bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel point mutations leading to type 1 antithrombin deficiency and thrombosis
Authors: Olds R. J. , Lane D. A. , Ireland H. , Leone G. , Stefano V. De , Wiesel M. L. , Cazenave J‐P. , Thein S. L.
Published: March 2008

Summary. Direct sequencing of antithrombin III (AT) gene fragments specifically amplified by the polymerase chain reaction was utilized to identify the molecular basis of type 1 AT deficiency in two unrelated kindreds, both with thrombotic disease. Two novel point mutations were identified, deletion of a T from the second position of codon 81 in one propositus and insertion of a G in codon 424 in the second kindred. The AT 81(‐T) frameshift mutation leads to a premature stop signal in codon 89, while the AT 424(+G) allele has a premature stop only one codon short of the normal gene. The latter mutation changes the eight carboxy terminal residues of AT, including 429Cys. and increases the proportion of polar amino acids in this region. We suggest that altered folding of the mutant protein may explain the AT deficiency.
Molecular basis for hereditary antithrombin III quantitative deficiencies: a stop codon in exon IIIa and a frameshift in exon VI
Authors: Gandrille S. , Vidaud D. , Emmerich J. , Clauser E. , Sié P. , Fiessinger J. N. , Alhenc‐Gelas M. , Priollett P. , Aiach M.
Published: March 2008

Summary. Antithrombin III (AT III) is an inhibitor of serine protease (serpin) comprising 432 amino acids. Quantitative AT III deficiencies are associated with a high risk of thrombotic disease. Although this risk is smaller in patients with qualitative AT III deficiencies, the molecular defects characterizing the latter have been the subject of many studies. However, in quantitative AT III deficiencies, only three mutations have been described: Pro 407 to Leu and Ala 404 to Thr (both located in the C‐terminal part of the AT III molecule) and also a frameshift in exon IIIa.

Using the asymmetric polymerase chain reaction (PCR) and genomic DNA analysis by direct sequencing, we detected two mutations in three unrelated families: (i) a C→T transition in exon IIIa in two families, leading to the replacement of the codon corresponding to Arg 129 by a stop codon, and (ii) in the third family, insertion of an adenine in the codon corresponding to Phe 408, a highly conserved serpin amino acid. This insertion altered the reading frame and led to the appearance of a premature stop signal. Patients of all three families were heterozygous for their abnormality.

These results show that asymmetric PCR and genomic DNA analysis by direct sequencing permit fast identification of the molecular basis of quantitative AT III deficiencies. It is concluded that in many cases the absence of AT III gene product probably results from point mutation, as previously observed for another serpin, alpha‐1‐antitrypsin.
Effect of subcutaneously administered human recombinant erythropoietin on erythropoiesis in patients with myelodysplasia
Authors: Kamp Harmen van , Prinsze‐Postema Tineke C. , Kluin Philip M. , Den Ottolander Gerard J. , Beverstock Geoffrey C. , Willemze Roel , Fibbe Willem E.
Published: March 2008

Summary In a phase II study, 12 patients with a myelodysplastic syndrome (MDS) and anaemia (nine transfusiondependent) were treated with recombinant human erythropoietin (rHuEpo) to assess the therapeutic effect on erythropoiesis and on transfusion requirement. Patients with a low risk of developing acute leukaemia were included, i.e. refractory anaemia (RA), RA with ringed sideroblasts (RARS) and RA with excess blasts (RAEB), providing the percentage of myeloblasts in the bone marrow did not exceed 10%. Recombinant HuEpo treatment was initated at a dose of 50 units/kg body weight and administered subcutaneously three times weekly. At 3‐week intervals the dose was increased with 50 units/kg per injection, until after 15 weeks a maximum dose of 250 units/kg three times weekly was reached. All patients completed the study. Recombinant HuEpo was well tolerated and no serious side effects were seen. There was no evidence of the emergence of a new malignant clone in response to rHuEpo as shown by sequential karyotyping. In none of the patients was an increase in haemoglobin level or a diminished red blood cell transfusion requirement seen. In four out of 10 evaluable sequential bone marrow smears, an increase in erythropoiesis was seen, suggesting stimulation of ineffective red cell production. One of these patients also showed a rise in reticulocyte count. The number of erythroid progenitor cells (BFU‐E and CFU‐E) in blood and bone marrow was not affected by rHuEpo treatment. Also no change in the number of myeloid progenitor cells (CFU‐GM) in blood and bone marrow was noted. In conclusion, subcutaneous treatment with rHuEpo at dosages up to 250 units/kg body weight (three times weekly) fails to increase the haemoglobin level or to diminish the transfusion requirement in patients with MDS and anaemia. It is unclear whether higher doses of rHuEpo are effective or whether patients with less severe anaemia who are transfusion independent, have a higher likelihood of response.
A newly‐characterized α‐thalassaemia‐1 deletion removes the entire α‐like globin gene cluster in an Italian family
Authors: Fortina Paolo , Dianzani Irma , Serra Anna , Gottardi Enrico , Saglio Giuseppe , Farinasso Loredana , Piga Antonio , Gabutti Vilma , Camaschella Clara
Published: March 2008

