bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the N-terminal fragment ofEscherichia coliLon protease
Authors: Li Mi , Gustchina Alla , Rasulova Fatima S. , Melnikov Edward E. , Maurizi Michael R. , Rotanova Tatyana V. , Dauter Zbigniew , Wlodawer Alexander
Published: July 2010

The structure of a recombinant construct consisting of residues 1–245 ofEscherichia coliLon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function fromBordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.
Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection
Authors: Bowler Matthew W. , Guijarro Matias , Petitdemange Sebastien , Baker Isabel , Svensson Olof , Burghammer Manfred , Mueller-Dieckmann Christoph , Gordon Elspeth J. , Flot David , McSweeney Sean M. , Leonard Gordon A.
Published: July 2010

Crystals of biological macromolecules often exhibit considerable inter-crystal and intra-crystal variation in diffraction quality. This requires the evaluation of many samples prior to data collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron-based facilities optimized for high-throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample-containing loop for microcrystals using an X-ray beam of 5 µm diameter and selecting the most ordered regions of relatively large crystals using X-ray beams of 5–50 µm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction-quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRF's structural biology beamlines is also presented.
The first crystal structure of a gramicidin complex with sodium: high-resolution study of a nonstoichiometric gramicidin D–NaI complex
Authors: Olczak A. , Główka M. L. , Szczesio M. , Bojarska J. , Wawrzak Z. , Duax W. L.
Published: July 2010

The crystal structure of the nonstoichiometric complex of gramicidin D with NaI has been studied using synchrotron radiation at 100 K. The limiting resolution was 1.25 Å and theRfactor was 16% for 19 883 observed reflections. The general architecture of the antiparallel two-stranded gramicidin dimers in the studied crystal was a right-handed antiparallel double-stranded form that closely resembles the structures of other right-handed species published to date. However, there were several surprising observations. In addition to the significantly different composition of linear gramicidins identified in the crystal structure, including the absence of the gramicidin C form, only two cationic sites were found in each of the two independent dimers (channels), which were partially occupied by sodium, compared with the seven sites found in the RbCl complex of gramicidin. The sum of the partial occupancies of Na+was only 1.26 per two dimers and was confirmed by the similar content of iodine ions (1.21 ions distributed over seven sites), which was easily visible from their anomalous signal. Another surprising observation was the significant asymmetry of the distributions and occupancies of cations in the gramicidin dimers, which was in contrast to those observed in the high-resolution structures of the complexes of heavier alkali metals with gramicidin D, especially that of rubidium.
Quantitive evaluation of macromolecular crystallization experiments using 1,8-ANS fluorescence
Authors: Watts David , Müller-Dieckmann Jochen , Tsakanova Gohar , Lamzin Victor S. , Groves Matthew R.
Published: July 2010

Modern X-ray structure analysis and advances in high-throughput robotics have allowed a significant increase in the number of conditions screened for a given sample volume. An efficient evaluation of the increased amount of crystallization trials in order to identify successful experiments is now urgently required. A novel approach is presented for the visualization of crystallization experiments using fluorescence from trace amounts of a nonspecific dye. The fluorescence images obtained strongly contrast protein crystals against other phenomena, such as precipitation and phase separation. Novel software has been developed to quantitatively evaluate the crystallization outcome based on a biophysical metric correlated with voxel protein concentration. In >1500 trials, 85.6% of the successful crystallization experiments were correctly identified, yielding a 50% reduction in the number of `missed hits' compared with current automated approaches. The use of the method in the crystallization of three previously uncharacterized proteins from the malarial parasitePlasmodium falciparumis further demonstrated.
Emerging from pseudo-symmetry: the redetermination of human carbonic anhydrase II in monoclinicP21with a doubledaaxis
Authors: Robbins Arthur H. , Domsic John F. , Agbandje-McKenna Mavis , McKenna Robert
Published: July 2010

The crystal structure of human carbonic anhydrase II in the monoclinicP21space group with a doubledaaxis from that of the usually observed unit cell has recently been reported, with one of the two molecules in the asymmetric unit demonstrating rotational disorder [Robbinset al.(2010),Acta Cryst. D66, 628–634]. The structure has been redetermined, with the coordinates of both pseudo-symmetrically related molecules in the crystallographic asymmetric unit translated byx′ =x± 1/4, and no rotational disorder is observed. This corresponds to a different choice of how the four molecules in the unit cell should be grouped into pairs that represent a single asymmetric unit.
Nest‐site fidelity in parental male bluegill Lepomis macrochirus: spatial patterns and the influence of prior mating success
Authors: Bartlett J. A. , Ward M. P. , Landsman S. J. , Epifanio J. M.
Published: July 2010

A 4 year mark–recapture study examined the pattern of nesting site fidelity of parental‐type male bluegill Lepomis macrochirus. The study results indicated that iteroparous male L. macrochirus choose new nest sites near their own previously used sites. The scale of site fidelity varied, but generally males choose to renest within shoreline areas rather than specific or exact nest locations (94% within‐year, 86% among‐years). Iteroparous males also displayed no preference to nest in proximity to neighbouring males from previous colonies to suggest social fidelity. Contrary to expectation, manipulating males' reproductive success had no significant effect on the pattern or scale of male reproductive site fidelity.
Rapid sequence spinal anaesthesia for category‐1 urgency caesarean section: a case series
Authors: Kinsella S. M. , Girgirah K. , Scrutton M. J. L.
Published: July 2010

