bluuebunny/crossref_metadata_2025 huggingface dataset:
Schumann resonance parameters calculated with a partially uniform knee model on Earth, Venus, Mars, and Titan
Authors: Pechony Olga , Price Colin
Published: October 2004

A partially uniform knee (PUK) model is a combination of two two‐dimensional telegraph equation (TDTE)‐based techniques: the “knee” model, which addresses the problem of approximating the knee‐like conductivity profile (on a semilogarithmic scale) of the Earth's ionosphere, and the global partially uniform day‐night model, which allows a convenient treatment of the day‐night asymmetry. Incorporation of the “knee” conductivity profile allows to overcome the shortcoming of the two‐exponential technique, widely used in extremely low frequency (ELF) work, too flat frequency dependence of quality factor in the Schumann resonance (SR) range (5–40 Hz). The PUK model predictions for Schumann resonance parameters reasonably represent observations in the SR frequency range. Propagation parameters for other planets were calculated on the basis of existing ionospheric models of the planets. To allow the approximation of structured conductivity profiles of Venus and Mars, the “knee” model was upgraded to a double‐“knee” by inserting an additional “knee” to the profile. In general, this technique allows to approximate very structured profiles by adding as many “knees” as necessary. Calculations show that the detection of Schumann resonances on Venus, Mars, and Titan is possible, though low‐quality factors on Mars and Titan imply that pronounced peaks are not to be presumed on these planets.
Targeting the A site RNA of the Escherichia coli ribosomal 30 S subunit by 2′-O-methyl oligoribonucleotides: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics
Authors: ABELIAN Arthur , WALSH Andrew P. , LENTZEN Georg , ABOUL-ELA Fareed , GAIT Michael J.
Published: October 2004

The bacterial ribosome comprises 30 S and 50 S ribonucleoprotein subunits, contains a number of binding sites for known antibiotics and is an attractive target for selection of novel antibacterial agents. On the 30 S subunit, for example, the A site (aminoacyl site) close to the 3′-end of 16 S rRNA is highly important in the decoding process. Binding by some aminoglycoside antibiotics to the A site leads to erroneous protein synthesis and is lethal for bacteria. We targeted the A site on purified 30 S ribosomal subunits from Escherichia coli with a set of overlapping, complementary OMe (2′-O-methyl) 10-mer oligoribonucleotides. An equilibrium dialysis technique was applied to measure dissociation constants of these oligonucleotides. We show that there is a single high-affinity region, spanning from A1493 to C1510 (Kd, 29–130 nM), flanked by two lower-affinity regions, within a span from U1485 to G1516 (Kd, 310–4300 nM). Unexpectedly, addition of the aminoglycoside antibiotic paromomycin (but not hygromycin B) caused a dose-dependent increase of up to 7.5-fold in the binding of the highest affinity 10-mer 1493 to 30 S subunits. Oligonucleotides containing residues complementary to A1492 and/or A1493 showed particularly marked stimulation of binding by paromomycin. The results are consistent with high-resolution structures of antibiotic binding to the A site and with greater accessibility of residues of A1492 and A1493 upon paromomycin binding. 10-mer 1493 binding is thus a probe of the conformational switch to the ‘closed’ conformation triggered by paromomycin that is implicated in the discrimination by 30 S subunits of cognate from non-cognate tRNA and the translational misreading caused by paromomycin. Finally, we show that OMe oligonucleotides targeted to the A site are moderately good inhibitors of in vitro translation and that there is a limited correlation of inhibition activity with binding strength to the A site.
Two related ARID family proteins are alternative subunits of human SWI/SNF complexes
Authors: WANG Xiaomei , NAGL Norman G. , WILSKER Deborah , VAN SCOY Michael , PACCHIONE Stephen , YACIUK Peter , DALLAS Peter B. , MORAN Elizabeth
Published: October 2004