Summary We describe a new deletional form of α thalassae‐mia which encompasses the entire α‐like globin gene cluster in a 15‐year‐old boy of Southern Italian descent. The deletion removes ∼31 kb, the 5′‐end point is located approximately 4 kb upstream of the ζ gene, while the 3′‐end point maps between the α1‐ and θ1‐globin genes. The interaction of this deletion with the common ‐α3.7 form gives origin to a classical form of haemoglobin (Hb) H disease in the propositus of this study. Deletional forms of ζα‐thalassaemia are uncommon in the Mediterranean basin; as for other unusual ζα‐thalassae‐mia forms, heterozygotes for this mutation may escape detection in population surveys based on ζ and α probes.
Erythroid 5‐aminolaevulinate synthase activity during normal and iron deficient erythropoiesis
Authors: Houston T. , Moore M. R. , McColl K. E. L. , Fitzsimons E.
Published: March 2008

Summary Reduced erythroblast 5‐aminolaevulinate (ALA) synthase activity was observed during iron/haem deficient erythropoiesis. Enzyme activity was reduced approximately threefold to levels similar to those previously detected during sideroblastic erythropoiesis. This response would appear to be erythroblast specific as haem deficiency is known to stimulate hepatic ALA synthase activity. It is, however, unclear as to whether this reduced enzyme activity relates to iron deficiency or to the consequent haem deficiency.
Iron absorption by hypotransferrinaemic mice
Authors: Simpson Robert J. , Lombard Martin , Raja Kishor B. , Thatcher Ray , Peters Timothy J.
Published: March 2008

Summary Iron absorption rates by homozygous and wild‐type mice from a hypotransferrinaemic mouse colony were examined with in vivo tied‐off duodenal sengments and in vitro incubated duodenal fragments. Enhanced initial rates of mucosal uptake and carcass transfer by homozygotes, compared to wild‐types, were observed. The changes in vivo and in in vitro uptake kinetics resemble changes seen in iron deficient or hypoxic mice, suggesting that the liver iron loading shown by homozygotes is due to a failure of the normal mechanism for regulation of iron absorption. In vivo mucosal uptake and carcass transfer of radioiron showed an inverse correlation with liver non‐haem iron content in homozygous hypotransferrinaemic mice, suggesting that some degree of control of absorption, albeit at markedly reduced sensitivity, can operate in these mice. No correlation between haemoglobin level and iron absorption was observed in homozygous hypotransferrinaemic mice, suggesting that this regulator of iron absorption does not function in these mice. The precise pathogenic mechanism of the enhanced iron absorption in hypotransferrinaemia remains to be determined. Mucosal transferrin levels were found to parallel serum transferrin levels in homozygotes. heterozygotes and wild‐type mice. This supports previous suggestions that mucosal transferrin is derived from plasma transferrin and that the enhancement of iron absorption, by physiological mechanisms, does not require the presence of mucosal transferrin.
The site of platelet destruction in thrombocytopenic purpura as a predictive index of the efficacy of splenectomy
Authors: Najean Y. , Dufour V. , Rain J. D. , Toubert M. E.
Published: March 2008

Summary The significance of the site of platelet sequestration in determining the indication for splenectomy in idiopathic thrombocytopenic purpura (ITP) is a controversial subject. However, most of the negative conclusions are based on 51chromium labelling of homologous platelets. We report here the results of an analysis of 222 cases in which the kinetic study of 111indium‐oxinate‐labelled autologous platelets was performed under homogeneous technical conditions. 103 of these patients subsequently underwent splenectomy.

This study demonstrates that the site of platelet sequestration in active ITP constitutes a variable independent of the patient's age, history of the disease and its severity (platelet count, lifespan). The sequestration site is a good predictive element of the short‐term efficacy of splenectomy (71/76 cases with splenic sequestration obtained a platelet count exceeding 100 × 109/1 versus 7/13 cases with mixed sequestration and 1/14 cases with hepatic sequestration), and the long‐term results (6 months to 5 years after splenectomy) do confirm the clinical value of this study.
Interleukin 6, a possible autocrine growth and differentiation factor for the human megakaryocytic cell line, CMK
Authors: Fuse Akira , Kakuda Harumi , Shima Yukichi , Van Damme Jo , And Alfons Billiau† , Sato Takeyuki
Published: March 2008

Summary. CMK is a human cell line derived from a megakaryoblastic leukaemia. It has characteristics of the megakaryocytic lineage, such as the presence of platelet peroxidase, membrane glycoproteins (GP)Ib and GPIIb/IIIa, α‐granules, and demarcation membranes. The cell line proliferates autonomously in serum‐containing medium. Here we report that the cell line expresses the gene for IL‐6 and releases small quantities of the cytokine into the medium. Addition of exogenous IL‐6 to cultures seeded into medium was found to promote growth of the cells. Conversely, addition of a neutralizing anti‐IL‐6 antibody inhibited cell growth. These data support the notion that autocrine IL‐6 is one of the factors accounting for autonomous growth of the cell line.
Quantification of platelet‐bound immunoglobulins of different class and subclass using radiolabeled monoclonal antibodies: assay conditions and clinical application
Authors: Tijhuis G. J. , Klaassen R. J. L. , Modderman P. W. , Ouwehand W. H. , Kr. vondem Borne A. E. G.
Published: March 2008

Summary. Quantification of platelet‐bound immunoglobulins (PBIg) with radiolabelled murine monoclonal antibodies (mAbs) has been described only for IgG so far. Here we describe some modifications of this mAb radioimmunoassay (MARIA) and show that by using a panel of radiolabelled specific mAbs it is possible to quantify not only PBIgG but also PBIgG subclasses and PBIgM. Analysis by gel filtration showed that all anti‐IgG and anti‐IgG‐subclass mAbs bound to their respective antigens in a ratio of about 1:1. However, the binding ratio for the anti‐IgM Mab could not be established. There was a good correlation between the antibody‐density per platelet as determined with the anti‐IgG mAb and determined as the sum of the IgG molecules of different subclass per platelet (r= 0·90). Platelet fragments did not interfere in the assay. 89 normal healthy controls had 140 IgG molecules per platelet and bound 269 anti‐IgM molecules per platelet (geometric means).

