bluuebunny/crossref_metadata_2025 huggingface dataset:
The in vivo kinetics of 111In‐ and 51Cr‐labelled platelets: a comparative study using both stored and fresh platelets
Authors: Wadenvik Hans , Kutti Jack
Published: March 2008

Summary The in vivo kinetics of simultaneously injected 51Cr‐ and 111‐In‐labelled platelets was investigated in 20 healthy male volunteers. The studies were carried out using both fresh platelets and platelets stored for 5 d at 22°C; the disappearance of platelet‐bound radioactivity was measured on whole blood samples as well as platelet pellets. Compared to 111In, the labelling efficiency was notably lower for 51Cr, and a higher amount of 51Cr was bound to contaminating red cells. As regards the fresh platelets, only small dissimilarities were observed in the in vivo kinetics obtained with the two labels. 51Cr yielded a slightly higher platelet recovery and longer T1/2 than 111In, when whole blood samples were used for calculations; no differences were seen when using platelet pellets. When stored platelets were studied, 51Cr gave significantly longer T1/2 and mean life‐span (MLS) than did 111In. This difference was present for all mathematical models used for the calculation of MLS, and when whole blood samples as well as platelet pellets were employed. It was demonstrated that the estimation of MLS was also highly dependent upon techniques of blood sampling and curve fitting. Calculations, in which the initial data points were excluded, gave consistently longer MLS (P<0.0001), as compared to when all data points were included. Furthermore, when all survival studies were grouped together, calculations using platelet pellets gave a significantly (P<0.001) shorter platelet MLS than calculations using whole blood samples. It is concluded that both 51Cr and 111In are acceptable as radiolabels for both fresh and stored platelets. However, it appears that the viability of stored platelets may be influenced by the choice of label, and caution must be taken when these isotopes are used together in dual tracer experiments. Also, our results show that a meticulously standardized processing of blood samples and experimental data is required to enable inter‐laboratory comparisons of the results.
Antigenic determinants responsible for the reactions of drug‐dependent antibodies with blood cells
Authors: Salama A. , Santoso S. , Mueller‐Eckhardt C.
Published: March 2008

Summary In an attempt to determine the antigenic combining sites of drug‐dependent antibodies in patients with drug‐related immune haemolysis, we have assessed the reactivity of 35 nomifensine‐induced antibodies against human red blood cells (RBC) in the presence of 11 closely related compounds: nomifensine, its three main metabolites including their methoxylated analogues and diclofensine with its three main metabolites. Three types of antibody reactivity patterns could be differentiated: Firstly, antibodies most strongly reactive with nomifensine (n= 12); secondly, antibodies primarily directed against one of its main metabolites (n= 7), and, thirdly, antibodies optimally reactive with its unknown metabolites (n= 16). The antibodies preferentially directed against nomifensine showed varying cross reactions with nomifensine‐related compounds and in almost all cases also with diclofensine and/or its metabolites. Those antibodies which were optimally reactive with metabolites reacted only with nomifensine‐derived compounds and only one of them was non‐crossreactive. The third group of antibodies showed no (n= 12) or only weak (n= 4) cross reactions with nomifensine and/or its metabolites. Although none of the substances used bound firmly to RBC, certain blood groups have been identified in previous studies to be defined. RBC antigens were required for the reaction in approximately 40% of all antibodies, independent of their reactivity with the compounds. Thus, even when the specificity of some antibodies appeared to be predominantly controlled by certain structural features of the compounds, the actual antigenic combining site of each antibody was different and seemed to comprise parts of the drug‐related determinants as well as different constituents on RBC membranes.

These findings indicate firstly that RBCs function as ‘carrier‐like’macromolecules, since they are directly involved in the reaction; secondly, that the drugs and their metabolites act as ‘pseudohaptens’, in as much as they do not bind tightly to the cells; and, thirdly, that the determinants which govern the immune response appear to result from an accidental attachment rather than from a predetermined selection of antigenic membrane structures, since each antibody shows a unique reaction pattern.
CD16 and CR3 receptors distinguish between the two mechanisms of tumour cytotoxicity in neutrophils
Authors: Gavioli Riccardo , Spisani Susanna , Giuliani Anna Lisa , Elisabetta  , Cosulich  , Risso Angela , Traniello Serena
Published: March 2008

Summary Previous studies have suggested that phorbol ester‐activated neutrophils kill both antibody non‐coated and antibody‐coated K562 target cells. In this report the contribution of the receptors FcγIII (CD16) and CR3 (CD11b/CD18) in the lytic process was investigated. In neutrophils CD16 and CR3 are up‐regulated by the phorbol ester up to 4 and 10 times, respectively. As expected, lysis of non‐immunized K562 targets is not affected by the treatment of neutrophils with anti CD16, AB8.28, whereas lysis of immunized targets is decreased by 50%. In addition, the interaction of CD16 and AB8.28 induces calcium mobilization and increases granule secretion. Surprisingly, the simultaneous binding of AB8.28 and anti‐CR3 0KM1 to neutrophils completely abolishes the lysis of antibody‐coated targets. Unlike CD16, CR3 does not possess a functional role and binding of 0KM1 to CR3 does not affect cytotoxicity of immunized K562 targets, but it blocks lysis of non‐coated target almost completely, indicating a function as adhesion protein for CR3.