General anaesthesia is the fastest method for anaesthetising a category‐1 caesarean section but is associated with increased maternal morbidity and mortality. We describe the ‘rapid sequence spinal’ to minimise anaesthetic time. This consists of a no‐touch spinal technique, consideration of omission of the spinal opioid, limiting spinal attempts, allowing the start of surgery before full establishment of the spinal block, and being prepared for conversion to general anaesthesia if there are delays or problems. We present a case series of 25 rapid sequence spinal anaesthetics for category‐1 caesarean section. The mean (SD [range]) decision‐delivery interval was 23 (6 [14–41]) min. After excluding cases where there was an identified delay, the median (IQR [range]) time to prepare and perform the spinal was 2 (2–3 [1–7]) min, and time to develop a ‘satisfactory’ block was 4 (3–5 [2–7]) min. The total time to induce spinal anaesthesia was 8 (7–8 [6–8]) min. There were three pre‐operative conversions to general anaesthesia and three women had pain during surgery that did not require treatment. Our data indicate that one might expect to establish anaesthesia in 6–8 min using a rapid sequence spinal. Careful case selection is crucial. While rapid anaesthesia is important, the reduction of the decision‐delivery interval also requires attention to other stages in the pre‐operative process.
Acceptability and characteristics of 124 human bioequivalence studies with active substances classified according to the Biopharmaceutic Classification System
Authors: Ramirez Elena , Laosa Olga , Guerra Pedro , Duque Blanca , Mosquera Beatriz , Borobia Alberto M. , Lei Suhua H. , Carcas Antonio J. , Frias Jesus
Published: July 2010

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• In USA and Europe the classical way to guarantee equivalence between different formulations is the bioequivalence (BE) study based on in vivo bioavailability.

• The Biopharmaceutic Classification System (BCS) classifies active substances into four different groups according to their aqueous solubility and intestinal permeability.

• Recently the European Medicines Agency (EMA) has released a new bioequivalence guideline for immediate release solid oral dosage forms that includes recommendations on BCS‐based biowaivers.

WHAT THIS STUDY ADDS

• As far as we know this is the first time that the probability of proving BE of more than one hundred BE studies with 80 active substances categorized according to BCS was evaluated.

• Despite the usually employed test dissolution methodology proposed as quality control, pharmaceutical products with active substances from the four classes of BCS showed non‐BE studies.

AIM

The aim of this study was to evaluate the acceptability of 124 bioequivalence (BE) studies with 80 active substances categorized according to the Biopharmaceutics Classification System (BCS) in order to establish if there were different probabilities of proving BE between the different BCS classes.

METHODS

We evaluated the differences between pharmaceutical products with active substances from different BCS classes in terms of acceptability, number of subjects in the study (n), the point estimates, and intra‐ and inter‐subject coefficients of variation data from BE studies with generic products.

RESULTS

Out of 124 BE studies 89 (71.77%) were performed with pharmaceutical products containing active substances classified by the BCS. In all BCS classes there were non‐bioequivalent pharmaceutical products: 4 out of 26 (15.38%) in class 1, 14 out of 28 (50%) in class 2, 3 out of 22 (13.63%) in class 3 and 1 out of 13 (7.69%) in class 4. When we removed those pharmaceutical products in which intra‐subject variability was higher than predicted (2 in class 1 active substances, 9 in class 2 and 2 in class 3) there were still non‐BE pharmaceutical products in classes 1, 2 and 3.

CONCLUSIONS

Comparisons between pharmaceutical products with active substances from the four BCS classes have not allowed us to define differential characteristics of each class in terms of n, inter and intra‐subject variability for Cmax or AUC. Despite the usually employed test dissolution methodology proposed as quality control, pharmaceutical products with active substances from the four classes of BCS showed non‐BE studies.
Comparison of the agonist‐antagonist interaction model and the pool model for the effect of remoxipride on prolactin
Authors: Ma Guangli , Friberg Lena E. , Movin‐Osswald Gunilla , Karlsson Mats O.
Published: July 2010

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Prolactin response is a common side effect of antipsychotic drugs.

• The pool model was proposed to describe the effect of remoxipride on prolactin.

• The agonist‐antagonist interaction (AAI) model, which incorporated a dopamine‐prolactin feedback loop mechanism and circadian rhythm, was used to depict the effects of risperidone and paliperidone on prolactin.

• The pool model could not characterize prolactin response in clinical studies of risperidone and paliperdone.

WHAT THIS STUDY ADDS

• The AAI model and the pool model were both reconstructed based on the remoxipride data set from which the original pool model was built. The pool model was extended with a circadian rhythm component which was employed in the AAI model.

• The objective function values calculated by NONMEM revealed that the pool model with a circadian rhythm component best fitted the data. However, predictive checks of both models were satisfactory and the AAI model was superior in describing the circadian rhythm in healthy male volunteers when compared with earlier studies.

• This study shows that the AAI model was superior as it worked well across different populations, study designs and drugs.

AIMS

The tolerance to the prolactin response following administration of antipsychotic drugs has been modelled as a depletion of a prolactin pool (pool model) and a model where the tolerance is explained by a feedback loop including the dopamine interaction of prolactin release (agonist‐antagonist interaction model, (AAI model)). The AAI model was superior to the pool model when analyzing data from clinical trials of risperidone and paliperidone. Here we evaluated the two models using the remoxipride data, designed to challenge the short‐term prolactin response, from which the original pool model was built.