In a study on the detection of PBIg in 147 thrombocytopenic patients, it appeared that the MARIA had a sensitivity of 61% and a specificity of 45% for the diagnosis of idiopathic thrombocytopenic purpura (ITP). Both in ITP and in secondary thrombocytopenia (STP), PBIgG1 and PBIgG3 were found more frequently (60% and 61%, respectively) than PBIgG2and PBIgG4 (13% and 9%, respectively). There was no relation between the amount of total PBIgG or PBIgM and the platelet count in either ITP or STP. Also, if IgG antibodies of only one subclass were found, there was no relation between the severity of the thrombocytopenia and the amount of PBIgG.

By applying the MARIA, it is possible to quantify PBIgG, all four PBIgG‐subclasses and PBIgM in ITP and STP in a reliable way.
K+ efflux in deoxygenated sickle cells in the presence or absence of DIOA, a specific inhibitor of the [K+, Cl‐] cotransport system
Authors: Olivieri Oliviero , Vitoux Dominique , Bachir Dora , Beuzard Yves
Published: March 2008

Summary. The ouabain bumetanide resistant (OBR) K+ efflux was investigated in deoxygenated sickle cells in comparison to oxygenated ones, by using a specific inhibitor of the [K+, Cl‐] co‐transport system, [(DihydroIndenyl)Oxy] Alkanoic acid (DIOA). A DIOA sensitive and a DIOA resistant K+ efflux were measured in deoxygenated sickle cells. The DIOA sensitive K+ efflux shared the properties of the [K+, Cl‐] co‐transport system, being stimulated by decreased pH and hypoosmolarity. This DIOA sensitive K+ efflux represented 70% of the total K+ efflux at pH 7·0 and at low pO2 (10‐15 mmHg). Thus, a small reduction in Ph effectively stimulated the [K+, Cl‐] co‐transport system in deoxygenated condition, and this may contribute significantly to the sickle cell dehydration. We conclude that at pH lower than 7·4, the [K+,Cl‐] co‐transport system is permanently activated in sickle cells and leads to sickle cell dehydration in both oxygenated and deoxygenated conditions.
The priming effects of the products of stimulated mononuclear cells on the response of neutrophils to C5a des arg
Authors: Crouch Sharon , Fletcher John
Published: March 2008

Certain recombinant human cytokines have been shown to enhance polymorphonuclear leucocyte (PMN) responses to subsequent stimulation. Mononuclear cells (MNC) from normal healthy individuals were stimulated for 5 h with 1 μg/ml bacterial lipopolysaccharide (LPS) in order to induce production and secretion of inflammatory cytokines into the surrounding medium. These mononuclear cell conditioned media (MNCM) were then used to prime PMN isolated from healthy volunteers. Preincubating the PMN with MNCM for 15 min at 4°C followed by washing and warming to 37°C caused a 344% increase (n= 26) in the rate of superoxide anion production in response to zymosan‐activated serum (ZAS), a source of C5a des arg. This effect could not be reproduced with recombinant human forms of interleukin 1 beta (Il‐1 beta) or granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), although, with the latter, there was some effect when the preincubation stage was carried out for 60 min at 37°C. Only recombinant human tumour necrosis factor‐alpha (rh‐TNF‐alpha) gave a similar PMN priming effect to that seen with MNCM. This effect could not be reversed by washing away either the MNCM or rh‐TNF‐alpha. The priming effect could be markedly reduced (74.8%, n= 6) by employing the use of polyclonal antibody to TNF‐alpha in the preincubation step; assaying for TNF‐alpha in these MNCMs showed that the degree of priming corresponded to the amount of TNF‐alpha present. Rh‐TNF‐alpha alone appeared to have very little direct stimulatory effect on respiratory burst activity.

The results show that TNF‐alpha produced by LPS stimulated MNC after 5 h binds to a PMN surface receptor in the cold and warming of the cells to 37°C allows for an immediate and dramatic response to ZAS stimulation. This suggests that TNF‐alpha is the important cytokine upregulating PMN responses to other physiological mediators, including C 5 a des arg during the early phases of an inflammatory reaction.
A fibroblast cell culture model to study vitamin K metabolism and the inhibition of vitamin K epoxide reductase by known and suspected antagonists
Authors: Ross P. J. , Shearer M. J. , Diplock A. T. , Schey S. A.
Published: March 2008