These studies demonstrate a distinct role of CD16 and CR3 in mediating antibody‐dependent and antibody‐independent cellular cytotoxicity, respectively.
Follow‐up of residual disease (MRD) in B lineage acute leukaemias using a simplified PCR strategy: evolution of MRD rather than its detection is correlated with clinical outcome
Authors: Nizet Y. , Martiat P. , Vaerman J. L. , Philippe M. , Wildmann C. , Staelens J. P. , Cornu G. , Ferrant A. , Michaux J. L. , Sokal G.
Published: March 2008

Summary Bone marrow samples of 16 patients (two adults and 14 children) with a B lineage acute lymphoblastic leukaemia (ALL), and in whom Ig heavy chain gene rearrangements were detectable at diagnosis using polymerase chain reaction (PCR), were studied during evolution using PCR. The VDJ junctional fragment of the Ig heavy chain rearranged gene was amplified at diagnosis. After length reduction by restriction digestion, the amplified fragment was recovered by chromatography, labelled using a specific hexamer as a primer and directly used as a clonospecific probe. The sensitivity of the PCR ranged from 1:104 to 1:105 cells, depending on the patient's rearrangement. Residual disease (MRD) was detected in most of the patients achieving a complete remission after induction therapy, regardless of the long‐term outcome of treatment. However, in patients remaining in complete remission, the level of MRD showed a tendency to decrease and ultimately become undetectable for variable periods of time, while in patients eventually relapsing there was a trend for MRD to persist at stable levels and even to increase before relapse was clinically evident. We conclude that the use of a simplified methodology for obtaining a clonospecific probe from the Ig heavy chain gene, though less sensitive than the sequencing methodology, is a valuable and readily available tool to monitor MRD in a high proportion of B lineage ALL.
Detection of mixed chimaerism in flow‐sorted cell subpopulations by PCR‐amplified VNTR markers after allogeneic bone marrow transplantation
Authors: Van Leeuwen J. E. M. , Van Tol M. J. D. , Bodzinga B. G. , Wijnen J. Th. , Keur M. van der , Joosten A. M. , Tanke H. J. , Vossen J. M. , Khan P. Meera
Published: March 2008

Summary The amplification of Variable Number of Tandem Repeats (VNTR) by the polymerase chain reaction (PCR) was used to determine the extent of chimaerism in flow sorted lymphoid and myeloid cell populations following allogeneic bone marrow transplantation (BMT). Pre‐BMT screening with a set of five VNTR revealed that at least one marker was maximally informative in 95% of donor‐recipient pairs. Mixing reconstruction experiments indicated that detection of 1–5% of the minor cell population in a sample of 5 ± 103 nucleated cells is feasible. Flow sorted post‐transplant peripheral blood B‐ and T‐lymphocyte, natural killer and monocyte cell populations were subjected to PCR‐VNTR marker analysis. It was shown that this procedure can be used for the early detection of engraftment and the identification of mixed chimaerism in various haematopoietic cell lineages in patients with leukaemia or severe combined immune deficiency, treated with allogeneic BMT.
T cell sensitization to factor VIII in haemophilia A?
Authors: Madhok Rajan , Smith John , Jenkins Andrew , Lowe Gordon D. O.
Published: March 2008

Summary Previous studies have indicated that factor VIII concentrates have a direct inhibitory effect on the proliferation of peripheral blood mononuclear cells (PBMC) obtained from normal volunteers. We have studied the effects of an intermediate and high purity factor VIII concentrate on haemophiliac PBMC. Intermediate purity factor VIII concentrate inhibited PBMC proliferation, the high purity factor VIII concentrate had no effect. The high purity factor VIII concentrate increased production of the lymphokine inter‐leukin 2, suggesting the presence of a clone of T cells sensitized to factor VIII.
The high molecular weight form of endothelial cell von Willebrand factor is released by the regulated pathway
Authors: Tsai Han‐Mou , Nagel Ronald L. , Hatcher Victor B. , Seaton Alison C. , Sussman Ira I.
Published: March 2008

Summary We have previously reported that two forms of von Willebrand factor (vWf) exist in cultured human umbilical vein endothelial cells: a high molecular weight (HMW) form that is released and can be proteolytically cleaved into a series of plasma‐like multimers, and a non‐secreted low molecular weight (LMW) form. In this study, the mode of vWf release and the relationship between the two forms were examined. As determined by two‐dimensional analysis as well as by immunoreactivity with an antibody to the propolypeptide, the LMW form of endothelial cell vWf consisted of a 260 kD pro‐vWf polypeptide, while the HMW form consisted of a 225 kD mature polypeptide. Only the 260 kD polypeptide was susceptible to digestion with endoglycosidase H. Release of the HMW form into the culture media was accompanied by a decrease in cellular vWf. Treatment of endothelial cells with cycloheximide or tunicamycin caused a decrease in the LMW form but did not affect the secretion of the HMW form. These results suggest that two pools of vWf exist in endothelial cells—a LMW form of pro‐vWf in the endoplasmic reticulum and a HMW form of mature vWf in the storage compartment. Released vWf derives only from the storage pool.
Elevated serum interleukin‐6 levels in patients with reactive thrombocytosis
Authors: Hollen Charles W. , Henthorn James , Koziol James A. , Burstein Samuel A.
Published: March 2008

Summary Interleukin‐6 (IL‐6) is known to promote mega‐karyocytopoiesis in vitro and raise platelet counts in vivo. To determine if there is a relationship between circulating IL‐6 and thrombocytosis in man, we measured bioactive IL‐6 in the serum of 13 patients with myeloproliferative disorders and 143 patients with reactive thrombocytosis having platelet counts > 600 × 109/I. IL‐6 activity was assayed using the IL‐6‐responsive B9 cell line. Seventy‐one controls with normal platelet counts had a mean IL‐6 level of 2.19 U/ml × 1.08 (SD). None of the 13 patients with myeloproliferative disorders had elevated IL‐6 levels (1.56 U/ml × 1.2). In contrast, serum IL‐6 levels of 143 patients (158 samples) with reactive thrombocytosis were significantly greater than controls (38.3 U/ml × 94.6; P < 0.001), with 83% of the samples showing elevated serum IL‐6. No significant correlation was observed between serum IL‐6 levels and platelet counts in the reactive thrombocytosis group. We conclude that elevated IL‐6 is associated with reactive thrombocytosis, and hypothesize that the increased platelet count in many cases is causally related to elevated IL‐6.
Characterization of murine monoclonal antibodies directed against the Kell blood group glycoprotein
Authors: Jaber Abdeslam , Loirat Marie‐Jeanne , Willem Catherine , Bloy Christian , Cartron Jean‐Pierre , Blanchard Dominique
Published: March 2008