METHODS

The remoxipride data were collected from a study where eight healthy male subjects received two remoxipride infusions on five occasions. The intervals between the first and second dose on each occasion were 2, 8, 12, 24 and 48 h, respectively. The pool and AAI models were fitted using NONMEM.

RESULTS

According to the objective function values the pool model with a circadian rhythm function fitted the data slightly better, while the AAI model was better in describing the circadian rhythm of prolactin. Visual predictive checks revealed that the models predicted the prolactin profiles equally well.

CONCLUSIONS

According to the analysis performed here, a previous analysis of several clinical studies and literature reports on prolactin concentrations, it appears that the dopamine feedback mechanism included in the AAI model is better than the storage depletion mechanism in the pool model to estimate the bio‐rhythm of prolactin time‐course and the tolerance development across different populations, drugs, treatment schedules and time.
Appropriate infliximab infusion dosage and monitoring: results of a panel meeting of rheumatologists, dermatologists and gastroenterologists
Authors: De Vries Hilbert S. , Van Oijen Martijn G. H. , Driessen Rieke J. B. , De Jong Elke M. G. J. , Creemers Marjonne C. W. , Kievit Wietske , De Jong Dirk J.
Published: July 2010

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

Infliximab is an effective treatment for rheumatoid arthritis, ankylosing spondylitis, Crohn's disease (both adult and paediatric), ulcerative colitis, psoriatic arthritis and plaque psoriasis and national and international guidelines have been developed for each indication.

WHAT THIS STUDY ADDS

This study is the first study which compared current international, national and local guidelines from the medical specialties involved in the treatment with infliximab on the following topics: indication, dosage, synergy and monitoring of vital signs.

AIMS Infliximab, an anti‐TNF biologic agent, is currently indicated and reimbursed for rheumatoid arthritis, ankylosing spondylitis, Crohn's disease (both adult and paediatric), ulcerative colitis, psoriatic arthritis and plaque psoriasis. Development of national and international guidelines for rheumatology, gastroenterology and dermatology, was mostly based on clinical studies and expert opinion. The aim of this study was to compare available guidelines and local protocols for rheumatology, dermatology and gastroenterology, regarding dosage of infliximab, synergy of infliximab with concomitant medication and monitoring of vital signs during infliximab administration, for achieving optimal care.

METHODS Current international, national and local guidelines on the use of infliximab were reviewed and compared, differences and shortcomings were identified, and optimal treatment schedules discussed during a meeting (July 2008) of clinical experts and researchers from three departments of a Dutch university hospital.

RESULTS Recommended dosages of infliximab are not equal for different indications. Loss of response to infliximab is a common problem encountered within the three medical specialties, but indications for adjustments in treatment schedules are lacking in all of the guidelines. Monitoring of vital signs (blood pressure, pulse, temperature) during infusion with infliximab is common practice and recommended by some guidelines. Routine measurement of vital signs is not of any value in predicting or recognizing acute infusion reactions, in our experience, and this is confirmed by literature on inflammatory bowel disease.

CONCLUSION Different indications encompass different dosing schedules. National and internal guidelines do not provide advice regarding loss of response. Routine measurement of vital signs during infusion is not valuable in detecting acute infusion reactions and should only be performed in case of an acute infusion reaction. These topics need to be studied in future studies and covered in future guidelines.
Characterization of Bone Marrow Stromal Cells in Suspension and Monolayer Cultures
Authors: Mendelow B. , Grobicki D. , La Hunt M. de , And J. Katz , Metz J.
Published: July 2010

Summary. Aspirated marrow fragments from healthy adult baboons were cultured in a simple liquid suspension tissue culture system. During the incubation period, haemopoietic cells were discharged from the fragments and settled to the floor of the culture vessels, thus allowing a separation of marrow stromal elements, retained within the fragments. Cells associated with the marrow stroma in vitro were of four main types: adipocytes, macrophages, plasma cells and unidentified lipid‐laden cells related to fibroblasts. These last cells were capable of DNA synthesis in vitro, and were morphologically and functionally distinguishable from typical marrow macrophages. Macrophages were characterized by their phagocytic properties. Plasma cells were capable of prolonged survival and immunoglobulin output in vitro.

In monolayer cultures of disaggregated fragment cell suspensions, the lipid‐laden stromal cells rapidly assumed fibroblast‐like morphology. Fibroblastic proliferation to confluent monolayers was seen within a few days. Plasma cells reorientated themselves around these cells to form rosettes, and bridge communications between the two cell types were seen frequently. Plasma cell immunoglobulin output rate per cell appeared to be potentiated by this association.
Characteristics of a Cell Line Established from a Child with Familial Acute Myeloid Leukaemia
Authors: Fischer Patricia , Karpas A. , Nacheva Elisabeth , Haas O. , Winterixitner Heide , Krepler P.
Published: July 2010