The metabolism and antagonism of vitamin K has been studied in cultured fibroblasts. Monolayers of 3T3 mouse fibroblasts (grown in the absence or presence of warfarin or other putative antagonists) were incubated for 24 h with [1′, 2′‐3H2]phylloquinone (K1) or [1′,2′‐3H2]phylloquinone epoxide (K1O), the cells harvested and lipid extracts fractionated by high performance liquid chromatography. [3H]K1 was converted to [3H]K1O (about 20% of [3H] lipids) and to unidentified polar metabolites (30%). [3H]K1O was converted to [3H]K1 (3%) and to polar metabolites (50%). Cells grown with warfarin showed a marked increase in the [3H]K1O:K1 ratio and in the proportion of polar metabolites. The metabolic interconversion of K1 and K1O and inhibitory response to warfarin provide evidence for a fibroblast pathway analogous to the vitamin K‐epoxide cycle in the liver. From the K1O:K1 ratios it was possible to grade the antagonism of vitamin K epoxide reductase activity by known and suspected inhibitors. Inhibitory ratios were seen for racemic warfarin down to 10−8m. S‐warfarin was a more potent antagonist than the R‐enantiomer. Consistently low K1O:K1 ratios were observed for N‐methyl‐thiotetrazole and antibiotics with (moxalactam) or without (cefotaxime) this side chain suggesting that none of these compounds are direct inhibitors of vitamin K epoxide reductase. Fibroblasts grown in cell culture provide a useful model to study the extrahepatic role of vitamin K and the mode of action of vitamin K antagonists.
Interleukin 2 infusion induces haemopoietic growth factors and modifies marrow regeneration after chemotherapy or autologous marrow transplantation
Authors: Heslop Helen E. , Duncombe Andrew S. , Reittie Joyce E. , Bello‐Fernandez Concha , Gottlieb David J. , Prentice H. Grant , Mehta Atul B. , Hoffbrand A. Victor , Brenner Malcolm K.
Published: March 2008

Administration of interleukin 2 (IL2) to patients with minimal residual malignant disease following myelo‐ablative chemo‐radiotherapy may augment immune reconstitution and reduce the risk of relapse by increasing cytotoxic effector function and cytokine dependent killing directed at residual malignant cells. The ability of IL2 generated activated killer cells to inhibit haemopoietic progenitor cells and to release gamma‐interferon (γIFN) and tumour necrosis factor (TNF) may, however, retard haemopoietic recovery, as both TNF and γIFN inhibit normal myelopoiesis in vitro. To determine the effect of IL2 infusion on myeloid regneration in vivo, we have examined haemopoietic recovery in patients receiving this cytokine following autologous marrow transplantation or ablative chemotherapy. We find that IL2 infusion accelerates neutrophil recovery and that granulocyte‐macrophage colony stimulating factor (GMCSF) and IL3 mRNA become detectable in circulating mononuclear cells. Induction of TNF by IL2 may also contribute to subsequent acceleration of myelopoiesis by initiation of GM‐CSF mRNA synthesis in patient marrow fibroblasts. These results show that IL2 infusion may facilitate myeloid recovery when administered during the period of haemopoietic regeneration following ablative chemoradiotherapy.
Anti‐GPIIb/IIIa (CD41) monoclonal antibody‐induced platelet activation requires Fc receptor‐dependent cell‐cell interaction
Authors: Anderson George P. , de Winkel Jan G. J. van , Anderson Clark L.
Published: March 2008

We studied platelet activation by UR1, a murine IgG1 anti‐CD41 mAb. Like thrombin and crosslinked anti‐FcγRII mAb IV3, UR1 initiates prompt aggregation and Ca2+ mobilization. UR1 F(ab′) fragments failed to activate, yet inhibited UR1 IgG‐mediated activation. UR1‐induced activation was blocked by anti‐FcγRII mAb. High viscosity (15% dextran or Ficoll), which impedes cell‐cell interaction, inhibited activation by UR1. Cell‐cell interaction was confirmed by cell‐mixing studies. UR1 binding to platelets of one pool was blocked with UR1 F(ab′)2 allowing UR1 binding only to FcγRII. IV3 Fab fragments blocked ligand binding to FcγRII on platelets of a second pool; thus, UR1 could bind only its epitope. UR1 initiated an immediate [Ca2+]i increase in the intermixed pools at low ionic strength. These studies indicate that UR1 IgG binds CD41 on one platelet to form immune complexes which then crosslink and stimulate FcγRII on nearby platelets. Two other anti‐CD41 mAb, 6C9 and C17, and two anti‐CD9 mAb, AG1 and mAb7, activated platelets in a UR1‐like manner. We propose that platelet FcγRII crosslinking that follows the interaction of IgG‐opsonized platelets may be a common mechanism by which antiplatelet antibodies activate platelets.
Two different forms of homozygous sickle cell disease occur in Saudi Arabia
Authors: Padmos M. A. , Sackey K. , Roberts G. T. , Kulozik A. , Bail S. , Morris J. S. , Serjeant B. E. , Serjeant G. R.
Published: March 2008

Haematological, clinical and some molecular genetic features of homozygous sickle cell (SS) disease in Saudi Arabia have been compared in 33 patients from the Eastern Province (Eastern) and 30 from the South Western Province (Western). Eastern patients all had the Asian beta globin haplotype whereas Western patients were more variable but predominantly of the Benin haplotype. Eastern patients had more deletional alpha thalassaemia, higher total haemoglobin and fetal haemoglobin levels, and lower HbA2, mean cell volume, reticulocytes, and platelet counts. Clinically, Eastern patients had a greater persistence of splenomegaly, a more normal body build and greater subscapular skin fold thickness, and Western patients had more dactylitis and acute chest syndrome. Painful crises and avascular necrosis of the femoral head were common and occurred equally in both groups. The disease in the Eastern province has many mild features consistent with the higher HbF levels and more frequent alpha thalassaemia but bone pathology (painful crises, avascular necrosis of the femoral head, osteomyelitis) remains common. The disease in the West is more severe consistent with the Benin haplotype suggesting an African origin.
A simple and rapid method of direct sequencing using Dynabeads
Authors: Thein S. L. , Hinton J.
Published: March 2008