Summary Murine monoclonal antibodies (MoAbs) were produced against the blood group KEL1 glycoprotein (93 kD component) immunopurified from human erythrocytes. One monoclonal antibody, 5A11 (IgGl, K), detects by immunoblotting a 93 and 184 kD component from KEL: 1, ‐2 or KEL: ‐ 1,2 red cell membrane preparations, separated by SDS polyacrylamide gel electrophoresis (PAGE) under non‐reducing conditions. The 184 kD component was not detected under reducing conditions, suggesting that it represented a dimer of the 93 kD KEL glycoprotein. Neither the 93 nor the 184 kD could be detected from KO or McLeod erythrocyte membrane preparations, indicating that the monoclonal antibody reacts with the KEL glycoprotein previously identified as a 93 kD species. Since this antibody does not agglutinate native or protease‐treated erythrocytes, it is likely that it reacts with the cytoplasmic domain of the KEL glycoprotein. This was also substantiated by showing that 5A11 could immunoprecipitate the 93 kD component from intact membranes and inside‐out vesicles but not from right‐side‐out vesicles. Immunostaining of membrane proteins prepared from human blood cells (platelets, lymphocytes, monocytes and granulocytes) and non‐human erythrocytes revealed that the 93 kD molecule was only present on human red cells. Several other murine monoclonal antibodies obtained from the same fusion experiment gave identical results, but competition analyses on immunoblots indicated that these antibodies reacted with distinct epitopes on the KEL glycoprotein.
Treatment of Diamond‐Blackfan anaemia with haematopoietic growth factors, granulocyte‐macrophage colony stimulating factor and interleukin 3: sustained remissions following IL‐3
Authors: Dunbar Cynthia E. , Smith David A. , Kimball Janice , Garrison Leslie , Nienhuis Arthur W. , Young Neal S.
Published: March 2008

Summary We have treated six transfusion‐dependent, steroid‐unresponsive, Diamond‐Blackfan anaemia (DBA) patients with the recombinant human growth factors granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐3 (IL‐3), administered sequentially with an interim rest period. GM‐CSF was given at a dose of 500 μg/m2/d subcutaneously for 6 weeks. Three patients increased their absolute reticulocyte counts 1.5‐35‐fold (mean 20.8‐fold) and into the normal range, but only one showed a reduction in transfusion requirements. Between 4 and 25 weeks after discontinuation of GM‐CSF, these six patients were treated with recombinant human IL‐3, at doses of 60 or 125 μg/m2/d subcutaneously for 4‐6 weeks. Three increased their absolute reticulocyte counts from 2‐ to 28‐fold (mean 10.6‐fold) and two required fewer transfusions. One of these two patients has remained transfusion independent for over a year since completion of IL‐3 therapy, and the second patient required infrequent transfusions for 9 months and then became transfusion independent for the subsequent 5 months. The sustained clinical remissions seen in two of the six patients after IL‐3 therapy is very encouraging and further studies in a larger cohort of DBA patients with IL‐3 alone or in combination with GM‐CSF or other growth factors should be carried out.
Treatment of acute promyelocytic leukaemia relapsing after allogeneic bone marrow transplantation with all‐trans‐retinoic acid: suppression of the leukaemic clone
Authors: Bashford John , Szer Jeff , Wiley James S. , Buckley Michael , Garson O. Margaret , Van der Weyden Martin B.
Published: March 2008

Summary We describe the clinical experience of a male patient with acute promyelocytic leukaemia, relapsing after sex‐mismatched allogeneic bone marrow transplantation and treated with all‐trans‐retinoic acid. Resolution of the coagulopathy was observed by day 7 of therapy. A complete remission was achieved by day 47 after a period of pancytopenia, dysplastic myeloid maturation and bone marrow hypo‐cellularity with necrosis and fibrosis. Serial cytogenetic analyses revealed a progressive loss of the male leukaemic clone [46XY, t(15;17)] and emergence of normal female (donor) cells [46XX] which became completely dominant with remission. Adverse effects of all‐trans‐retinoic acid included bone pain and a prominent leucocytosis requiring leukaphereses and hydroxyurea therapy. All‐trans‐retinoic acid can induce complete remission of recurrent acute promyelocytic leukaemia following bone marrow transplantation. The data suggest that remission is due to differentiation and suppression of the leukaemic clone while allowing repopulation of the marrow with non‐malignant cells.
In vivo effects of macrophage colony‐stimulating factor on human monocyte function
Authors: Khwaja Asim , Johnson Beryl , Addison Ian E. , Yong Kwee , Ruthven Karen , Abramson Stephen , Linch David C.
Published: March 2008