p270 (ARID1A) is a member of the ARID family of DNA-binding proteins and a subunit of human SWI/SNF-related complexes, which use the energy generated by an integral ATPase subunit to remodel chromatin. ARID1B is an independent gene product with an open reading frame that is more than 60% identical with p270. We have generated monoclonal antibodies specific for either p270 or ARID1B to facilitate the investigation of ARID1B and its potential interaction with human SWI/SNF complexes in vivo. Immunocomplex analysis provides direct evidence that endogenous ARID1B is associated with SWI/SNF-related complexes and indicates that p270 and ARID1B, similar to the ATPase subunits BRG1 and hBRM, are alternative, mutually exclusive subunits of the complexes. The ARID-containing subunits are not specific to the ATPases. Each associates with both BRG1 and hBRM, thus increasing the number of distinct subunit combinations known to be present in cells. Analysis of the panels of cell lines indicates that ARID1B, similar to p270, has a broad tissue distribution. The ratio of p270/ARID1B in typical cells is approx. 3.5:1, and BRG1 is distributed proportionally between the two ARID subunits. Analysis of DNA-binding behaviour indicates that ARID1B binds DNA in a non-sequence-specific manner similar to p270.
Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes
Authors: ABOULAICH Nabila , VAINONEN Julia P. , STRÅLFORS Peter , VENER Alexander V.
Published: October 2004

Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.
Phosphorylation of androgen receptor isoforms
Authors: WONG Hao Yun , BURGHOORN Jan A. , van LEEUWEN Marije , de RUITER Petra E. , SCHIPPERS Esther , BLOK Leen J. , LI Ka Wan , DEKKER Henk L. , de JONG Luitzen , TRAPMAN Jan , GROOTEGOED J. Anton , BRINKMANN Albert O.
Published: October 2004

Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser–Ala substitution mutant S650A (Ser-650→Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed.
C-terminus of mitotic centromere-associated kinesin (MCAK) inhibits its lattice-stimulated ATPase activity
Authors: MOORE Ayana , WORDEMAN Linda
Published: October 2004

Mitotic centromere-associated kinesin (MCAK) is a microtubule (MT)-destabilizing molecular motor. In the present study we show that the final 8 amino acids of the C-terminus of MCAK inhibit lattice-stimulated ATPase activity of the motor. Surprisingly, loss of this C-terminal ‘tail’ (MCAK-Q710) leads to more rapid depolymerization of MTs relative to full-length MCAK (wt-MCAK). Biochemical and microscopic assays revealed that MCAK-Q710 bound to the MT lattice with higher apparent affinity as compared with wt-MCAK. End-stimulated depolymerization was similar for both enzymes. These data suggest that lattice-bound MCAK can increase the rate of MT depolymerization, but at an energy cost. The function of the C-terminus of MCAK may be to selectively inhibit lattice-stimulated ATPase activity, resulting in limited interactions of the motor with the MT lattice. This increases the coupling between ATP hydrolysis and tubulin dimer release, but it also limits MT depolymerization.
Development of a chimaeric receptor approach to study signalling by tumour necrosis factor receptor family members
Authors: XU Duorong , SHI Zhenqi , McDONALD Jay , PAN George , CAO Xuemei , YU Xueqing , FENG Xu
Published: October 2004

Members of the tumour necrosis factor receptor family play a pivotal role in cell differentiation, function and apoptosis. However, signalling by many members of the family remains to be elucidated. In the present study, we developed a chimaeric receptor approach for studying signalling by receptors belonging to this family. The chimaeric receptor comprises the human Fas external domain linked to the transmembrane and cytoplasmic domains of a tumour necrosis factor receptor family member of interest. When the chimaera is expressed in mouse cells, the clustering of the chimaera induced by a human Fas-activating antibody activates the intracellular domain of the chimaera without affecting its endogenous counterpart. Since the antibody recognizes only human Fas, this approach can be used to dissect signalling by any tumour necrosis factor family member using any type of mouse cell including those endogenously expressing Fas. Moreover, we also showed that the chimaeric receptor approach can be used to study signalling at any stage of cell differentiation or function.
Molecular triangulation: Bridging linkage and molecular-network information for identifying candidate genes in Alzheimer's disease
Authors: Krauthammer Michael , Kaufmann Charles A. , Gilliam T. Conrad , Rzhetsky Andrey
Published: October 2004