Highly specific regions of genomic DNA can now be produced in large quantities by enzymatic amplification using the polymerase chain reaction (PCR) and a thermostable Taq polymerase (Saiki et al. 1988). This has encouraged the development of direct sequencing of the amplified DNA. Apart from the obvious advantage of the avoidance of cloning of DNA, direct sequencing enables both alleles to be analysed simultaneously. The latter is particularly advantageous in heterozygous states where the mutant allele cannot be differentiated from the normal. However, direct dideoxy sequencing of PCR‐amplified DNA is still not consistently satisfactory. The difficulty arises from the nature of the PCR‐amplified DNA which consists of short, linear double‐stranded DNA. The two strands of the DNA can rapidly reanneal during DNA sequencing, blocking or displacing the sequencing primer from the template strand, thereby decreasing the amount of specific termination products formed in the sequencing reactions.
IL‐2 abolishes fibroblast proliferation in long‐term bone marrow culture by inhibition of an accessory cell
Authors: Carron J. A. , Cawley J. C.
Published: March 2008

Addition of interleukin‐2 (IL‐2 (>250 U/ml) during the first 3 d of long‐term bone marrow culture (LTBMC) permanently abolished the fibroblast component of the stromal layer, even when IL‐2 was removed after the 3 d culture period. When IL‐2 was added at more than 72 h after initiation of culture, no effect was observed. Stromal growth in IL‐2‐treated culture was restored by addition of irradiated bone marrow, indicating that the IL‐2 inhibited an accessory cell rather than the fibroblast directly. Accessory cells were shown to be necessary for fibroblast proliferation at low cell densities and were also inhibited by IL‐2. The accessory cell effect could not be replaced by LTBMC supernatants or extracellular matrix. It is suggested that these observations are relevant to the suppression of haemopoiesis observed in patients receiving IL‐2.

Addition of interleukin‐2 (IL‐2 (>250 U/ml) during the first 3 d of long‐term bone marrow culture (LTBMC) permanently abolished the fibroblast component of the stromal layer, even when IL‐2 was removed after the 3 d culture period. When IL‐2 was added at more than 72 h after initiation of culture, no effect was observed. Stromal growth in IL‐2‐treated culture was restored by addition of irradiated bone marrow, indicating that the IL‐2 inhibited an accessorycell rather than the fibroblast directly. Accessory cells were shown to be necessary for fibroblast proliferation at low cell densities and were also inhibited by IL‐2. The accessory cell effect could not be replaced by LTBMC supernatants or extracellular matrix. It is suggested that these observations are relevant to the suppression of haemopoiesis observed in patients receiving IL‐2.
Autologous blood stem cell harvesting and transplantation in patients with advanced ovarian cancer
Authors: Menichella G. , Pierelli L. , Foddai M. L. , Paoloni A. , Vittori M. , Serafini R. , Panici P. Benedetti , Scambia G. , Baiocchi G. , Greggi S. , Laurelli G. , Salerno G. , Mancuso S. , Mango G. , Bizzi B.
Published: March 2008

We investigated the feasibility of a programme of autologous blood stem cell (ABSC) harvesting and transplantation in 13 patients with advanced ovarian cancer, previously untreated by chemotherapy or radiotherapy and entering a phase II study of high‐dose cisplatin, etoposide and carboplatin with haematopoietic stem cell rescue. Prior to high‐dose treatment all patients underwent two courses of cisplatin and cyclophosphamide. An 8‐fold increase of the peripheral colony forming unit granulocytic‐macrophage (CFU‐GM) was observed during recovery from myelosuppression after the first chemotherapy course. The second course determined a 2·5‐fold increase of peripheral CFU‐GM. In 70% of enrolled patients (nine patients) we were able to perform ABSC harvesting by leukaphereses; in the apheresed patients we harvested an average of 20·8 × 104/kg CFU‐GM (range 10·9–37·0). Haematopoietic trilineage engraftment, established as the number of days necessary to reach white blood cells (WBC) >1·0 × 109/1, polymorphonuclear leucocytes (PMN) >0·5 × 109/1 and platelets (PLT) >50·109/1. occurred very promptly and was sustained in the same series after high‐dose cisplatin, carboplatin and etoposide, followed by autologous blood stem cell transplantation (ABSCT). In our experience we found a significant correlation (r = 0·77; P<0·05) between CFU‐GM infused dose and the engraftment speed of PMN. We conclude that the combination of cisplatin and cyclophosphamide is effective in mobilizing haematopoietic progenitors in the peripheral blood of patients with advanced ovarian cancer, previously untreated by chemora‐diotherapy. Moreover, ABSCT is capable of rapidly restoring the haematopoietic function after high‐dose treatment and for this reason it represents a particularly advisable therapeutic option for the treatment of solid tumours because these patients are commonly older than 50 and can be excluded from bone marrow transplantation.
A monoclonal antibody to a 67 kD cell membrane glycoprotein directly induces persistent platelet aggregation independently of granule secretion
Authors: Zuzel M. , Walton S. , Burns G. F. , Berndt M. C. , Cawley J. C.
Published: March 2008

