless, whereas adult BFU‐E showed a maximal growth in the presence of Epo plus IL‐3 or Epo plus SCF, fetal BFU‐E showed an optimal growth in the presence of Epo alone, the sensitivity of fetal BFU‐E to suboptimal concentrations of Epo being approximately 10–15‐fold higher than that of adult BFU‐E. Addition of optimal concentrations of IL‐3, GM‐CSF or SCF, alone or in various combinations, to Epocontaining cultures induced a significant increase in both the number and size of fetal CFU‐GEMM, and CFU‐GM, and a parallel decrease of fetal BFU‐E. Finally, SCF potently syner‐gized with IL‐3 in increasing the growth of both classes of fetal megakaryocyte progenitors, BFU‐meg and CFU‐meg.
Serum levels of major basic protein in patients with or without eosinophilia: measurement by enzyme‐linked immunosorbent assay
Authors: Yamamoto Harumi , Ninomiya Haruhiko , Yoshimatsu Kentaro , Uchiyama Yasuo , Shibasaki Masanao , Enokihara Hideo , Tachibana Shinro , Abe Tsukasa
Published: March 2008

Summary. A bone marrow proteoglycan (BMPG) has been purified which consists of the same amino acid sequence as that of pro‐MBP, and produced two anti‐BMPG mAbs. Serum levels of major basic protein (MBP), a cationic protein rich in the eosinophil granule, were measured in patients with eosinophilia or allergic diseases by an enzyme‐linked immunosorbent assay (ELISA) using these mAbs. The serum levels of MBP in patients with eosinophilia (n= 64) and in those with allergic diseases without eosinophilia (n= 32) were elevated significantly (P < 0·001 and P= 0·038, respectively). There was a weak positive correlation between the serum levels of MBP and the eosinophil counts in the patients with eosinophilia (r= 0·38). Among these patients, extremely high serum levels of MBP were found in those with hypereosinophilic syndrome (HES) and Kimura's disease. Serum levels of MBP decreased more slowly than the eosinophil counts in patients with eosinophilia when treated by glucocorticoids. We conclude that measurement of serum levels of MBP is useful in evaluating the total‐body proliferation and infiltration of eosinophils more accurately than following‐up the eosinophil counts alone.
Clonal history of a human follicular lymphoma as revealed in the immunoglobulin variable region genes
Authors: Zhu D. , Hawkins R. E. , Hamblin T. J. , Stevenson F. K.
Published: March 2008

Summary. The variable region genes used to encode the immunoglobulin expressed by tumour cells of a patient with follicular lymphoma have been identified and sequenced. Initially, a lymph node biopsy was analysed and revealed usage of VH and Vκ genes which had numerous substitutions as compared with the closest germ line genes. The pattern of mutations in VH was consistent with a role for positive selection by antigen. In addition, there was evidence in both VH and Vκ sequences for clonal heterogeneity.

After 5 years, which included treatment with chemotherapy, the patient relapsed with tumour cells present in the blood. Analysis of the V‐genes used by the emerging tumour revealed a single homogeneous sequence for both VH and VL, which, in each case, matched closely one of the sequences in the original lymph node biopsy.

These results indicate that selection. possibly mediated by antigen, can operate on a cell destined to give rise to lymphoma, and that intraclonal variation can occur after the neoplastic event. However, in this case, late relapse in the blood is dominated by a single clone.
Anaplastic large cell lymphoma (CD30+/Ki‐1+): results of a prospective clinico‐pathological study of 69 cases
Authors: Pileri Stefano , Bocchia Monica , Baroni Carlo D. , Martelli Maurizio , Falini Brunagelo , Sabattini Elena , Gherlinzoni Filippo , Amadori Sergio , Poggi Simonetta , Mazza Patrizio , Burgio Vito , Zinzani Pier Luigi , Melilli Giuseppe , Benni Monica , Saragoni Luca , Martelli Massimo F. , Stein Harald , Mandelli Franco , Tura Sante
Published: March 2008