Summary. The properties of an unusual new cell line from a child with acute myeloid leukaemia (AML) is described. The patient, his mother and grandmother all died within 2 years of AML. Karyotypic analysis suggests that the cells which grow in vitro represent a proliferation of the child's leukaemic cells. The presence of myeloid alkaline phosphatase in these cells suggests that they represent a proliferation of malignant myeloblasts. During the first year in culture a significant percentage of the cells formed E‐rosettes, but the cells failed to react with monoclonal anti‐T‐cell antibodies. The cells are SmIg, Fc, C3 and EBNA negative. Ultrastructural examination of the cells revealed cytoplasmic inclusion bodies which contained virus‐like particles. These inclusion bodies and particles were similar to those observed by us in two other human leukaemic cell lines and not unlike those in fresh human leukaemic cells, as reported by others.
The triplicated α gene locus and β thalassaemia
Authors: Kanavakis E. , Metaxotou‐Mavromati A. , Kattamis C. , Wainscoat J. S. , Wood W. G.
Published: July 2010

Summary. In five families, the coinheritance of β thalassaemia and an additional α gene (ααα/αα) has been observed. Among the β thalassaemia heterozygotes, no phenotypic effect of the triplicated α gene was detected clinically or at the haematological level. Unexpectedly, however, four out of five β thalassaemia homozygotes with the ααα/αα gene complement had the milder clinical condition of thalassaemia intermedia and in at least one case there was evidence to suggest that this might be due to the ααα gene arrangement acting as an α thalassaemia allele.
5‐NUCLEOTIDASE ACTIVITY OF MEGAKARYOBLASTS IN A CASE OF ACUTE MEGAKARYOBLASTIC LEUKAEMIA
Authors: El‐Mohandes E. , Hayhoe F.G. J.
Published: July 2010

Summary. The enzyme 5′‐nucleotidase (5′‐N) was demonstrated cytochemi‐cally in the blast cells from a case of acute megakaryoblastic leukaemia. No activity of 5′‐N could be detected in blasts of other types of acute leukaemia.
Platelet density analysis: a tool for the detection of acquired storage pool disease in man
Authors: Boneu B. , Sié P. , Eche N. , Caranobe C. , Hugo B. , Nouvel C.
Published: July 2010

Summary. This study was designed to evaluate the usefulness of platelet density analysis in the detection of acquired storage pool defects in human patients. Two groups of patients were investigated: 19 subjects affected with a myeloproliferative disorder (group I) where abnormal platelets are released from the megakaryocytes and 11 patients hospitalized in an intensive care unit (Group II) where normal platelets are injured in the circulation. Platelet density distribution after isopycnic centrifugation on a discontinuous stractan density gradient, dense granule markers (serotonin, ATP and ADP) and alpha granule markers (intraplatelet beta‐thromboglobulin and platelet factor 4) were simultaneously determined. An increased proportion of the percentage of light platelets was observed in 16 patients of group I and nine of group II; an increased ATP/ADP ratio was observed in 12 patients of group I and 10 of group II. Both the tests were abnormal in 11 patients of group I and nine of group II. In group I, the level of serotonin was low and was related to the percentage of light platelets. The alpha granule specific proteins were normal in the two groups. These results indicate that platelet density analysis may serve as a screening test to detect exhausted platelets in human diseases.
The molecular basis of HbH disease in Greece
Authors: Tzotzos S. , Kanavakis E. , METAXOTOU‐MAVROMATI A. , Kattamis C.
Published: July 2010

Summary. Globin gene mapping in 16 Greek individuals with HbH disease and their parents has demonstrated the occurrence of several HbH genotypes brought about by the interaction of two α+‐thalassaemia and two α+‐thalassaemia haplotypes. Eight of the 16 patients had the genotype Med/ ‐α3.7, four the genotype –(a)20.5/–α3.7 and three the genotype Med/aαT. In one patient the restriction data are consistent with two possible genotypes aαT/αTor /aαT. It is demonstrated that HbH disease in Greece is heterogeneous, with the deletion haplotypes Med and –α3.7 being more prevalent than the ‐(a)20.5 and non‐deletion (ααT) haplotypes.
Platelet prothrombin converting activity in hereditary disorders of platelet function
Authors: Bevers E. M. , Comfurius P. , Nieuwenhuis H. K. , LEVY‐TOLEDANO S. , Enouf J. , Belluci S. , Caen J. P. , Zwaal R. F. A.
Published: July 2010

Summary. Prothrombinase activities of platelets have been measured in diluted platelet‐rich plasma using a chromogenic substrate assay and purified coagulation factors. No abnormalities in prothrombinase activities were found for platelets from patients with storage pool disease (dense‐body deficiency), grey platelet syndrome, and Glanzmann's thrombasthenia. It is concluded that neither release of dense bodies and a‐granules nor aggregation of platelets are essential prerequisites for exposure of a procoagulant surface. Platelets from patients with Bernard‐Soulier syndrome, however, have approximately 10‐fold higher prothrombinase activities in the non‐stimulated form than normal non‐stimulated platelets. The increased procoagulant activity cannot be completely ascribed to an increase in platelet size. It is suggested that the increased prothrombinase activity reflects an increased exposure of phosphatidylserine at the outer surface of non‐stimulated Bernard‐Soulier platelets, earlier described byPerretetaI(1983).
The haematological puzzle of Hb J Cape Town is partly solved
Authors: Lambridis Alkes J. , Ramsay Michele , Jenkins Trefor
Published: July 2010