The transition from reversible to persistent platelet aggregation has been difficult to study because of interference both from preceding primary aggregation and from the events associated with granule secretion during secondary aggregation. As a result it remains unclear whether the persistence of aggregation involves some secretion‐independent specific platelet surface reactions. Here we show that a monoclonal antibody (MAb), LeoAl, against a newly described 67 kD platelet membrane glycoprotein induced active platelet aggregation consisting of two distinct phases. The first secretion‐independent phase was in several respects (extracellular protein, divalent cation, and pH dependence) different from primary aggregation, but closely resembled the transition from primary to secondary aggregation observed at certain concentrations of physiological agonists. The second, faster phase was indistinguishable from secretion‐dependent aggregation to various stimulants. It was shown that p67, GPIIb‐IIIa and FCγRII are all involved in the observed aggregation, probably through their close topographical association. It is suggested that LeoAl‐induced aggregation can be used as a model to study the receptors, ligands and metabolic pathways specifically involved in the transition from reversible to persistent platelet aggregation.
Haemorheological changes in the very short term after abstention from tobacco by cigarette smokers
Authors: Rothwell M. , Rampling M. W. , Cholerton S. , Sever P. S.
Published: March 2008

A study was made of the haemorheological changes that took place in the days immediately following tobacco withdrawal from cigarette smokers. In both males and females substantial and persistent reductions in blood viscosity occurred within 2 d, the fall at high shear rate corresponding to about 8% while at low shear rate it was approximately twice as much. These changes were due partly to a fall in packed cell volume (PCV), but also to reductions in total plasma protein and fibrinogen concentrations which led to reductions in plasma viscosity and rouleaux formation. These plasma protein‐related changes were less pronounced in the female group. The results indicate that, even in the very short‐term, abstention from cigarettes leads to substantial improvements in the haemorheological profile of heavy smokers.
Chromosome 17p and p53 changes in lymphoma
Authors: Rodriguez M. A. , Ford R. J. , Goodacre A. , Selvanayagam P. , Cabanillas F. , Deisseroth A. B.
Published: March 2008

The clinical course of lymphoma patients in whom rearrangements or deletions of the short arm of chromosome 17 (17p) were evident by cytogenetics was rapidly progressive with a short survival. The gene for the protein designated p53 resides in 17p. We studied four lymphoma cell lines derived from human tumours, and 25 tumour samples of patients with lymphomas, for any evidence of p53 genomic changes by Southern blot technique. The four cell lines and four of the 25 tumour samples showed numerical changes of chromosome 17 or structural abnormalities of 17p (translocations or deletions). Allelic loss of the p53 gene was found in two of the four cell lines, and one of these in addition showed a rearrangement of the 3’end of the gene. Of the four tumours known to have chromosome 17 abnormality, one specimen showed allelic loss of the p53 gene. None of the remaining tumour samples showed any significant change. These studies suggest that acquisition of changes in the short arm of chromosome 17, which may be interrelated with the p53 gene, may carry a poor prognosis in patients with non‐Hodgkin's lymphoma.
No activation of the common pathway of the coagulation cascade after a highly purified factor IX concentrate
Authors: Mannucci P. M. , Bauer K. A. , Gringeri A. , Barzegar S. , Santagostino E. , Tradati F. C. , Rosenberg R. D.
Published: March 2008

Purer factor IX concentrates, containing very little or no factor II or X, have been developed in an attempt to avoid the thromboembolic complications that occur with prothrombin complex concentrates (PCC), which also contain factors II and X and variable amounts of factor VII. To evaluate ex vivo the thrombogenic potential of one of these purer concentrates, we studied whether large single doses produced signs of activation of the coagulation cascade in patients with haemophilia B, and compared the results with those obtained after infusion of a PCC. Seven patients were infused with 50 IU/kg of factor IX concentrate and seven additional patients were subsequently infused with 100 IU/kg of the same concentrate. After the infusions, factor IX levels rose in proportion to the administered dose while the concentrations of factor II and factor X did not rise at all. At both doses of concentrate, we did not observe significant postinfusion increments in the levels of the factor X activation peptide (a measure of the activity of the factor VIla‐tissue factor complex and/or the factor IXa‐VIIIa‐activated surface complex), prothrombin fragment 1 + 2 (a measure of factor Xa activity), and fibrinopeptide A (a measure of thrombin activity). We also infused 10 patients with a PCC (50 IU/kg). After the infusions, significant rises in the concentrations of the factor X activation peptide and prothrombin fragment were observed. Therefore, it appears that the infusion of a PCC to patients with haemophilia B can augment factor X activation and subsequently thrombin generation in vivo and that this process can be abrogated by the administration of more pure factor IX concentrate.
Platelet activation and alpha granule secretion in type IIB von Willebrand's disease
Authors: Gralnick Harvey R. , Williams Sybil , McKeown Laurie P. , Connaghan Gerard , Shafer Brenda , Hansmann Kristin , Magruder Louise , Vail Mike
Published: March 2008

Type IIB von Willebrand disease is characterized by enhanced ristocetin‐induced platelet aggregation, spontaneous platelet aggregation, thrombocytopenia and the absence of the largest plasma von Willebrand factor (vWf) multimers. The absence of the largest plasma vWf multimers is related to their enhanced binding to platelets. The abnormal affinity of the IIB von Willebrand factor to platelets results in thrombocytopenia, but the mechanism is not known. We have studied the platelets from three patients with type IIB von Willebrand disease and have found evidence of platelet activation and alpha granule secretion as defined by increased amounts of von Willebrand factor, fibrinogen and the alpha granule protein PADGEM/GMP‐140 on the surface of these platelets. The degree of thrombocytopenia appears to be directly related to the number of platelets with fibrinogen bound to the surface. PADGEM/ GMP‐140, an alpha granule membrane protein, fuses with the platelet plasma membrane after activation and is a site on platelets which binds to neutrophils or monocytes. This alpha granule protein may play an additional role in platelet clearance and thrombocytopenia in type IIB von Willebrand disease. This may, in part, explain the absence of thromboembolic phenomena despite the presence of activated platelets in patients with type IIB von Willebrand disease.
Postnatal transition of γ‐globin gene expression in normal Japanese population
Authors: Enoki Y. , Ohga Y. , Sakata S. , Kohzuki H.
Published: March 2008