Summary. Sixty‐nine anaplastic large cell lymphomas (ALCLs) were selected from an Italian comparative trial on MACOP‐B and F‐MACHOP. As no significant difference in effectiveness of the protocols emerged, they were considered homogenously treated. The ALCLs were divided into two groups according to previously defined criteria: 41 were common type (ALCLs‐CT) and 28 Hodgkin‐related (ALCLs‐HR). T‐cell phenotype was most common (58%), while B‐cell, null and hybrid forms accounted for 27%, 13% and 2%. Clinically, ALCLs CT and HR differed as to mean age (27 v 34·3 years) and presentation; all ALCLs‐HR showed mediastinal involvement, with bulky disease in 57%, and more frequent occurrence in stage II. In contrast, ALCLs‐CT showed mediastinal masses in 58·5%, infrequently revealed bulky disease (24%), and were not specifically associated to stage. Among the ALCLs‐CT, 68·4% achieved complete remission (CR), 24·4% partial remission (PR), one (2·4%) was resistant to therapy, and two (4·8%) had fatal drug toxicity. Of the ALCLs‐HR, 67·8% reached CR, 14·3% PR, and 17·9% did not respond. In CR, ALCLs‐CT showed a greater tendency to relapse (32·1%v 14·2%). At present, 65·8% of ALCLs‐CT and 67·8% of ALCLs‐HR are alive with overall survival/disease‐free survival averages of 31/27 and 29/24 months respectively. Our data emphasize that, independently of subtype, ALCLs benefit from the application of third‐generation protocols for high‐grade non‐Hodgkin's lymphomas.
Quantitation of multidrug resistant MDR1 transcript in acute myeloid leukaemia by non‐isotopic quantitative cDNA‐polymerase chain reaction
Authors: Lyttelton M. P. A. , Hart S. , Ganeshaguru K. , Hoffbrand A. V. , Mehta A. B.
Published: March 2008

Summary. Drug resistance in acute myeloid leukaemia (AML) may be caused by overexpression of the P glycoprotein (PGP), an efflux pump encoded by the multidrug resistance mdr1 gene. Previous studies have suggested that increased PGP expression in the leukaemic blasts is of prognostic significance, and that use of PGP antagonists may be beneficial in treatment. We describe preliminary results with a non‐isotopic quantitative MDR1 cDNA‐PCR assay, using an artificial RNA construct sharing primer recognition sites with the target MDR1 mRNA (MDR1 nucleic acids 483–504 and 624–644) as an internal control. KB 3.1 parent and KB 8.5 MDR positive cell lines expressed 0·004 and 1·96 molecules MDR1 mRNA/pg total RNA. Semiquantitative screening of 60 RNA samples from 53 AML cases detected MDR1 transcript ranging from 0 to 1·81 molecules per pg RNA. The median value at presentation (33 patients) was 0·055 and was higher in 14 patients at relapse (0·13) and in seven patients with refractory disease (0.14). Quantitation of MDR1 transcript in serial samples in seven treated patients between presentation and relapse showed the decrease in three patients (0·18–0·02x) to be as marked as the increase in three other patients (3–16x). The method described is well suited for the study of clinical samples because it is sensitive, specific, rapid and requires small amounts of clinical material.
The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules
Authors: McNicol Archibald , Israels Sara J. , Robertson Catherine , Gerrard Jon M.
Published: March 2008

Summary. Two sisters with lifelong bleeding tendencies were examined to determine whether their condition was associated with a platelet defect. Their platelet aggregation in response to epinephrine and collagen was abnormal, and the secretion of serotonin and ATP was markedly reduced. The platelet contents of serotonin, ADP, and ATP were all diminished and the ATP:ADP ratio was increased. Direct enumeration by whole‐mount and quinacrine‐fluorescence techniques demonstrated that the platelets from both sisters had significantly fewer dense granules than controls. These characteristics are similar to an individual with Hermansky‐Pudlak syndrome and are consistent with a platelet dense granule deficiency. In contrast, immunofluorescence studies using an antibody against the dense granule membrane protein granulophysin suggested that both sisters had numbers of granules within the normal range. Evaluation by immunoblotting and ELISA indicated the presence of normal levels of granulophysin in the platelets from both sisters: FACS analysis demonstrated the surface expression of granulophysin under conditions of selective dense granule release. These results are consistent with these sisters having a form of dense granule storage pool deficiency where the granular membranes are present but the granules have reduced contents. This observation represents a novel form of storage pool disease which we have termed the empty sack syndrome.
The role of lipids in the detection of lupus anticoagulant by the dilute Russell Viper venom test: are platelets or reagents containing hexagonal HII phases necessary?
Authors: Stevenson K. J. , Seddon J. M.
Published: March 2008