Summary.Molecular studies have shown that the mutation giving rise to Hb J Cape Town (α92 arg→gln) is situated on a chromosome from which the other α‐globin gene has been deleted. The −α3.7deletion has resulted from crossing‐over within segment I of the Z region of homology. There appears to be an unusually high proportion of the variant haemoglobin in heterozygotes which cannot be explained by gene dosage alone.
Type IIB von Willebrand's disease presenting as thrombocytopenia during pregnancy
Authors: Giles Alan R. , Hoogendoorn Hugh , Benford Kerry
Published: July 2010

Type IIb von Willebrand's disease has been found to be associated with the development of thrombocytopenia following the infusion of DDAVP (desmopressin). It has also been associated with sporadic thrombocytopenia and evidence of spontaneous platelet aggregation. A family with documented Type IIb von Willebrand's disease is described, where two of the affected females presented with moderate to severe thrombocytopenia developing during pregnancy with reversal to normal or minimally reduced platelet counts in the early post gestational period. In each case, the levels of factor VIII: C, von Willebrand factor antigen and von Willebrand factor ristocetin co‐factor activity rose during pregnancy but there were notable discrepancies between the levels of each in any one individual. It is suggested that pregnancy resulted in increased synthesis of the variant form of von Willebrand factor resulting in progressively increasing platelet/variant form von Willebrand factor interaction and subsequent thrombocytopenia. Whether this reflects consumption or sequestration remains uncertain. Although spontaneous platelet aggregation was observed in some family members, the majority did not exhibit this phenomenon. Circulating platelet aggregates could not be detected. Both pregnancies were relatively uneventful and there is no history of unusual bleeding associated with pregnancy in the family. These observations suggest that Type IIb von Willebrand's disease should be considered in the differential diagnosis of thrombocytopenia developing during pregnancy, particularly in those individuals where evidence supporting the diagnosis of immune mediated thrombocytopenia is not forthcoming. Where the diagnosis of Type IIb von Willebrand's disease is established, active intervention other than confinement in a hospital with experience in haemostatic disorders is probably not required as the development of thrombocytopenia does not appear to exert an additive effect on the underlying defect relating to the variant form of von Willebrand's disease.
The platelet alloantigen Zwa or PlA1 is expressed by cultured endothelial cells
Authors: Leeksma O. C. , Giltay J. C. , Zandbergen‐Spaargaren J. , Modderman P. W. , Van Mourik J. A. , Kr. von dem Borne A. E. G.
Published: July 2010

Recently, the synthesis by cultured human endothelial cells of a membrane protein complex immunologically related to platelet glycoprotein (GP) IIb/IIIa complex was demonstrated. Since platelet GP IIIa is known to carry the platelet alloantigen Zwa or PaA1, studies were performed to establish whether this antigen is also expressed on endothelial cells. The present report describes the results of these studies, which provide evidence for the presence of the Zwa or PlA1 antigen on the surface of cultured human endothelial cells. This evidence is based on the following observations: (1) cultured endothelial cells react with anti‐Zwa (PlA1) antibodies as shown by indirect immunofluorescence: (2) two proteins are precipitated by anti‐Zwa (PlA1) antibodies from lysates of 125I‐labelled endothelial cells with an electrophore‐tic mobility corresponding with that of GP IIb and IIIa; (3) anti‐Zwa (PlA1) reacts specifically, as shown by immunoblotting of sodium‐dodecylsulphate polyacrylamide gels of solubilized endothelial cells, with a protein with a mobility similar to that of platelet GP IIIa.
Inhibition of interleuk:in‐2 secretion by factor VIII concentrates: a possible cause of immunosuppression in haemophiliacs
Authors: Thorpe Robin , Dilger Paula , Dawson Nicholas J. , Barrowcliffe Trevor W.
Published: July 2010

Summary The inhibitory effect of factor VIII concentrate products on IL‐2 secretion by human T‐cells was investigated. The six products used widely in the lJ.K. showed very different activities varying from almost total inhibition to no significant effect. There appeared to be no obvious relationship between inhibitory activity and protein composition but factor VIII itself was not responsible for the effect as affinity purified products were entirely non‐inhibitory. The two wetheated products were most inhibitory whereas dry‐heated products were less inhibitory or non‐inhibitory. However, a wet‐heated version of a non‐inhibitory dry‐heated product was also non‐inhibitory, suggesting that the composition of the concentrate rather than anti‐viral treatment is important for immunosuppressive activity. A product treated by the solvent/detergent procedure showed considerable inhibitory activity. Immunoglobulin and albumin products did not inhibit IL‐2 secretion to any significant extent, but factor IX concentrates were inhibitory. We suggest that inhibition of IL‐2 secretion by factor VIII concentrates may be related to the immunosuppression observed in haemophiliacs treated with high dose factor VIII products and that our results should be considered by clinicians and manufacturers of factor VIII products.
Serum levels of soluble CD30 molecule (Ki‐1 antigen) in Hodgkin's disease: relationship with disease activity and clinical stage
Authors: Pizzolo G. , Vinante F. , Chilosi M. , Dallenbach F. , Josimovic‐Alasevic O. , Diamantstein T. , Stein† H.
Published: July 2010