A major challenge in human genetics is identifying the molecular basis of common heritable disorders. In contrast to rare single-gene diseases, multifactorial disorders are thought to arise from the combined effect of multiple gene variants, such that any single variant may have only a modest effect on disease susceptibility. We present a method to identify genes that may harbor a significant proportion of the genetic variation that predisposes individuals to a given multifactorial disorder. First, we perform an automated literature analysis that predicts physical interactions (edges) among candidate disease genes (seed nodes, selected on the basis of prior information) and other molecular entities. We derive models of molecular networks from this analysis and map the seed nodes to them. We then compute the graph-theoretic distance (the minimum number of edges that must be traversed) between the seed nodes and all other nodes in the network. We assume that nodes that are found in close proximity to multiple seed nodes are the best disease-related candidate genes. To evaluate this approach, we selected four seed genes, each with a proven role in Alzheimer's disease (AD). The method performed well in predicting additional network nodes that match AD gene candidates identified manually by an expert. We also show that the method prioritizes among the seed nodes themselves, rejecting false-positive seeds that are derived from (noisy) whole-genome genetic-linkage scans. We propose that this strategy will provide a valuable means to bridge genetic and genomic knowledge in the search for genetic determinants of multifactorial disorders.
Aspirin triggers antiinflammatory 15-epi-lipoxin A
4
and inhibits thromboxane in a randomized human trial
Authors: Chiang Nan , Bermudez Edmund A. , Ridker Paul M. , Hurwitz Shelley , Serhan Charles N.
Published: October 2004

There is increasing evidence that aspirin initiates biosynthesis of novel antiinflammatory mediators by means of interactions between endothelial cells and leukocytes. These mediators are classified as aspirin-triggered 15-epi-lipoxins. Such compounds may account at least in part for aspirin's clinical benefits, which are distinct from the well appreciated action of aspirin as a platelet inhibitor. Here, we addressed whether aspirin-triggered 15-epilipoxinA
4
(ATL) formation is aspirin-dependent in humans and its relationship to aspirin's antiplatelet activity. We conducted a randomized clinical trial among 128 healthy subjects allocated to placebo or to 81-, 325-, or 650-mg daily doses of aspirin for 8 weeks. Plasma thromboxane (TX)B
2
, an indicator of platelet reactivity, and ATL were assessed from blood collected at baseline and at 8 weeks. Plasma ATL levels significantly increased in the 81-mg aspirin group (0.25 ± 0.63 ng/ml,
P
= 0.04), with borderline increases in the 325-mg group (0.16 ± 0.71 ng/ml) and no apparent significant changes in the 650-mg group (0.01 ± 0.75 ng/ml,
P
= 0.96). When ATL and TXB
2
were compared, levels changed in a statistically significant and opposite direction (
P
< 0.01) for all three aspirin doses. These results demonstrated that low-dose aspirin (81 mg daily) initiates production of antiinflammatory ATL opposite to the inhibition of TX. Monitoring ATL may represent a simple clinical parameter to verify an individual's vascular leukocyte antiinflammatory response with low-dose aspirin treatment. These results also emphasize the importance of cell-cell interactions in the modulation of hemostatic, thrombotic, and inflammatory processes.
Bacteriolytic therapy can generate a potent immune response against experimental tumors
Authors: Agrawal Nishant , Bettegowda Chetan , Cheong Ian , Geschwind Jean-Francois , Drake Charles G. , Hipkiss Edward L. , Tatsumi Mitsuaki , Dang Long H. , Diaz Luis A. , Pomper Martin , Abusedera Mohammad , Wahl Richard L. , Kinzler Kenneth W. , Zhou Shibin , Huso David L. , Vogelstein Bert
Published: October 2004