Summary. Liposomes prepared from rabbit brain extracts (RBE) and individual pure lipids (high phosphatidyl serine content, HIPS) were compared with frozen‐thawed platelets (PLTS) in the dilute Russell Viper venom time (dRVVt). While all three preparations demonstrated sensitivity to lupus anticoagulant (LA) the highest detection rate was seen with RBE. For confirmation of LA, high concentration RBE achieved the most efficient correction of the defect. Electron microscopy and particle sizing showed RBE to be small, discrete liposomes, whereas HIPS and PLTS were aggregates of larger diameter particles. Low‐angle X‐ray diffraction showed no evidence of hexagonal HII phase. There appears to be no specific requirement either for platelets or for reagents containing hexagonal HII phases in the dRVVt. The dRVVt can be optimized by incorporating a simple dilute rabbit brain lipid mixture for detection of LA and a concentrated mixture as a correcting reagent.
In situ hybridization for the detection of cytomegalovirus in blood or bone marrow leucocytes after allogeneic bone marrow transplantation
Authors: Fest T. , Deschaseaux M. , Mougin C. , Cahn J. Y. , Dupond J. L. , Herve P.
Published: March 2008

Summary. We report a study of CMV detection by a non‐radioactive in situ hybridization (ISH) technique using a biotinylated CMV‐DNA probe. The method was applied to blood and bone marrow cells and its results were compared with those of other currently used techniques. First, we analysed peripheral leucocytes on blood donors, and, after comparison with PCR, we obtained 75% sensitivity and 87% specificity rates. We then studied four BMT recipients during the first 105 d post‐transplant. A positive signal was detected as a predominantly nuclear staining. The detection of an ISH‐positive signal occurred at least 8 d before CMV isolation from viral culture. The number of positive cells was variable according to the patients’clinical evolution, showing a dramatic increase of labelled cells in viraemic patients and then a decrease until complete disappearance of labelled cells when an efficient anti‐viral treatment was initiated. This study suggests that the detection of CMV‐DNA by ISH performed on peripheral leucocytes is a valuable tool for the early diagnosis of CMV infection and could lead to the initiation of optimal ganciclovir treatment.
Erythrocyte deformability has no influence on the rate of erythrophagocytosis in vitro by autologous human monocytes/macrophages
Authors: Baerlocher G. M. , Schlappritzi E. , Straub P. W. , Reinhart W. H.
Published: March 2008

Summary. Erythrocytes with decreased deformability are known to be rapidly removed from the circulation by splenic macrophages. The exact mechanism is, however, not well understood. We have analysed the phagocytosis of less‐deformable erythrocytes by macrophages in vitro. Human monocytes/macrophages were isolated from peripheral blood and cultured for a total time of 6 h at 37°C with 5% CO2. Autologous erythrocytes of the rhesus positive donor were rigidified by heat treatment (47°C for 1 h). The change in erythrocyte deformability was assessed with a filter aspiration technique; the membrane elastic modulus was found to be increased about 2·5‐fold. For controls, untreated erythrocytes and erythrocytes incubated with anti‐RhD‐antibodies were prepared. The rate of phagocytosis during 2 h at 37°C and 5% CO2 was 0·74·0±59 (erythrocytes per monocyte/macrophage) for controls, 3·58·2±72 for anti‐RhD‐loaded erythrocytes and 0·82·0±74 for heat‐treated erythrocytes, respectively. We conclude that decreased erythrocytes deformability does not cause an increased rate of phagocytosis by monocytes/macrophages compared to normally deformable erythrocytes in our in vitro model. This suggests that the preferential removal of rigid cells in vivo is probably not a specific process, but is due to the increased splenic transit time of rigid erythrocytes and hence longer interaction time between erythrocytes and phagocytes.
A successful pregnancy after bone marrow transplantation for severe aplastic anaemia with pre‐transplant conditioning of total lymph‐node irradiation and cyclophosphamide
Authors: Eliyahu S. , Shalev E.
Published: March 2008