In all cases of Hodgkin's disease (HD) Reed‐Sternberg (RS) cells express the CD30 antigen. It has been recently demonstrated that this molecule can be released from the cell membrane of CD30+ neoplastic cells in a soluble form (sCD30), detectable in culture supernatants and body fluids. In this paper we investigated by an immunoassay the serum levels of sCD30 in 58 patients with HD, in order to define the possible relationship of this molecule to the clinical and pathological findings. sCD30 molecule was found at detectable levels in 24 out of 50 patients (48%) with active disease, whereas it was always absent in control sera and in cases in complete remission. Among the patients with active HD, the incidence and mean values of detectable levels (±SEM) of sCD30 were higher in cases with progressive or relapsing disease (61.5%, mean 458±190 U/ml) as compared to those at presentation (43.2%, mean 116 ± 3 3 U/ml). Among the cases at presentation, detectable levels were observed more often in patients with advanced stages (III+IV: 61%) and constitutional symptoms (‘B’: 61‐5%) than in early stages (I+11: 26%) and without symptoms (‘A’: 33.3%). In addition, higher mean values were found in stages III+IV (182±60 U/ml) and in ‘B’cases (208±73 U/ml) than in stages I+II (65 ± 30 U/ml) or ‘A’patients (64 ± 26 U/ ml).

The above findings suggest a possible role for the sCD30 as a tumour marker in HD.
Evaluation of leukaemic contamination in peripheral blood stem cell harvests by reverse transcriptase polymerase chain reaction
Authors: Nagafuji Koji , Harada Mine , Takamatsu Yasushi , Eto Tetsuya , Teshima Takanori , Kamura Takumi , Okamura Takashi , Hayashi Shin , Akashi Koichi , Murakawa Masahiro , Niho Yoshiyuki
Published: July 2010

Summary. A major issue in autologous blood stem cell transplantation (ABSCT) for leukaemia is whether peripheral blood stem cell (PBSC) harvests are less contaminated with leukaemic cells than bone marrow mononuclear cells (BMMNC). We compared leukaemic contamination in PBSC harvests and BMMNC, obtained simultaneously, by using reverse transcriptase polymerase chain reaction (RT‐PCR) of leukaemia‐specific chimaeric messenger RNA (mRNA), in three patients with Philadelphia chromosome (Ph)‐positive acute lymphoblastic leukaemia (ALL), one with Ph‐positive acute myelogenous leukaemia (AML), and two with acute promyelocytic leukaemia (APL). Our two‐step PCR method employed ‘nested primers’ in the second step and can detect one leukaemic blast diluted into 106 HL‐60 cells. In three of four patients with Ph‐positive ALL and AML we detected leukaemic contamination in both PBSC harvests and BMMNC. In the remaining patient with ALL, both PBSC harvests and BMMNC were PCR‐negative. Both PBSC harvests and BMMNC from one patient with APL were PCR‐positive. In contrast, PBSC harvests from another patient with APL, whose BMMNC could not be obtained because of bone marrow necrosis, were PCR‐positive after the first course of consolidation chemotherapy, but became PCR‐negative after the second course. The present study does not support the hypothesis that PBSC harvests are less contaminated by leukaemic cells than BMMNC, but suggests that PBSC harvests are contaminated when BMMNC are contaminated.
Parvovirus B319‐related lymphadenopathy
Authors: Tsuda Hiroyuki , Maeda Yosuke , Nakagawa Kazumi
Published: July 2010

Summary. We tested whether parvovirus B19 (PVB19) is involved in benign lymph node swelling as a causative agent. The serum specimens from 13 patients with lymph node swelling of unidentified cause were tested for IgM and IgG antibody against PVB19 by Western blot analysis. Five patients had IgM antibody, and PVB19 DNA was detected in the serum from one of them by dot‐blot analysis, suggesting the recent PBV19 infection and its involvement in lymph node swelling of these patients. Clinical and haematological features of the patients are also discussed.
Long‐term outcome of acquired aplastic anaemia in children: comparison between immunosuppressive therapy and bone marrow transplantation
Authors: Kojima Seiji , Horibe Keizo , Inaba Jun , Yoshimi Ayami , Takahashi Yoshiyuki , Kudo Kazuko , Kato Koji , Matsuyama Takaharu
Published: July 2010

A total of 100 children under the age of 17 years with acquired aplastic anaemia (AA) were initially treated with immunosuppressive therapy (IST) (n = 63) or bone marrow transplantation (BMT) (n = 37) from an HLA‐matched family donor. The projected 10‐year survival rates were 55 ± 8% and 97 ± 3% respectively (P = 0·004). Because the IST group included 11 non‐responders who were salvaged by BMT from an HLA‐matched unrelated donor, we compared failure‐free survival (FFS) between the groups. The probability of FFS at 10 years was 97 ± 3% for the BMT group, compared with 40 ± 8% for the IST group (P = 0·0001). Seven patients evolved to myelodysplastic syndrome (MDS) with monosomy 7 and the estimated cumulative incidence of MDS 10 years after diagnosis was 20 ± 7% in the IST group. We compared the outcome of children treated with IST during the two consecutive periods of 1983–91 (group A, n = 40) and 1991–8 (group B, n = 23) to assess the impact of combined therapy with anti‐thymocyte globulin and cyclosporin. The probability of FFS at 7 years follow‐up was the same in the two groups (50 ± 8% vs. 40 ± 15%, P = 0·40). We recommend BMT as first‐line therapy in paediatric severe AA patients with an HLA‐matched family donor. Alternative donor BMT is recommended as salvage therapy in patients who relapse or do not respond to initial IST.
Interleukin 10‐induced thrombocytopenia in normal healthy adult volunteers: evidence for decreased platelet production
Authors: Sosman Jeffrey A. , Verma Amit , Moss Steven , Sorokin Patricia , Blend Michael , Bradlow Basil , Chachlani Nasir , Cutler David , Sabo Ronald , Nelson Mary , Bruno Edward , Gustin David , Viana Marlos , Hoffman Ronald
Published: July 2010