Temporal course of postnatal changes in the γ isoform composition of human fetal haemoglobin (Hb F) was studied in 259 cord, 272 infantile and 216 adult Japanese blood samples. Reversed phase high‐performance liquid chromatography was used for determination of the three isoforms of the γ chain (AγT, Aγ1 and cγ), and the adult samples, usually with less than 1% of Hb F, were enriched for Hb F by an alkali denaturation‐salting out procedure. The results show that the cγ gene expression, kept at a higher and strictly‐controlled level in the neonatal period, undergoes a gradual change during 1 year, beginning 3–4 months after birth, to the adult stage which is characterized by a generally lower and loosely‐controlled expression of the gene. Further change seems to occur after 1 year, settling to the final adult level. The AγT gene frequency is estimated as 0‐1 39 in the present Japanese adult population, and is essentially identical to the value for the newborns in the same population (0‐141).
Iron metabolism in patients with the anaemia of end‐stage renal disease during treatment with recombinant human erythropoietin
Authors: Kooistra M. P. , Es A. Van , Struyvenberg A. , Marx J. J. M.
Published: March 2008

Iron metabolism was studied in 21 patients with the anaemia of end‐stage renal disease during 40 weeks of treatment with recombinant human erythropoietin (rhEPO). Oral iron was prescribed to all patients. Initial serum iron concentrations and transferrin saturation levels were subnormal, decreased during the correction period of treatment, and increased thereafter. In 81% of patients in whom pretreatment transferrin saturation was below 0–25, transferrin saturation decreased below 0–16, despite sufficiently high serum ferritin levels. Serum ferritin concentrations decreased significantly. There was no correlation between serum ferritin levels and serum iron or transferrin saturation.

Ferrokinetic studies, performed before and during treatment, showed an increase in plasma iron turnover, in erythron transferrin uptake, and in the flux of iron binding sites through the plasma.

The rhEPO dose needed to keep the haematocrit at the target level during the maintenance period of treatment was significantly correlated with transferrin saturation, and iron binding capacity, but not with serum ferritin concentrations. This suggests that the functional availability of iron in plasma, rather than the size of body iron stores, is a major factor in the determination of the response to rhEPO treatment in end‐stage renal disease.
Molecular cloning of interferon receptors: a short review
Authors: Aguet Michel
Published: March 2008

Interferons (IFNs) exert their pleiotropic effects through at least two different receptors. IFNs α/β (type I IFNs) crossreact with presumably common receptors, while IFNγ (type II IFN) initiates its biological effects through its own specific receptor system. Transmembrane proteins corresponding to the ligand binding chains of these receptors have recently been cloned. But transfection experiments revealed that additional components are probably involved in high affinity binding of some type I IFN subtypes and that a species specific putative signal transducer is required to confer biological responsiveness to IFNγ. Although some elements involved in transcriptional regulation of interferon inducible genes have been identified, the pathways involved in these regulatory processes are not understood as yet. The current knowledge on the structure of IFN receptors is reviewed in the context of some recent advances on other cytokine receptors.
Interferon alpha in lymphoma
Authors: Rohatiner A. Z. S.
Published: March 2008

A number of studies are currently in progress to evaluate the use of interferon alpha, alone and in combination with conventional chemotherapy in patients with follicular lymphoma. The background to these is reviewed, together with currently available data about the potential role of interferon alpha in cutaneous T cell lymphoma.
The molecular pathology of chronic myelogenous leukaemia
Authors: Kurzrock Razelle , Talpaz Moshe
Published: March 2008

The first consistent karyotypic abnormality found to be associated with neoplastic disease was the Philadelphia (Ph) chromosome (Nowell & Hungerford, 1960). Furthermore, the best‐studied example of translocation‐mediated gene activation occurs in leukaemia patients bearing this abnormality (reviewed by Kurzrock et al, 1988). In these individuals, the Ph translocation (t(9;22)(q34;q11)) results in transposition of the ABL proto‐oncogene from chromosome 9q34 to 22q11, where it is fused with part of the BCR gene. It is now known that as a result of the Ph translocation, p160BCR and p145ABL (the normal BCR and ABL gene products) are replaced by p210BCR‐ABL. This aberrant protein constitutes the molecular fingerprint of CML. The enhanced tyrosine phosphokinase enzymatic activity (a property possessed by some growth factor receptors and transformation‐inducing oncogenes) of p210BCR‐ABL implicates a direct role for this molecule in the pathogenesis of CML. Because the Ph translocation is present in the early chronic phase, the union of the BCR and ABL genes is probably involved in the initiation of the leukaemic process. The secondary molecular forces driving progression of CML to blast crisis are however unknown, and may differ from patient to patient.

Approximately 10% of CML patients lack a Ph chromosome. One‐half of these individuals have bcr rearrangement and express p210BCR‐ABL. Ph + and Ph ‐ bcr + (p210 +) CML are identical and should be treated the same. Molecular follow‐up of diploid bcr + CML patients is essential for detection of persistent malignancy after therapy.