When spores of the anaerobic bacteriumClostridium novyi-NTare systemically injected into animals, they germinate exclusively within the hypoxic regions of cancers. The germinated bacteria destroy adjacent tumor cells but spare a rim of well oxygenated tumor cells that subsequently expand. Surprisingly, we found that ≈30% of mice treated with such spores were cured of their cancers despite the viable tumor rim initially remaining after spore germination. The mechanism underlying this effect was shown to be immune-mediated, because cured animals rejected a subsequent challenge of the same tumor. Similar effects were observed in rabbits with intrahepatic tumors. It was particularly notable that the induced immune response, when combined with the bacteriolytic effects ofC. novyi-NT, could eradicate large established tumors.
What Role for the EPAs in the Caribbean?
Authors: Gasiorek Michael , Winters L. Alan
Published: October 2004

The aim of this paper is to consider the possible implications of an EPA between the EU and the Caribbean. The focus is on the Caribbean economies, and on the question of what form of EPA might be pro‐development and pro‐poor for the region. The discussion outlines the specificities of the Caribbean region, and some of the economies therein as well as detailing the key analytical issues which need to be considered. The empirical analysis focuses on examining patterns of trade by product and geographical source at a highly detailed level of disaggregation. The analysis suggests that future EPA arrangements are more likely to lead to significant trade diversion as opposed to trade creation or trade reorientation. MFN liberalisation would serve to minimise trade diversion, but in turn is likely to lead to greater adjustment costs. If the EPAs are to be pro‐development and pro‐poor than maintaining/increasing levels of market access to the EU, and ensuring appropriate levels of assistance and aid will be critical.
Trade Policy Making in a Small Island Economy: The WTO Review of the Maldives
Authors: Athukorala Prema‐chandra
Published: October 2004

The purpose of this paper is to examine trade policy regime and trade‐related development issues in the Maldives on the basis of the Trade Policy Review Maldives 2003 of the WTO. The key theme of the paper is that, given the narrow resource base and small domestic market, openness to foreign trade and investment remains the ‘natural’ policy choice for a small developing economy like the Maldives. Since the late 1980s, the Maldivian government has made considerable progress in implementing policy reforms driven by this conviction. However, the reform process is far from complete. High import tariffs maintained predominantly on revenue considerations, a large direct role played by the public sector in foreign trade and some key sectors of the economy, lack of transparency in duty concessions and other investment incentives, failure to incorporate environmental concerns as part of the national development policy, and delays in meeting reform commitments under the WTO are among the key items of the unfinished reform agenda.
Beyond Border Barriers: The Liberalisation of Services Trade in Tunisia and Egypt
Authors: Konan Denise Eby , Kim Karl E.
Published: October 2004

Tunisia and Egypt have both recently undertaken significant steps toward trade reform. They have committed to a partnership agreement with the European Union. Both countries have also joined the WTO and are participating in Doha Round discussions on the liberalisation of non‐tariff barriers on both goods and services trade. These developments provide an interesting context within which to investigate not only the changes in welfare associated with reforms affecting the trade in goods, but also the impacts of services liberalisation. Using open‐economy computable general equilibrium models for both Tunisia and Egypt, this paper explores the reasons why structural differences in these two economies imply different opportunities and challenges with trade reform and services liberalisation. The gains from eliminating barriers at the border for goods trade are significantly greater for Tunisia than Egypt. Both countries, however, gain substantially from liberalisation of foreign direct investment in services. Furthermore, economic growth is more evenly distributed across sectors than with liberalisation of trade in goods alone. In addition to reporting on the impact of alternative policies on income, output, employment and trade, sector‐level effects are also considered.
Functional recovery after bleeding episodes in haemophilia
Authors: De Kleijn P. , Gilbert M. , Roosendaal G. , Poonnose P. M. , Narayan P. M. , Tahir N.
Published: October 2004