Summary. Macrophage colony‐stimulating factor (M‐CSF) is reported to enhance a variety of functions of mature monocyte/macrophages in vitro. We have examined the effects of a 2 h intravenous infusion of M‐CSF obtained from human urine (hM‐CSF) on haematological parameters and selected monocyte functions. There was a rapid, small, but consistent reduction in Hb concentration (mean 6·5 ± 2·3%, P <0·0005 by paired t test) by the completion of the hM‐CSF infusion and small, transient falls in platelet, monocyte and neutrophil counts were noted in the 2 h following the end of the infusion. No effect on monocyte or neutrophil CD11b cellular adhesion molecule expression was detected. Exposure to hM‐CSF in vivo did not directly stimulate the monocyte respiratory burst, but increased the percentage of monocytes responding to f‐met‐leu‐phe from 9·8 ± 2·5 to 16·6 ± 4·2 (P < 0·01). The number of Candida ingested and degraded per 100 monocytes increased from 101 ± 14 pre‐infusion to 160 ± 22 post‐infusion (P < 0· 01). There was a rapid increase in the numbers of monocytes entering a skin window membrane from a mean of 226±71 pre‐infusion to 1064 ± 404 at the end of the infusion, with no effect on neutrophil migration. These data show that the administration of hM‐CSF enhances several of the functions of peripheral blood monocytes in vivo, and this may be of benefit in the treatment of selected infections.
Acute myeloid leukaemia relapsing following interleukin‐2 treatment expresses the alpha chain of the interleukin‐2 receptor
Authors: Macdonald D. , Jiang Y. Z. , Swirsky D. , Vulliamy T. , Morilla R. , And J. Bungey , Barrett A. J.
Published: March 2008

Summary. Immunotherapy with recombinant human Interleukin‐2 (rhIL‐2) was given to nine patients in first complete remission from acute myeloid leukaemia (AML). Five patients relapsed. The median time to relapse after commencing rhlL‐2 was 26 weeks (range 2‐44).

Four patients were studied at relapse. The morphological and cytochemical features at relapse and presentation were similar. Cytogenetic analysis at relapse in patients 1 and 3 showed a normal karyotype. At relapse, patient 4 had the abnormality 46,XY, t(2;3). Patient 2 had the chromosomal abnormality t(8;21) at presentation and relapse.

Patients 3 and 4 with M5 AML relapsed rapidly at 2 and 9 weeks after starting rhIL‐2 treatment. Relapse leukaemia cells had features normally associated with lymphoid development. Patient 3 was TdT positive, with rearranged immunoglobulin genes, and a proportion of cells expressing the CD7 antigen; patient 4 also expressed the CD7 antigen.

Relapse leukaemic cells from three of four patients expressed the α chain of the IL‐2 receptor as assessed by flow cytometry. After overnight incubation and removal of T‐lymphocytes the proportion of cells from these patients expressing the α chain increased from 15% to 61% (P < 0·01). Using tritiated thymidine uptake to assess cell proliferation, two of three patients who expressed the IL‐2 receptor α chain proliferated in response to 1000 u/ml of rhIL‐2 in vitro, with a stimulation index > 1·95 (P < 0·05).

Following rhIL‐2 immunotherapy for AML, relapse cells may express an inducible form of the a chain of the IL‐2 receptor, which can mediate a proliferative response. It is possible that rhIL‐2 when administered to AML patients in remission, may induce relapse. This may be a particular risk in patients with the M5 subtype.
Evaluation of CR1 expression in neutrophils from chronic myeloid leukaemia: relationship between prognosis and cellular activity
Authors: Lanza Francesco , Fagioli Franca , Gavioli Riccardo , Spisani Susanna , Malavasi Fabio , Luigi Castoldi Gian , Traniello Serena
Published: March 2008

Summary. The expression of the complement receptor CR1 has been evaluated using an immunoalkaline phosphatase staining method on peripheral blood neutrophils and granulocyte precursors from 22 patients with chronic myeloid leukaemia (CML) and 15 healthy subjects. The immunocytochemical labelling pattern of CR1 was evaluated semi‐quantitatively on cell smears using three different anti‐CR1 monoclonal antibodies. The scoring method showed that seven patients with CML had a marked reduction in CR1 expression which did not change with in vitro stimulation of neutrophils with phorbol‐myristate‐acetate (PMA) whereas control cells responded to PMA, increasing the receptor level two‐fold. In addition, functional analysis of neutrophils with low CR1 expression from CML patients showed a very low cytolytic activity against K562 tumour target, suggesting a relationship between the cellular content of CR1 and neutrophil tumouricidal activity. The involvement of CR1 in neutrophil‐mediated lysis is consistent with complete lack of tumour toxicity following receptor neutralization by anti‐CR1 monoclonal antibodies.

Interferon therapy improved CR1 expression and the cytolytic response of neutrophils in three out of five CML patients with a moderately low CR1 score. CML patients non‐responding to interferon therapy and those with a very low CR1 score, independent of the clinical stage, progressed more rapidly into the advanced clinical stage and blastic crisis.
Role of leucocyte integrins in phagocyte responses to granulocyte‐macrophage colony stimulating factor (GM‐CSF): in vitro and in vivo studies on leucocyte adhesion deficiency neutrophils
Authors: Yong K. , Addison I. E. , Johnson B. , Webster A. D. B. , Linch D. C.
Published: March 2008