Summary. Pregnancy after bone marrow transplantation (BMT) for severe aplastic anaemia (SAA) is now an encouraging reality. We present a successful pregnancy, 4 years after BMT for SAA, in a woman being immunosuppressed before transplantation with cyclophosphamide and total lymphnode irradiation (TLI). No fetal complication was diagnosed.
Cytokine activity after allogeneic bone marrow transplantation, V. Analysis of IL‐2 and IFN production by isolated CD4+ and CD8+ cells
Authors: Cooley Margaret A. , Wright Louise , Atkinson Kerry
Published: March 2008

Summary. Previous studies from this laboratory have shown that PBMC from recipients of an HLA‐identical sibling bone marrow transplant produce levels of IL‐2 which are 10–100‐fold lower than those produced by the same number of PBMC from healthy controls, whereas production of IFN‐γ is normal. The present study examined IL‐2 and IFN production over a range of cell numbers for PBMC and for isolated CD4+ and CD8+ cells for controls and marrow transplant recipients. There was a 5‐fold lower IL‐2 production in marrow transplant recipient CD8+ cells compared with equivalent numbers of control cells, whereas no difference was found in IL‐2 production by CD4+ cells. In contrast, IFN production by CD4+ cells from marrow transplant recipients was 4‐fold higher than in controls, whereas CD8+ cells from both populations produced similar amounts of IFN. When the observed production of cytokine by PBMC was compared with the expected production based on the CD4+ and CD8+ content of the PBMC, control values were similar, but the expected values for both cytokines were approximately 2‐fold higher than the observed values for marrow transplant recipients. The results suggest that the capacity of T cells from marrow transplant recipients to produce IL‐2 and IFN is not impaired, but that the frequency of cytokine‐producing cells may be reduced, and that a negative interaction present in recipient PBMC, eliminated by isolating T‐cell subsets, is responsible for the observed low levels of cytokine production.
Expression of IFN A genes in subpopulations of peripheral blood cells
Authors: Brandt Evelyn R. , Linnane Anthony W. , Devenish Rodney J.
Published: March 2008

Summary. The polymerase chain reaction (PCR) was used to detect the expression of IFN A genes in general (with ‘universal’ primers) and specifically the expression of mRNA transcripts encoding the subtypes IFN‐α‐1, ‐α‐2, ‐α‐4, ‐α‐5 and ‐α‐14 (with gene specific primers) in normal human peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. Our examination revealed that all transcripts tested for could be detected not only following induction with inducers such as Sendai virus, Semliki Forest virus and poly(I):poly(C), but also in the absence of induction. IFN A1, IFN A2 and IFN A4 mRNAs were found to constitute the major transcripts of Sendai virus and poly(I):poly(C) induced PBMC. Fractionation of PBMC into T cells, B cells, adherent cells, mononuclear (MN) cells and polymorphonuclear (PMN) cells revealed that these cell populations all contain specific IFN A mRNA transcripts both in the absence of an inducer and following induction with Sendai virus. The proportion of IFN A transcripts detected was dependent on the cell type investigated. IFN A1, IFN A2 and IFN A4 transcripts constituted the major RNA species present in PBMC and PMN cells. In MN cells IFN A5 transcripts were also present as a major IFN RNA species. Expression of the IFN A transcripts tested for in T cells, B cells and adherent cells did not vary significantly. These results emphasize the importance of identifying IFN A subtype expression in order to further our understanding of the biological significance of differential regulation and expression of particular IFN‐α subtypes.
Phenotypic, functional and molecular analysis of CD3− LGL expansions indicates a relationship to two different CD3− normal counterparts
Authors: Cantoni Claudia , de Totero Daniela , Lauria Francesco , Raspadori Donatella , Conte Romana , Ferrini Silvano , Tazzari Pier Luigi , Biassoni Roberto
Published: March 2008