Summary.  Haemophilia is characterized by intra‐articular and intramuscular bleeding episodes. Although much work has been carried out into post‐bleeding mechanisms in both synovial joints and muscles, the picture is not yet clear. A firm post‐bleeding rehabilitation protocol is of utmost importance for people with haemophilia. The use of the International Classification of Functioning Disability and Health reveals that information worldwide is on body level, whereas the goals of patients with haemophilia implicitly aim at optimal functional recovery. This interferes much more with activities and participation in society. The Functional Independence Score for Haemophilia and the Post‐bleeding Protocol Based on Functional Milestones are examples of post‐bleeding approaches that deal more with function. Two cases will emphasize the value of this approach.
Physical therapy and imaging outcome measures in a haemophilia population treated with factor prophylaxis: current status and future directions
Authors: Manco‐Johnson M. J. , Pettersson H. , Petrini P. , Babyn P. S. , Bergstrom B‐M. , Bradley C.‐S. , Doria A. S. , Feldman B. M. , Funk S. , Hilliard P. , Kilcoyne R. , Lundin B. , Nuss R. , Rivard G. , Schoenmakers M. A. G. C. , Van den berg M. , Wiedel J. , Zourikian N. , Blanchette V. S.
Published: October 2004

Summary.  Routine infusions of factor VIII to prevent bleeding, known as prophylaxis, and other intensive therapies are being more broadly applied to patients with haemophilia. These therapies differ widely in replacement product usage, cost, frequency of venous access and parental effort. In order to address residual issues relating to recommendations, implementation, and evaluations of prophylaxis therapy in persons with haemophila, a multinational working group was formed and called the International Prophylaxis Study Group (IPSG). The group was comprised of haemophilia treaters actively involved in studies of prophylaxis from North America and Europe. Two expert committees, the Physical Therapy (PT) Working Group and the Magnetic Resonance Imaging (MRI) Working Group were organized to critically assess existing tools for assessment of joint outcome. These two committees independently concluded that the WFH Physical Examination Scale (WFH PE Scale) and the WFH X‐ray Scale (WFH XR Scale) were inadequately sensitive to detect early changes in joints. New scales were developed based on suggested modifications of the existing scales and called the Haemophilia Joint Health Score (HJHS) and the International MRI Scales. The new scales were piloted. Concordance was measured by the intra‐class correlation coefficient of variation. Reliability of the HJHS was excellent with an inter‐observer co‐efficient of 0.83 and a test‐retest value of 0.89. The MRI study was conducted using both Denver and European scoring approaches; inter‐reader reliability using the two approaches was 0.88 and 0.87; test‐retest reliability was 0.92 and 0.93. These new PT and MRI scales promise to improve outcome assessment in children on early preventive treatment regimens.
WFH: back to the future
Authors: O'Mahony B.
Published: October 2004

Summary.  This paper outlines the history of the World Federation of Hemophilia (WFH) and its work. The WFH produces a number of publications each year, and conducts regular surveys and data collection. Two of its most important roles are in the provisions of programmes to developing countries, and in promoting the views of the haemophilia community on issues that impact on the lives of people with haemophilia. Further initiatives are planned in the future to increase the length and quality of life for those living with haemophilia.
Preclinical and clinical gene therapy for haemophilia
Authors: Chuah M. K. L. , Collen D. , Vandendriessche T.
Published: October 2004

Summary.  The goal of all haemophilia therapy is to prevent bleeding and its associated complications. Replacement by factor concentrates can only ever be suboptimum, and efforts are being made to correct the genetic cause of the disorder. Haemophilia is an ideal candidate for gene therapy, as it is caused by mutations in a single gene. A number of vectors have been used in an attempt to obtain therapeutic levels of factor VIII and factor IX in animal models, with some success. A number of phase 1 clinical trials have been conducted, and, although connection of the bleeding disorder was neither complete nor long‐lasting, they do offer hope for a permanent gene‐therapy cure for the disease.
How to evaluate phenotype–genotype relationship in rare coagulation haemorrhagic disorders: examples from FVII deficiency
Authors: Bernardi F. , Marchetti G. , Dolce A. , Mariani G.
Published: October 2004