The role of leucocyte integrins in phagocyte function has been studied by comparing normal neutrophils with those from a patient with partial leucocyte adhesion deficiency (LAD), in whom the levels of CD11b and CD11c were 10% of controls, whereas CD11a levels were normal. Unstimulated LAD neutrophils exhibited defective adhesion to plastic (4·4 ± 1·5% cf. 14·4 ± 3·8% in controls), but not to human umbilical vein endothelial cells (HUVECs). The adhesion to HUVECs could be further upregulated by granulocyte‐macrophage colony stimulating factor (GM‐CSF), but not by 12‐0‐tetradecanoylphorbol 13‐acetate (TPA) which, in normal cells, is a more potent ‘pro‐adhesive agonist’. The normal neutrophil‐endothelial interaction induced by GM‐ CSF in LAD neutrophils was confirmed in vivo when administration of GM‐CSF resulted in rapid phagocyte marginatum. Neutrophil migration and phagocytosis/killing were defective in LAD neutrophils, and some improvement in phagocytosis/killing was seen following in vivo administration of GM‐CSF. These studies illustrate that the degree to which the leucocyte integrins mediate adherence‐related phagocyte functions varies not only with the particular function, but also with the conditions of stimulation. High levels of CD11b and CD11c expression appear not to be required for unstimulated or GM‐CSF‐stimulated neutrophil‐endothelial interactions, either In vitro or in vivo. Other neutrophil functions, on the other hand, such as migration and phagocytosis/killing are much more dependent on the leucocyte integrins.
Immunophenotypic heterogeneity of multiple myeloma: influence on the biology and clinical course of the disease
Authors: San Miguel J. F. , Gonzáalez M. , Gascón A. , Moro Ma J. , Hernáandez J. M. , Ortega F. , Jimenez R. , Guerras L. , Romero M. , Casanova F. , Sanz M. A. , Sáanchez J. , Portero J. A. , Orfao A.
Published: March 2008

In 112 untreated myeloma patients we have analysed the immunophenotype of plasma cells both by immunofluorescence (IF) and immunocytochemistry (APAAP). Both techniques yielded similar results pointing to aH important degree of heterogeneity in antigenic expression not only between different patients but also within the same patient. The expression of CD38 and Han‐PCl antigens (Ags) was almost constant (> 90% positive cases), while CD9 was detected in 66% of the cases. On the other hand, less than one third of patients were positive for CD 10, CD20 and HLA‐DR and generally with a weak expression (< 30% positive plasma cells). In occasional cases plasma cells were weakly positive for the myelomonocytic markers CD13 (9%), CD15 (25%) and CD14 (6%). The possibility that this heterogeneity might be the result of different stages of differentiation of the neoplastic clone is suggested both by the positive correlation in the expression of some of these antigens (CD10, CD9, CD20, HLA‐DR) and by the relationship between CD10 and myeloid antigens with immature plasma cell morphology. Finally, the cALLA antigen does not seem to be of significant value in predicting survival. Moreover, none of the other markers explored showed a clear influence in the course of the disease, although the tendency towards a lower survival found for the CD20+ cases as well as the association of the expression of some antigens and advanced clinical stage, may warrant further studies in a larger series of patients.
Electrokinetic properties and surface membrane sialic acid status of platelets in idiopathic thrombocytopenic purpura (ITP)
Authors: Crook M. , Machin S. , Crawford N.
Published: March 2008

Continuous flow electrophoresis has been used to investigate heterogeneity in the circulating platelets from patients with idiopathic thrombocytopenia (ITP) and from age‐ and sex‐matched control donors. The ITP patients’platelet counts ranged from 15 to 80 × 109/1 and they had much higher mean volumes and significantly higher (twofold) total cell and neuraminidase‐labile surface sialic acid contents expressed per unit cell than normal. After volume normalization the mean difference between the two groups was not statistically significant. The relationships of these findings to abnormal thrombopoiesis and to molecular recognition for reticuloendothelial system (RES) removal are discussed.
Intestinal transferrin receptors and iron absorption in the neonatal rat
Authors: Anderson Gregory J. , Walsh Michael D. , Powell Lawrie W. , Halliday June W.
Published: March 2008

The transferrin receptor is a major protein found on the basolateral membranes of intestinal epithelial cells, yet its possible role in intestinal iron metabolism and also in iron absorption is unclear. We have studied intestinal transferrin receptor expression during the peri‐ and postnatal development of the small intestine of the rat using immunohisto‐chemistry with a monoclonal antibody to the rat receptor. Two major changes in transferrin receptor expression in the developing small intestine were found, a decrease in receptor expression associated with birth, and an increase at the time of weaning. Around the time of weaning there was a large decrease in iron absorption, but there was no direct correlation between absorption and transferrin receptor expression. However, at both birth and weaning there were major changes in intestinal cell kinetics, and the distribution of receptor correlated well with the distribution of proliferating cell populations. In addition, as the intestinal epithelial cells differentiated and stopped dividing, there was a redistribution of transferrin receptors from the cell surface to intracellular sites. These data suggest that the most likely role of the transferrin receptor in the neonatal intestine is in the supply of iron to the developing epithelial cells in the crypts, and that the receptor does not play a direct role in iron transit across the intestinal epithelium.
Interleukin‐4 prevents the induction of G‐CSF mRNA in human adherent monocytes in response to endotoxin and IL‐1 stimulation
Authors: Vellenga Edo , Dokter Wim , de Wolf Joost Th. M. , van de Vinne Bernard , Esselink Mariet T. , Halie M. Ruud
Published: March 2008

Human recombinant interleukin‐4 (IL‐4) was studied for its effects on the expression of granulocyte‐colony stimulating factor (G‐CSF) mRNA in human adherent monocytes in the absence and presence of endotoxin and interleukin 1 (IL‐1), IL‐4 (15 ng/ml) did not induce G‐CSF transcripts in monocytes but suppressed the endotoxin‐induced G‐CSF expression when added simultaneously. Sequential treatment of monocytes with IL‐4 followed by endotoxin suppressed G‐CSF mRNA induction totally. This effect was independent of the presence of fetal bovine serum but dependent of the IL‐4 dose. Comparable results were obtained with IL‐1, IL‐1 (50 U/ml) induced G‐CSF expression in human adherent monocytes which could be counteracted by IL‐4 pretreatment. In addition, it was shown that the induction of G‐CSF mRNA by the calcium‐ionophore A23187 or by c‐AMP elevating agents could be blocked by IL‐4. These suppressive effects of IL‐4 were not related to changes in the half‐life of G‐CSF mRNA and were independent of protein synthesis. Finally it was demonstrated that IL‐4 had comparable effects on the G‐CSF secretion of endotoxin and IL‐1 stimulated human monocytes by using a murine bone marrow assay. These results indicate that IL‐4 down‐regulates the expression of G‐CSF gene and secretion of proteins in human activated monocytes.
Analysis of multidrug resistance (MDR‐1) gene expression in chronic lymphocytic leukaemia (CLL)
Authors: Shustik C. , Groulx N. , Gros P.
Published: March 2008