Summary. In this study we have analysed the CD3 and TCR transcript expression in three CD3− large granular lymphocyte (LGL) expansions. These LGL populations show a heterogenous pattern of expression for CD2, CD8, CD16, CD56 and CD57 antigens. LGL1 is CD2+CD8−CD16+CD56+CD57+, while LGL2 is CD2−CD8+CD16−CD56−CD57−; LGL3 is similar to LGL1, except for CD8 antigen expression. Functional analysis has revealed a different behaviour of these LGL expansions in a cytotoxicity assay against the NK‐sensitive K562 cell line. LGL1 and 3 display a significant NK‐like activity, while LGL2 is inefficient against K562 target cells. TCR and CD3 transcript characterization of LGL expansions 1 and 3 showed that they expressed multiple TCRδ transcripts, a nonfunctional TCRβ transcript, CD3‐ and ‐ mRNA, but they lacked CD3δ transcript and they lacked or expressed at very low levels of CD3γ transcript. On the other hand. LGL2 expressed TCRδ, CD3‐γ, ‐ and ‐ transcripts, while it lacked CD3δ mRNA. On the basis of these data, LGL1 and 3 seem to be closely related to peripheral blood mature natural killer (NK) cells, whereas LGL2 displays a pattern of TCR and CD3 expression similar to that found in CD1−2−3−4−8−16− thymocytes.
Poor response to intensive chemotherapy in de novo acute myeloid leukaemia with trilineage myelodysplasia
Authors: Kuriyama Kazutaka , Tomonaga Masao , Matsuo Tatsuki , Kobayashi Tohru , Miwa Hiroshi , Shirakawa Shigeru , Tanimoto Mitsune , Adachi Koichi , Emi Nobuhiko , Hiraoka Akira , Tominaga Nobuhiko , Imai Kuniyuki , Asou Norio , Tsubaki Kazuo , Takahashi Isao , Minami Saburoh , Yoshida Minoru , Murakami Hirokazu , Minato Keisuke , Oshima Toshiteru , Furusawa Shinpei , Ohno Ryuzo
Published: March 2008

Summary. The findings of morphologically dysplastic features in haemopoietic cells in de novo acute myeloid leukaemia (AML) has been named AML with trilineage myelodysplasia (AML/TMDS). We analysed the clinical data, karyotypes, and treatment outcomes of 230 de novo AML patients treated with the Japan Adult Leukaemia Study Group AML‐87 protocol. 40 (17%) patients had AML/TMDS. Platelet count was significantly higher (F=0·006) and bone marrow blasts were fewer (P=0·01) in the AML/TMDS group than in the AML without TMDS. Abnormal karyotype was shown in 12/30 patients (40%) analysed.

The complete remission (CR) rate for AML/TMDS was significantly lower than AML without TMDS (63%v 81%) (P=0·01). The overall survival curves showed that the 40 patients with TMDS had a significantly worse survival than the 190 without TMDS (P=0·0005). AML/TMDS also showed significantly worse disease‐free survival (DFS) (P=0·0001).

Multivariate analysis revealed that the absence of TMDS in AML was the most significant factor in obtaining CR (P=0·01) and a significant factor in predicting longer DFS (P=0·04). Our data suggest that AML/TMDS responds poorly to intensive chemotherapy. Further study is required to determine the best treatment strategy for AML/TMDS and the biological differences between AML/TMDS and other types of AML.
Protein C, antithrombin III and plasminogen: effect of age, sex and blood group
Authors: Dolan G. , Neal K. , Cooper P. , Brown P. , Preston F. E.
Published: March 2008

Summary. We conducted a cross‐sectional study of antithrombin III (ATIII), protein C (PC) and plasminogen (Plg) concentrations in a population of healthy plasma donors in the Trent Region. The distribution of values for protein C was log normal whereas for ATIII and Plg the distributions were positively skewed and differed significantly from normal and log normal. Males had higher antithrombin III concentrations (mean 1·10 iu/ml, range 0·72–1·65) than females (mean 1·07 iu/ml, range 0·75–1·69) (P=0·001) and levels increased with age in women. Younger women aged 25–34 had significantly lower plasma concentrations of ATIII compared to males of similar age. For protein C, concentrations were higher in males (mean 1·07 u/ml, range 0·37–2·11) than in females (mean 1·01 u/ml, range 0·59–1·61) (P<0·001) and levels increased with age in both sexes (P<0·001). In women, a novel difference in protein C concentration between ABO blood groups was noted. There was no significant difference in plasminogen concentration between males and females, and in women plasminogen decreased with age (r=–0·205, P<0·001).