The presence of a specific marker—the BCR‐ABL message—permits the development of new diagnostic approaches for CML. For instance, detection of a BCR‐ABL message with the use of the highly sensitive polymerase chain reaction, a technique capable of detecting up to one leukaemia cell amongst one million normal cells, yields important information about minimal residual disease. Finally, the use of therapy directed against the BCR‐ABL product may be a worthwhile strategy which deserves investigation, and may prompt a new era of tumour‐specific treatment.
Interferon in essential thrombocythaemia
Authors: Gisslinger Heinz , Chott Andreas , Scheithauer Werner , Gilly Bettina , Linkesch Werner , Ludwig Heinz
Published: March 2008

In the present investigation, 20 patients with ET were treated with recombinant interferon alfa‐2c (IFN) for up to 4 years. Initially, IFN was administered subcutaneously at a dosage of 6–45 MU/week. The dosage was adjusted according to individual tolerance and response. The median dose during induction was 20 MU/week, 10 MU/week during the remaining first year, 6 MU/week during the second year and 2 MU/week thereafter.

13 patients (65%) achieved complete remission (platelet count <440/nl), four patients (20%) had partial remission (>440/nl but a reduction by more than 50% of the initial count). The median platelet count remained steady throughout the 4‐year period of treatment, in spite of extreme dose reductions. After withdrawal of IFN, however, platelet counts again increased. The white blood cells showed a marked decrease similar to that of platelet counts, whereas the haemoglobin level remained fairly stable. In the bone marrow, a significant decrease in megakaryocyte density and size could be observed.

Concurrently with the improvement of haematological parameters, clinical symptoms improved, but reappeared after withdrawal of IFN. During induction, fever, bone and/or muscle pain, fatigue, lethargy and psychological symptoms were the most prominent side‐effects in the majority of patients. In three patients these symptoms led to discontinuation of the treatment. With repeated dose reductions, excellent long‐term tolerance was achieved, and during late maintenance treatment the only observed side‐effect was an induction of thyroid autoimmunity in three patients.

IFN is an effective, well‐tolerated alternative in the long‐term treatment of symptomatic ET. However, since withdrawal of IFN leads to recurrence of thrombocytosis, continued treatment is to be recommended.
Rational basis for the metabolic modulation of 5‐fluorouracil by leucovorin and interferon alpha
Authors: Rustum Youcef M.
Published: March 2008

Although 5‐fluorouracil (FUra) has been the drug of choice for the treatment of patients with advanced colorectal carcinoma, the response rates are in the range of 15% and the median survival times do not exceed 9 months. Interferon alpha (IFNα) has limited antitumour activity in this disease. Recent clinical trials have demonstrated that both the response rate and the survival time of patients with advanced colorectal cancer can be significantly improved by the addition of leucovorin (CF) to FUra. In a randomized phase III trial, the median response to FUra and CF was about 30% with a range of 20–55%, dependent on the dose and schedule of administration of CF. Due to multiplicity of mechanisms of action postulated for IFNα, the role of IFNα in the modulation of the cytotoxicity and therapeutic efficacy of FUra alone and in combination with CF has been evaluated in patients with advanced colorectal cancer and also in a variety of human cell lines in culture, including human colorectal cell lines, HCT‐8, human bladder cell line RT‐4 and leukaemia CEM cells.

The results obtained in patients demonstrated that indeed the antitumour activity of FUra can be improved by the addition of IFNα with a response range from 26% to 76%. The initial clinical use of this combination, however, has been limited in some cases by the severe host toxicity. In vitro results demonstrated that: (1) in vitro cytotoxicity potentiation by IFNα is cell‐type dependent; (2) in these cells where modulation was possible, αIFN was used as a low and noncytotoxic doses (10–50 μg/ml for 3–5 d exposure), and (3) the cytotoxicity of IFNα was dose dependent, indicating a direct antiproliferative cellular effect.
Treatment of advanced malignant melanoma with recombinant interferon alfa‐2a in combination with DTIC: long‐term follow‐up of two phase II studies
Authors: Hersey P. , McLeod G. R. C. , Thomson D. B.
Published: March 2008

The present studies were designed to examine whether treatment of disseminated melanoma with a combination of recombinant interferon alpha (rIFNα‐2a) and DTIC may provide better results than either agent alone. Two dose levels of rIFNα‐2a were examined with the same dose and schedule of DTIC administration. In the first study 76 patients were treated with rIFNα‐2a (3 × 106 IU days 1–3, 9 × 106 IU days 4–70 then 9 × 106 IU TIW) plus DTIC (i. v. 200 mg/m2, escalating to 800 mg/m2 each 21 d) for 6 months or longer or until failure. Responses were seen in 26% of patients overall (7CR, 13PR) with the highest responses being seen in those assessed as having a good prognosis and in patients with skin and/or lung metastases. It was notable that males showed a better response than famales and that patients with metastases in lymph nodes showed low responses. Response rates in 30 patients treated with high‐dose rIFNα‐2a (18 × 106 IU days 7–70) were not superior to that seen at the lower dose and was accompanied by significant increase in bone marrow toxicity. The duration of responses appeared longer than expected from responses to DTIC alone and four patients remain progression free at periods in excess of 3 years. Failure in nine of the responders was due to the development of brain metastases which emphasizes the need for additional treatment approaches to treat micrometastases at this site. The results of a randomized control study comparing DTIC alone with DTIC plus‘low’dose rIFNα‐2a are awaited with interest.