Multidrug resistance (MDR) in cultured human cells is caused by the overexpression of the MDR‐1 gene. This gene codes for P‐glycoprotein, a proposed ATP‐dependent drug efflux pump, which reduces the net intracellular accumulation of a large group of chemotherapeutic agents in resistant cells. We have measured the level of expression of the human MDR‐1 gene in a series of patients with chronic lymphocytic leukaemia (CLL). Forty‐eight patients included in the study were at different stages of disease and were either untreated or had been treated with alkylating agents or alkylating agents in combination with drugs of the MDR spectrum, and were tested over a period of 3 years. The level of MDR‐1 expression was monitored by Northern blotting analysis using a specific cDNA hybridization probe and also after polymerase chain reaction (PCR) amplification of MDR‐1 complementary DNA (cDNA). Four of 28 previously untreated patients showed intrinsically high levels of MDR‐1 mRNA while 5/19 treated patients had elevated MDR‐1 expression. Elevated MDR‐1 expression in treated patients was unrelated to the type of chemotherapy and was independent of previous exposure to drugs of the MDR spectrum. Intrinsic MDR‐1 gene expression in positive patients did not appear to influence their response to chemotherapy with non‐MDR drugs such as alkylating agents.
Molecular defects in haemophilia B: detection by direct restriction enzyme analysis
Authors: Chan Vivian , Yip B. , Tong T. M. F. , Chan T. P. T. , Lau K. , Yam Irene , Chan T. K.
Published: March 2008

The common restriction fragment length polymorphisms (RFLPs) associated with the FIX gene: 5’BamH I, Dde I, BamH I (2), Taq 1 and 3’Hha I were absent or of low incidence in Southern Chinese and are therefore not useful for linkage analysis. No deletion was detected amongst seven consecutive unrelated haemophilia B patients, but one had an insertion of a 15 kb Pvu II fragment containing exon d. Using an alternate strategy of polymerase chain reaction (PCR) amplification and direct sequencing, the molecular defect in the other six patients was defined. The four novel mutations characterized were: nucleotide (nt) 6410 G→C (Gly12→Ala): nt 31261 Δ T (stop codon 31 bp downstream); nt 31260 C→G (Thr380→Ser) and nt 31122 C→A (Ala34→Asp). Two patients had the same mutation at nt 6365, G→A (Arg−4→Gln), identical to one previously described in other ethnic groups, suggesting that this is a hotspot for mutation. Each of the mutations was found to affect an enzyme recognition site and could thus be identified by direct visualization of abnormal restriction fragments in amplified genomic DNA. This allows rapid and accurate DNA diagnosis of haemophilia B in an ethnic group which otherwise shows little or no polymorphism for the common RFLP sites.
Book Review
Authors: Testa N. G.
Published: March 2008

Clinical Applications of TGF‐β. Ciba Foundation Symposium 157. Edited by Gregory R. Bock (Organizer) and Joan Marsh.
Drug‐induced agranulocytosis: prognostic factors in a series of 168 episodes
Authors: Juliá A. , Olona M. , Bueno J. , Revilla E. , Rosselló J. , Petit J. , Morey M. , Flores A. , Font Ll. , Maciá J.
Published: March 2008

The prognostic value of 36 clinical and analytical parameters at diagnosis in patients with drug‐induced agranulocytosis was analysed in an adult population. This multicentre, retrospective study examined possible prognostic factors by multiple logistic regression analysis in a series of 168 clinical episodes. The overall mortality was 16%. Renal insufficiency at diagnosis and the development of bacteraemia were associated with a poor prognosis. Advanced age, decreased leucocyte count, lymphocytopenia, bone marrow myeloid hypoplasia, increased percentage of bone marrow plasma cells and shock were found to be associated with a poor prognosis only in the univariate analysis. An independent analysis of the myeloid cellularity at diagnosis showed an inverse correlation with the time to recovery of the granulocyte counts (r=−0·43; P= 0·001). Our data indicate that despite some important clinical differences (higher incidence of infections of the oropharynx, shorter period of neutropenia and almost exclusive presence of gram‐negative organisms), the infections complicating the treatment of cancer patients have the same prognostic features than those seen in patients with acute agranulocytosis. Therefore the established therapeutic guidelines for neutropenia after cancer chemotherapeutic agents are applicable to patients with acute agranulocytosis.
Proliferation of double‐negative (CD4−CD8−) T cells bearing T‐cell receptor‐αβ in a haemophiliac with human immunodeficiency virus type 1 infection and factor VIII inhibitor: functional properties of double‐negative T‐cell receptor‐αβ+ T cells
Authors: Yasukawa Masaki , Hato Takaaki , Matsumoto Mitsuru , Takada Kiyonori , Fujita Shigeru , Kobayashi Yuzuru
Published: March 2008