We conclude that these variations in ATIII and protein C with age and sex are important considerations in the determination of reference ranges for these proteins.
Possible factors influencing the haemoglobin and fetal haemoglobin levels in patients with β‐thalassaemia due to a homozygosity for the IVS‐I‐6 (T→C) mutation
Authors: Efremov D. G. , Dimovski A. J. , Baysal E. , Ye Z. , Adekile A. D. , Ribeiro M. L. S. , Schiliro G. , Altay Ç. , Gürgey A. , Efremov G. D. , Huisman T. H. J.
Published: March 2008

Summary. We have collected haematological, haemoglobin (Hb) and DNA sequence data for 29 patients with a homozygosity for the IVS‐I‐6 (TC) mutation with the intention of identifying factors contributing to the observed variability in the severity of the disease. None of the patients had received blood transfusion therapy for at least 6 months prior to the study. Hb levels varied from 5·0 to 9·9 g/dl. Patients with high Hb F (more than 1·5 g/dl or <20%) had high total Hb levels (7·5–9·7 g/dl) but some with low Hb F also had high total Hb levels; two had a concomitant α‐thalassaemia‐2 (α‐thal‐2) heterozygosity. An inverse correlation between the Hb F and Hb A2 levels was observed. The majority of the patients were homozygous for haplotype VI (49/58 chromosomes) but haplotypes IV (2/58) and VII (7/58) were also present. The only haplotype IV homozygote had high Hb F levels with high Gγ values and the CT mutation at position – 158 in the Gγ promoter, while both high and low Hb F levels were observed among patients with haplotypes VI and VII. Analysis of sequence variations in regulatory regions included the 5 hypersensitive sites (HS) 4, 3 and 2 of the locus control region (LCR), the Gγ and Aγ 5 flanking regions, the second intervening sequence (IVS‐II), and the 5 β‐globin gene region in two patients with high Hb F (one homozygote each for haplotypes VI and IV), and in two patients with low Hb F levels (one homozygote each for haplotypes VI and VII). Haplotype specific differences were observed in the LCR 5 HS‐2 and in the Gγ and Aγ flanking and IVS‐II regions; however, no differences were present between the low and high Hb F‐producing haplotype VI chromosomes, suggesting a major role for factors which are not linked to the β‐globin gene cluster in mediating γ‐globin gene expression in patients with this type of β‐thal.
Molecular characterization of glucose‐6‐phosphate dehydrogenase deficiency in Chinese infants with or without severe neonatal hyperbilirubinaemia
Authors: Lo Y. S. , Lu C. C. , Chiou S. S. , Chen B. H. , Chang T. T. , Chang J. G.
Published: March 2008

Summary. To characterize mutations in the glucose‐6‐phosphate dehydrogenase (G6PD) gene in Chinese infants, we studied 213 G6PD‐deficient infants without blood exchange transfusion (BET) therapy, and 34 patients who required BET therapy for their severe hyperbilirubinaemia after birth. Nine different point mutations were characterized in all infants. Of these mutations, the G to T substitution at cDNA nucleotide (nt) 1376, which accounts for the mutations in 131 (53.0%) neonates, followed by G to A substitution at nt 1388 in 18 (10.5%) infants, A to G substitution at nt 493 in 17 (6.9%) infants, A to G substitution at nt 95 in 10 (4.1%) infants, C to T substitution at nt 1024 in six (2.4%) infants, and G to T substitution at nt 392 in three (1.2%) infants, G to A substitution at nt 487 in two (0.8%) infants, C to T substitution at nt 1360 in two (0.8%) infants and C to T substitution at nt 592 in two (0.8%) infants. Mutations in 48 (19.5%) G6PD‐deficient infants were not characterized. Most (64.7%) mutations in the G6PD‐deficient infants who required BET therapy after birth result from a G to T substitution at nt 1376. The enzyme activity of G6PD deficient infants who required BET therapy is significantly lower than for those who did not, even in a group with the same variant (as in 1376 mutation). Severe neonatal jaundice requiring BET therapy can take place with the majority of variants encountered in this area.
Metastatic papillary carcinoma of thyroid in a patient undergoing allogeneic BMT for CML
Authors: Morrison A. E. , O'Grady J. , Parker A. C. , McLaren K.
Published: March 2008