Summary.  The study of the molecular pathogenesis of several single‐gene disorders, such as coagulation‐factor deficiencies, has revealed the variability of phenotypic expression, even of the same mutations in single genes. These studies underline the complexity of research dealing with the definition of the molecular bases of disorders. Sequence variations provide only the starting point to define pathological genotype–phenotype relationships.
Clinical picture and management of congenital factor VII deficiency
Authors: Mariani G. , Dolce A. , Marchetti G. , Bernardi F.
Published: October 2004

Summary.  In patients with congenital FVII deficiency, bleeding manifestations and clinical presentation vary widely, ranging from asymptomatic subjects to patients with haemorrhages that may cause important handicaps. Owing to menorrhagia, which occurs in about two‐thirds of women of fertile age, bleeding is more frequent in women than in men. Gum bleeding and easy bruising are also more frequent in females. FVII:C levels are not a good predictor of bleeding tendency as there is a wide overlap between bleeders and asymptomatic patients. We propose a three‐grade system of classification based on clinical considerations. Therapy for congenital FVIII bleeding is discussed, with the advantages and disadvantages of each treatment, and the suggested single dose given.
New observations on factor XI deficiency
Authors: Salomon O. , Seligsohn U.
Published: October 2004

Summary.  Factor (F) XI is an injury‐related bleeding tendency that commonly occurs when trauma involves tissues rich in fibronolytic activators. Severe FXI deficiency is defined when the activity of FXI in plasma is less than 15 U dL−1. The disorder is inherited as an autosomal recessive trait manifesting in homozygotes or compound heterozygotes, and infrequently in heterozygotes. So far 53 mutations in the gene of FXI have been described and four of them were found to be prevalent in Ashkenazi Jews, Iraqi Jews, Basques or the English population. For each of the four mutations a founder effect was discerned. Inhibitors can develop in patients with FXI level < 1U dL−1 who were exposed to plasma which seriously complicates their management during surgery. No correction of a prolonged aPTT by normal plasma is indicative of the presence of an inhibitor. In contrast to patients with haemophilia A, severe FXI deficiency provides no protection against myocardial infarction. In patients with severe FXI deficiency undergoing surgery, fresh frozen plasma is the treatment of choice. FXI concentrates can also be used but cause thrombosis in approximately 10% of patients, particularly those with cardiovascular disease. Recombinant FVIIa has successfully prevented bleeding during or after surgery in patients with FXI inhibitors.
Evidence‐based treatment of haemophilia
Authors: Bolton‐Maggs P. H. B. , Stobart K. , Smyth R. L.
Published: October 2004

Summary.  Haemophilia care is effective but therapy is expensive. Despite concentrates being available in some areas for more than 30 years, many scientific questions concerning the management of haemophilia, such as what doses and duration of factor are required for different bleeding episodes or for surgical procedures, remain inadequately answered. Modern rigorous methods of assessment of the evidence both assist in determining what facts are available, how reliable the evidence is, and also help to define what studies need to be done. Cochrane methodology can be applied to haemophilia, and an assessment of prophylaxis provides an example. International collaborative studies would be valuable and require enthusiastic participants.
New high‐technology products for the treatment of haemophilia
Authors: Pipe S. W. , Saint‐Remy J‐M. , Walsh C. E.
Published: October 2004