Recombinant human interleukin 10 (rhuIL‐10) inhibits the production of proinflammatory cytokines and has shown promise in the treatment of inflammatory bowel disease. Clinical trials have been accompanied by a reversible decline in platelet counts. We conducted a randomized, double‐blinded, placebo‐controlled, parallel group trial in 12 healthy volunteers to investigate the aetiology of rhuIL‐10‐induced thrombocytopenia. Eight volunteers received 8 μg/kg/d of rhuIL‐10 subcutaneously, while four subjects received a placebo alone for 10 d. A reversible decline in the platelet counts from a mean of 275 × 109/l to 164 × 109/l was observed in the IL‐10‐treated cohort (P = 0·012). A fall in the haemoglobin mean levels was also observed in the IL‐10‐treated cohort from 13·7 to 11·7 g/dl (P = 0·011). No significant change was observed in the bone marrow cellularity or myeloid/erythroid ratio or in the number of megakaryocytes per high‐powered field (HPF). A fall was observed in the number of megakaryocyte colony‐forming units (CFU‐MKs) after the administration of IL‐10 compared with those receiving the placebo (P = 0·068). No difference in the change in granulocyte–macrophage CFUs (CFU‐GMs), mixed lineage CFUs (CFU‐GEMMs) or erythroid burst‐forming units (BFU‐Es) was observed when comparing the IL‐10‐ vs. placebo‐treated groups (P > 0·465). Serum cytokine levels of thrombopoietin (TPO), IL‐6 and granulocyte–macrophage colony stimulating factor (GM‐CSF) were not decreased following IL‐10 administration. In fact, both TPO and GM‐CSF appeared to be slightly increased in the serum. All subjects underwent In111‐labelled platelet survival studies with liver/spleen scans to assess splenic sequestration prior to and then on day 7 of treatment. A significant reduction in splenic sequestration of platelets (P = 0·012) was observed in the IL‐10‐treated group, but not in the placebo‐treated subjects.
Expression of MUM1/IRF4 selectively clusters with primary effusion lymphoma among lymphomatous effusions: implications for disease histogenesis and pathogenesis
Authors: Carbone Antonino , Gloghini Annunziata , Cozzi Maria Rita , Capello Daniela , Steffan Agostino , Monini Paolo , De Marco Luigi , Gaidano Gianluca
Published: July 2010

Primary effusion lymphoma (PEL) is a peculiar B‐cell lymphoma characterized by infection by human herpesvirus type‐8/Kaposi sarcoma‐associated herpesvirus (HHV‐8/KSHV) and by preferential growth in the serous body cavities. Histogenetic studies have suggested that PEL originates from B cells at a late stage of differentiation. In this study, we have investigated PEL for the expression status of MUM1/IRF4 (multiple myeloma 1/interferon regulatory factor 4) protein, which is involved in physiological B‐cell maturation and represents a histogenetic marker of late B‐cell differentiation. Using multiple detection assays, all cases of PEL (n = 22) were found to express MUM1/IRF4 molecules. MUM1/IRF4 expression was a selective feature of PEL among lymphomas involving the serous body cavities as secondary lymphomatous effusions generally failed to express the protein. In reactive lymphoid tissues, MUM1/IRF4 expression clustered with advanced stages of B‐cell differentiation. Comparison of MUM1/IRF4 expression with that of other histogenetic markers defined two phenotypic variants of PEL, i.e. MUM1/IRF4+, CD138/syndecan‐1+, B‐cell antigen− (20 out of 22 cases) and MUM1/IRF4+, CD138/syndecan‐1−, B‐cell antigen+ (2 out of 22 cases), suggesting a certain degree of heterogeneity in the disease histogenesis. The implications of these data are threefold. First, MUM1/IRF4 expression corroborates the notion that PEL originates from post‐germinal centre, preterminally differentiated B‐cells. Second, MUM1/IRF4 may help in the differential diagnosis of PEL among other lymphomas involving the serous body cavities. Finally, MUM1/IRF4 may interact with HHV‐8/KSHV‐encoded interferon regulatory factors (IRFs) and thus contribute to PEL escape from interferon‐mediated control of viral infection.
Ontogeny of natural killer cells and T cells by analysis of BCR–ABL rearrangement from patients with chronic myelogenous leukaemia
Authors: Cho Eun Kyung , Heo Dae Seog , Seol Jae Goo , Seo Eul Ju , Chi Hyun Sook , Kim Eun Shil , Lee Young Yiul , Kim Byoung Kook , Kim Noe Kyeong
Published: July 2010