Summary. A 30‐year‐old woman underwent HLA‐compatible bone marrow transplant for chronic myeloid leukaemia. Her post‐transplant course was complicated by disseminated aspergillus and CMV infections resulting in death 65 d post transplant. At post‐mortem, multiple pulmonary metastases of papillary carcinoma of the thyroid were found with a small (< 8 mm) primary carcinoma in the thyroid. It is suggested that the progression of this tumour was related to the patient's immunocompromised state.
An atypical myeloproliferative disorder with t(8;13) (p11;q12): a third case
Authors: Macdonald D. , Sheerin S. M. , Cross N. C. P. , Spencer A. , Goldman J. M.
Published: March 2008

Summary. A 43‐year‐old male presented with a myeloproliferative disorder with prominent lymphadenopathy. Examination of the bone marrow showed almost complete replacement by a population of cells with an acquired chromosomal translocation t(8;13) (p11;q12). There are two other case reports describing a similar clinical syndrome with t(8;13) (p11;12) as the sole chromosomal aberration in bone marrow cells, suggesting a role for this translocation in the pathogenesis of this myeloproliferative disorder.
Acute promyelocytic leukaemia in a patient treated with etoposide for Langerhans cell histiocytosis
Authors: Matsuzaki Akinobu , Inamitsu Takeshi , Watanabe Toshiaki , Ohga Shouichi , Ishii Eiichi , Nagotoshi Yoshihisa , Tasaka Hideko , Suda Masahiro , Ueda Kohji
Published: March 2008

Summary. We report a child with acute promyelocytic leukaemia (APL) who was treated with etoposide (VP16) for Langerhans cell histiocytosis (LCH). A 3‐year‐old Japanese girl was diagnosed as having LCH. She was treated with combination chemotherapy using VP16 and prednisolone. 56 months after beginning the chemotherapy she developed APL. Her bone marrow was occupied with atypical promyelocytes including giant granules and multiple Auer bodies. A cytogenetic analysis of the leukaemic cells showed 46, XX,11–,14q +, t(15,17). The cumulative dose of the administered VP16 was 12120 mg/m2, which suggested that VP16 may be responsible for the development of APL. The risk of developing secondary leukaemia after the administration of VP16 should therefore be considered when managing patients with LCH.
Polycythaemia associated with homozygosity for the abnormal haemoglobin Sherwood Forest (β104 (G6) Arg → Thr)
Authors: Williamson D. , Beresford C. H. , Langdown J. V. , Anderson C. C. , Green A. R.
Published: March 2008

Summary. We describe a 22‐year‐old Pakistani male with polycythaemia associated with homozygosity for a highaffinity haemoglobin mutant, Hb Sherwood Forest. This haemoglobin variant has an amino acid substitution in the β globin chain at position 104, Arg → Thr. In the two previously reported instances of this haemoglobin mutant the individuals were heterozygotes and were haematologically normal. We show here that the homozygous state for the mutation is associated with a compensatory erythrocytosis resulting from decreased delivery of oxygen to the tissues. A family study showed that both parents and two siblings are heterozygotes for the haemoglobin mutant and are haematologically normal. To our knowledge, this represents the first example of a β‐globin mutation producing polycythaemia in homozygotes but not in heterozygotes.
Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene‐transfer to murine lympho‐haemopoietic stem cells
Authors: Bernad Antonio , Varas Florencio , Gallego Jesúas M. , Almendral Jose M. , Bueren Juan A.
Published: March 2008

An efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro, expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5‐FU treatment were incubated under IL‐3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU‐GM and CFU‐S12 progenitor pools was observed (130‐ and 15‐fold, respectively), with no apparent impairment in long‐term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU‐GM susceptibility to the infection by supernatants containing a Moloney‐derived ecotropic retroviral vector carrying the neo’gene. The designed protocol thus involved the infection of freshly harvested 5‐FU‐treated bone marrow, followed by expansion under IL‐3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU‐GM and 50 CFU‐S12 per 105 bone marrow cells, free from non‐genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neo’gene and expressing resistance to G418 5 months after the transplantation indicates that long‐term lympho‐haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self‐renewal and differentiation properties. This gene‐transfer methodology may improve the development of gene therapy protocols where the purging of non‐transduced precursors would guarantee a lasting and uniform expression of exogenous genes.