Summary.  This review will focus on new technologies in development that promise to lead to further advances in haemophilia therapeutics. There has been continued interest in the bioengineering of recombinant factor VIII (rFVIII) and factor IX (rFIX) with improved function to overcome some of the limitations in current treatment, the high costs of therapy and to increase availability to a broader world haemophilia population. Bioengineered forms of rFVIII, rFIX or alternative haemostatic molecules may ultimately have an impact on improving the efficacy of therapeutic strategies for the haemophilias by improving biosynthesis and secretion, functional activity, half‐life and immunogenicity. Preventing and suppressing inhibitors to factor (F) VIII remain a challenge for both clinicians and scientists. Recent experiments have shown that it is possible to obtain anti‐idiotypic antibodies with a number of desirable properties: (i) strong binding avidity to FVIII inhibitors; (ii) neutralization of inhibitory activity both in vitro and in vivo; (iii) cross‐reactivity with antibodies from unrelated patients, and (iv) no interference with FVIII function. An alternative, although complementary approach, makes use of peptides derived from filamentous‐phage random libraries. Mimotopes of FVIII can be obtained, which bind to the paratope of inhibitory activity and neutralize their activity both in vitro and in vivo. In this paper, we review advanced genetic strategies for haemophilia therapy. Until recently the traditional concept for gene transfer of inherited and acquired haematological diseases has been focused on how best to obtain stable insertion of a cDNA into a target‐cell genome, allowing expression of a therapeutic protein. However, as gene‐transfer vector systems continue to improve, the requirement for regulated gene transcription and hence regulated protein expression will become more critical. Inappropriate protein expression levels or expression of transferred cDNAs in non‐intended cell types or tissues may lead to target‐cell toxicity or activation of unwanted host immune responses. Regulated protein expression requires that the transferred gene be transferred with its own regulatory cassette that allows for gene transcription and translation approaching that of the normal gene in its endogenous context. New molecular techniques, in particular the use of RNA molecules, now allow for transcription of corrective genes that mimic the normal state.
Human immunodeficiency and hepatitis virus infections and their associated conditions and treatments among people with haemophilia
Authors: Goedert J. J. , Brown D. L. , Hoots K. , Sherman K. E.
Published: October 2004

Summary.  Treatment with contaminated plasma products before 1990 resulted in extraordinary prevalence rates of human immunodeficiency virus (HIV) and hepatitis B and C viruses (HBV, HCV). In the Second Multicentre Haemophilia Cohort Study (MHCS‐II) during 2001–03, 30% of HCV‐seropositive survivors had HIV and 4.6% were HBV carriers. Highly active antiretroviral therapy (HAART) radically altered the consequences of HIV/HCV coinfection. Whereas opportunistic infections predominated previously, current major complications are liver failure and bleeding (exacerbated by decreased clotting factor synthesis, hypersplenic thrombocytopenia, and oesophageal varices). Most HIV‐positives in MHCS‐II were HIV RNA‐negative and had > 200 CD4+ cells µL−1, but only 59% were on HAART. With HIV, especially after 41 years of age, liver disease was apparent (jaundice in 5%, ascites 7%, hepatomegaly 9%, splenomegaly 19%). HAART increases survival but may contribute to various comorbidities. Without HIV, sustained HCV clearance is obtained in > 50% with combined pegylated interferons plus ribavirin, but data in haemophilic populations, especially with HIV, are limited. In MHCS‐II, HCV RNA negativity was 41% following standard interferon plus ribavirin; among interferon‐naïve participants (implying spontaneous HCV clearance), HCV RNA negativity was 12% with and 25% without HIV. Without HIV, spontaneous HCV clearance was much more likely with early age at infection and particularly with recent birth (late 1970s or early 1980s) but not with bleeding propensity or its treatment. Most (72%) participants had received no anti‐HCV therapy. Hepatic and haematological conditions are likely to increase during the coming years unless most adult haemophiliacs are successfully treated for HIV, HCV or both.
Optimizing factor prophylaxis for the haemophilia population: where do we stand?
Authors: Blanchette V. S. , Manco‐Johnson M. , Santagostino E. , Ljung R.
Published: October 2004