We present a patient with haemophilia A showing human immunodeficiency virus type 1 (HIV‐1) infection and factor VIII inhibitor in whom a novel T‐cell subpopulation, double‐negative (CD4−CD8−) T cells bearing T‐cell receptor (TCR)αβ, proliferated polyclonally in the peripheral blood. An interleukin‐2‐dependent T‐cell line with a CD4−CD8−TCR‐αβ+ phenotype was established from the peripheral blood lymphocytes of the patient, and its biological functions were studied. It was found that the CD4−CD8−TCR‐αβ+ T cells possessed both HLA‐unrestricted cytotoxicity and helper function for immunoglobulin production by B cells. In addition, these T cells were found to produce interferon‐γ and interleukin‐2 following activation via CD3‐TCR complexes. These data demonstrating the multifunction of these newly defined CD4−CD8−TCR‐αβ+ T cells thus suggest that these cells play an important role in protection against HIV infection. The mechanism of production of factor VIII inhibitor in the present case is also discussed focusing on the CD4−CD8−TCR‐αβ+ T cells.
Expression of cytoadhesion molecules (CD56, CD54, CD18 and CD29) by myeloma plasma cells
Authors: Van Riet Ivan , Waele Mark De , Remels Linda , Lacor Patrick , Schots Rik , Camp Benjamin Van
Published: March 2008

Recently we reported the expression of the human natural killer cell associated antigen CD56 (Leu 19/NKH1) in plasma cells of a majority of multiple myeloma (MM) patients. CD56 is known to be an isoform of the human neural adhesion molecule N‐CAM which is involved in homotypic adhesive interactions. By immunophenotyping using four CD56 specific monoclonal antibodies and immunoprecipitation analysis we here confirm that the Leu 19 antigen expressed by myeloma plasma cells is identical to N‐CAM and corresponds to the 145 kDa isoform. Because of the possible biological role of adhesion molecules on myeloma cells, we compared the expression of N‐CAM with the intercellular adhesion molecule 1 (ICAM‐1) and the β1 and β2 integrins. By immunogold‐silver staining of cytospin preparations of mononuclear cell suspensions, bone marrow plasma cells of 17 MM patients were analysed. Plasma cells expressed N‐CAM (CD56) in 14 patients, ICAM‐1 (CD54) in 16 patients, and β2 intergrins (CD18) in eight patients. β1 integrins (CD29) were expressed in all patients. The expression of β2 integrins was always very weak while N‐CAM, ICAM‐1 and the β1 integrins showed a moderate to strong positivity. The plasma cells of five haematological normal individuals lacked significant N‐CAM expression but were positive for ICAM‐1 and both integrin subgroups. One plasma cell leukaemia patient and two out of four end‐stage MM patients showed no expression of N‐CAM or β2 integrins on their circulating plasma cells. Among 11 previously established myeloma cell lines, surface expression of ICAM‐1 and the integrins was detected in most cases, while N‐CAM was present in only four lines. Most cell lines showed coexpression of the fibronectin receptors (VLA‐4 and VLA‐5) and the laminin receptor (VLA‐6). The collagen receptor (VLA‐2) was not expressed. The N‐CAM negative cell lines included four cell lines that were derived from plasma cell leukaemia patients. These results indicate that the expression of adhesion molecules is an intrinsic part of the biology of multiple myeloma.
An alloimmunized, thrombocytopenic patient successfully transfused with acid‐treated, random‐donor platelets
Authors: Shanwell A. , Sallander S. , Olsson I. , Gulliksson H. , Pedajas I. , Lerner R.
Published: March 2008

Alloimmunized, thrombocytopenic patients, refractory to random‐donor platelet transfusion, often respond to HLA‐identical single‐donor platelets. HLA‐compatible platelets are expensive, take time to prepare, and donors are sometimes not to be found.

We have used random‐donor platelets and ‘peeled’ the HLA‐antigen off the platelets, using a modified laboratory method (incubation of platelets with citric acid solution at 0°C). Platelet recovery in two healthy subjects was 72·0% for acid‐treated platelets, and 73·5% for untreated control platelets, using 111In‐labelled autologous platelets. Survival time (multiple hit) was 6·25 and 7·95 d, respectively. Random‐donor platelets that were strongly positive in the crossmatch with serum from a patient became negative after treatment with the acid solution. Furthermore, transfusion of these platelets gave a post‐transfusion, platelet‐count increment comparable with transfusion of HLA‐compatible single‐donor platelets.
Shift in the megakaryocyte ploidy in MDS patients: microcytofluorometry with DAPI staining after destaining of Wright‐Giemsa stain
Authors: Kobayashi Yutaka , Kimura Shinya , Tanaka Koji , Wada Katyuya , Ozawa Masaru , Horiuchi Hirohiko , Maruo Naoyuki , Kondo Motoharu
Published: March 2008

We applied DAPI (4′,6‐diamidino‐2‐phenylindole) staining to the determination of nuclear DNA content in single megakaryocytes in 12 normal subjects and 12 patients with myelodysplastic syndrome (MDS). After the megakaryocytes had been identified on Wright‐Giemsa stained smear and classified according to modified Feinendegen's classification, they were photographed. Then Wright‐Giemsa stain was removed by immersion in 50% ethanol at 37°C for 1 h and 100% methanol at 37°C for 1 h. The specimens were then stained with DAPI solution (DAPI 0.01 mg/ml, pH 7.4 Tris‐EDTA‐2Na buffer solution and 0.01 M 2‐mercaptoethylamine hydrochloride mixed at the ratio of 0.5:98.5:1.0) for more than 30 min. The amount of nuclear DNA in the previously identified megakaryocytes was measured by microcytofluorometry.