Chronic myelogenous leukaemia (CML) is a haematological malignant disorder characterized by the Philadelphia chromosome (Ph) and BCR–ABL gene rearrangement. This abnormal fusion gene can be considered to serve as a marker for the transformed cell clone in CML and is found in all cells arising from the same malignant precursor cell. It has been detected in CML cells of the myeloid, monocytic, erythroid and B‐lymphocytic lineages. However, it is still arguable as to whether T lymphocytes or natural killer (NK) cells carry this marker. Answering this question would clarify the ontogenic relationship between NK cells and T cells. We examined 12 CML patients and studied the expression of BCR–ABL rearrangement by fluorescence in situ hybridization (FISH) in both NK cells and T cells sorted by flow cytometry. The purity of T cells was 95·6–99·8% and that of NK cells was 95·3–99·3% after sorting. Neither NK cells nor T cells showed any positive BCR–ABL signal with the exception of one patient who recovered from a lymphoid blastic crisis. We speculate that T cells and NK cells originate from BCR–ABL‐negative stem cells.
Platelet and leucocyte activation in childhood sickle cell disease: association with nocturnal hypoxaemia
Authors: Inwald David P. , Kirkham Fenella J. , Peters Mark J. , Lane Rod , Wade Angie , Evans Jane P. , Klein Nigel J.
Published: July 2010

We hypothesized that vaso‐occlusive events in childhood sickle cell disease (SCD) may relate to inflammatory cell activation as well as interactions between sickle erythrocytes and vascular endothelium. Peripheral blood was examined from 24 children with SCD, of whom 12 had neurological sequelae and seven had frequent painful crises, and 10 control subjects. Platelet (CD62P and CD40L expression) and granulocyte (CD11b expression) activation and levels of platelet–erythrocyte and platelet–granulocyte complexes were determined by flow cytometry. Platelets (P = 0·019), neutrophils (P = 0·02) and monocytes (P = 0·001) were more activated and there were increased platelet–erythrocyte complexes (P = 0·026) in SCD patients compared with controls. Platelet–granulocyte complexes were not raised. There were no differences between the different groups of SCD. As hypoxia activates monocytes, platelets and endothelial cells and causes sickling of SCD erythrocytes, we also investigated 20 SCD patients with overnight pulse oximetry. Minimum overnight saturation correlated with the level of platelet–erythrocyte complexes (Spearman's ρ−0·668, P < 0·02), neutrophil CD11b (Spearman's ρ−0·466, P = 0·038) and monocyte CD11b (Spearman's ρ−0·652, P = 0·002). These findings provide important clues about the mechanism by which SCD patients may become predisposed to vaso‐occlusive events.
OKT‐3‐based reconditioning regimen for early graft failure in HLA‐non‐identical stem cell transplants
Authors: Schlegel P. G. , Eyrich M. , Bader P. , Handgretinger R. , Lang P. , Niethammer D. , Klingebiel T.
Published: July 2010

Primary non‐engraftment or early rejection after transplantation of haematopoietic stem cells represent life‐threatening complications of allogeneic stem cell transplantation. Management of early graft failure has been problematic, as the risk of fatal infectious complications increases with the time of pancytopenia and as a second transplant preceded by a conventional myeloablative conditioning regimen has been associated with high rates of cumulative organ toxicity. For paediatric patients with early graft failure following the transplantation of highly purified major histocompatibility complex (MHC)‐disparate haematopoietic stem cells, we have evaluated an immunosuppressive OKT‐3/methylprednisolone‐based reconditioning regimen with low toxicity in preparation for a secondary transplant of purified haematopoietic stem cells from the same donor. This report presents the results from a 4‐year pilot study including six patients with early graft failure. The results demonstrate that this antibody‐based regimen can be used effectively to prepare patients for secondary transplantation. Successful engraftment after a second transplant procedure was achieved in five of these six high‐risk patients. The median interval between first and second transplant was 27 d (range 22–51 d), and the median time for engraftment was 10 d (range 9–13 d). Chimaerism analysis of microsatellite regions by polymerase chain reaction (PCR) demonstrated complete donor chimaerism in four of these patients within the first month after secondary transplant and revealed mixed chimaerism in one patient who converted to complete chimaerism after T‐cell add‐back.
Insulin‐like growth factor induces the survival and proliferation of myeloma cells through an interleukin‐6‐independent transduction pathway
Authors: Ferlin Martine , Noraz Nelly , Hertogh Catherine , Brochier Jean , Taylor Naomi , Klein Bernard
Published: July 2010

Multiple myeloma (MM) is a B‐cell neoplasia that is associated with an increased level of bone resorption. One important mediator of bone remodelling, insulin‐like growth factor (IGF‐I), has been shown to stimulate the proliferation of human myeloma cells. However, the mechanisms of action of IGF‐I in these cells have not been determined. Using interleukin (IL)‐6‐dependent myeloma cell lines, we show IGF‐I to be as potent a survival and proliferation factor as IL‐6. We demonstrated that IGF‐I functions independently of the IL‐6 transducer gp130 and that these two cytokines have additive effects. Moreover, inhibition of the IGF‐I pathway did not modulate the proliferative effect of IL‐6. Accordingly, we found that IL‐6 and IGF‐I activated distinct downstream signalling molecules: IL‐6 activated STAT3 phosphorylation, whereas IGF‐I treatment resulted in the phosphorylation of IRS‐1. Interestingly, these signalling pathways appear to converge as both cytokines activated the ras/MAPK pathway. Thus, IGF‐I acts as a potent survival and proliferation factor for myeloma cells by stimulating an IL‐6‐independent signalling cascade. These data, together with the finding that, in vivo, IGF‐I is normally expressed in close proximity to myeloma cells within the bone matrix, strongly suggest a role for this cytokine in the pathophysiology of multiple myeloma.