The maximum population of megakaryocytes ploidy was in 16N in normal subjects, 8N in 10/12 MDS patients, and 4N in the remaining two patients. These findings suggest impairment of the development of the megakaryocytes nucleus in the MDS patients.
Is interferon alpha in cutaneous T‐cell lymphoma a treatment of choice?
Authors: Papa G. , Tura S. , Mandelli F. , Vegna M. L. , Defazio D. , Mazza P. , Zinzani P. L. , Simoni R. , DePita O. , Ferranti G. , Ghetti P. L. , Criscuolo D.
Published: March 2008

This study was designed to evaluate the therapeutic efficacy and toxicity of recombinant interferon alpha‐2a (rIFN alfa‐2a) given as initial systemic therapy in untreated mycosis fungoides and/or Sezary's syndrome patients, at a slowly escalating schedule up to the maximal tolerated dose. At the same time this schedule was administered in patients who had relapsed or were refractory to previous treatment; 28 newly diagnosed and 15 previously treated patients entered the study. IFN was given daily with dose escalation from 3 to 18 MU. The last follow‐up in June 1990 indicates that 90% of previously untreated patients who obtained a complete remission remain in continuous complete remission after 18 to 40 months and that 75% of previously untreated patients who obtained partial remission remain in partial remission after 20–44 months. The event‐free survival projected, calculated using the Kaplan and Meier product limit technique, was 21% of all patients at 54·7 months (40% in the previously untreated groups and 14% in the previously treated group:P=0·12).

In conclusion, interferon is very effective as a single agent in cutaneous T‐cell lymphomas.
Fluorouracil and recombinant interferon alfa‐2a in advanced gastrointestinal neoplasms
Authors: Pazdur Richard
Published: March 2008

Based on preclinical studies which demonstrated synergy between recombinant interferon alfa‐2a (rIFNα‐2a) and 5‐fluorouracil (5FU), clinical studies have been initiated to investigate this combination. The initial study conducted by investigators from the Albert Einstein Cancer Center reported a response rate of 76% with 13/17 patients with advanced colorectal carcinoma responding. To further evaluate this regimen, two clinical trials have been conducted in previously untreated advanced colorectal carcinoma patients with measurable disease. The regimen consisted of 5FU administered as a continuous infusion, 750 mg/m2/d for 5 consecutive days. Intravenous bolus administration of 5FU 750 mg/m2 was given weekly for 7 weeks starting 1 week after completion of the continuous infusion. rIFNα‐2a, 9 MU, was administered subcutaneously three times weekly. In The University of Texas M. D. Anderson Cancer Center trial, 15/45 evaluable patients experienced partial response, and one patient achieved a complete response for an overall response rate of 35%. Another trial of this regimen conducted by Memorial Sloan‐Kettering has reported a 26% response rate with 9/34 evaluable patients experiencing a partial response. Current randomized trials comparing this schedule of 5FU with rIFNα‐2a to 5FU plus folinic acid or single‐agent 5FU may clarify its role in the treatment of advanced colorectal carcinomas.
Immunoglobulin gene ‘fingerprinting’: an approach to analysis of B lymphoid clonality in lymphoproliferative disorders
Authors: Deane Miriam , Norton John D.
Published: March 2008

Rearrangement of the immunoglobulin heavy chain (IgH) gene is widely exploited as a marker of B cell lineage and clonality in the pathology of lymphoproliferative disorders. We have developed a simple, polymerase chain reaction (PCR) based method for detecting IgH gene rearrangement which relies on the observation that by using a panel of PCR amplimers specific for each of the six heavy chain variable region families in conjunction with a common joining region amplimer, clonal rearrangement can be detected in over 90% of cases of B lymphoid malignancy. By using radiolabelled amplimers and exploiting the size heterogeneity resulting from independent IgH rearrangement events. we show that high resolution gel electrophoresis can be used to generate a‘fingerprint’representing the spectrum of B cell clonality in complex populations of B lymphocytes. The method effectively scans the entire IgH gene rearrangement repertoire and is capable of detecting rare clonal or oligoclonal B lymphoid cell populations. In normal bone marrow mononuclear cells. clonal IgH rearrangement could be readily detected at a sensitivity of 10−3. We illustrate the application of the method in assessing the spectrum of B cell clonality occurring in an autoimmune condition, Hashimoto's thyroiditis, and in a malignant B cell disorder, chronic lymphocytic leukaemia. In addition, we explore the potential application of the technique in tracking minimal residual disease and for monitoring clonal evolution in acute lymphoblastic leukaemia.
Analysis of ras gene mutations in childhood myeloid leukaemia
Authors: Farr Christine , Gill Robert , Katz Fay , Gibbons Barbara , Marshall Christopher J.
Published: March 2008

Previous studies have shown that approximately 30% of adult acute myeloid leukaemias and 20% of adult acute lymphoid leukaemias contain point mutated ras oncogenes. In order to assess whether ras oncogenes are also involved in childhood leukaemias, we have used polymerase chain reaction (PCR) amplification and synthetic oligonucleotide probes to study the nature and frequency of ras gene mutations in childhood leukaemias, concentrating largely on the acute myeloid leukaemias (AML). Thirty‐four childhood presentation AML DNAs were screened for mutations in and around codons 12, 61 and 117 of N‐. K‐ and H‐ras. Eight of these samples (24%) contained ras mutations. As in the adult disease, the gene predominantly involved was N‐ras (6/8), with occasional activation of K‐ras (2/6). The most common base change was a G→A transition at codon 12 or 13 (4/8). Of the patients with mutant ras. 4/8 were diagnosed as AML FAB subtype M5. Five of the 34 childhood AMLs analysed displayed abnormalities of chromosome 7. However, none of these cases contained a mutant ras gene.

One AML patient was studied at relapse, 14 months after initial presentation. The presentation mutation (N61p3) was not detectable, although a new mutation (N13Cys) was readily identified. This observation extends our original finding with presentation and relapse samples of adult AML, in which it was uncommon for the relapse sample to contain the same ras mutation as the presentation DNA.

In addition, two out of five patients diagnosed as juvenile CML were found to harbour mutant ras.