Summary.  The hallmark of severe haemophilia, defined as a circulating level of factor (F) VIII (haemophilia A cases) or FIX (haemophilia B cases) of < 1%, is recurrent bleeding into muscles and joints (haemarthroses) from an early age of life. The inevitable result of such bleeding is progressive joint damage, leading to disabling arthritis that is typically evident within the first 2 decades of life in people with haemophilia who have limited or no access to regular factor replacement therapy, or those in whom factor replacement therapy is ineffective because of the presence of high‐titre inhibitors. For children with severe haemophilia and no evidence of inhibitors, the unwanted musculoskeletal complications of severe haemophilia can be effectively prevented by the early initiation of a programme of long‐term factor prophylaxis. In order to achieve the best outcome (a perfect musculoskeletal status for age) the programme of prophylaxis should be started before the onset of joint damage (primary prophylaxis). The gold standard primary prophylaxis regimen (the Malmö protocol) was pioneered and tested in Sweden and involves the infusion of 20–40 IU of FVIII per kg body weight on alternate days (minimum three times per week) for haemophilia A cases, and 20–40 IU kg−1 of FIX twice weekly for haemophilia B cases. This protocol is, however, demanding on peripheral veins and very expensive. Modifications of the parent protocol such as starting primary prophylaxis with once‐weekly infusions via peripheral veins with rapid escalation to full‐dose prophylaxis or dose escalation based on frequency of bleeding are increasingly implemented in haemophilia treatment centres in countries that can afford the high cost of such programmes. These modified programmes can be achieved in the majority of young children with severe haemophilia without the need for central venous access devices (e.g. Port‐a‐Caths) and with avoidance of device‐associated complications such as infection and thrombosis. In at least one centre, experience with arteriovenous fistulae as a strategy to ensure reliable venous access is being accumulated. The issues of compliance (adherence) to recommended prophylaxis protocols and when, if ever, to stop a programme of primary prophylaxis once started are real and require ongoing prospective studies. Such studies should incorporate outcome measures such as health‐related quality‐of‐life and economic analyses.
What is a cure and how do we get there?
Authors: Skinner M. W. , Lillicrap D. P. , McMillan J. , Castro Ozelo M. , Pierce G. F.
Published: October 2004

Summary.  The absence of adequate treatment for most of the world's 400 000 individuals with haemophilia makes the development of a cure compelling. Advances in the basic molecular sciences over the past 20 years have resulted in the feasibility of curing haemophilia through the application of gene therapy. However, the reality of this therapeutic strategy is highly complex. In addition, challenges to achieving a cure exist beyond the basic scientific hurdles. Thoughtful attention must also be given to a number of interrelated issues, including ethical considerations in patient recruitment, informed consent and geographical variables of global clinical trials. The global inequalities in healthcare mean that the ethics of international medical research, especially when it includes countries where people usually do not receive quality care, become much more complicated. The majority of haemophiliacs lives in developing countries and is a valuable resource of human subjects who could be enrolled in clinical trials. When recruiting subjects globally, investigators must be ever mindful that the patient population is a precious resource, which must be treated with respect and care. This presents a major challenge for investigators engaged in trials of haemophilia gene therapy to ensure that the informed consent process is current and comprehensive, that therapeutic misconceptions are appropriately managed, and that the roles of the researcher and physician are clear. Global clinical gene‐therapy trials are an important and appropriate component in the quest to achieve a cure for haemophilia. When trials follow identical internationally accepted standards, a successful outcome can be achieved for trials including developing countries, if country specific cultural and economic aspects are considered.
Risks and benefits of sports and fitness activities for people with haemophilia
Authors: Mulder K. , Cassis F. , Seuser D. R. A. , Narayan P. , Dalzell R. , Poulsen W.
Published: October 2004

Summary.  Physical activity is a key component of a healthy lifestyle. Exercise and physical activity have been shown to help maintain a healthy body weight, reduce stress, increase self‐esteem and feelings of wellbeing, control blood pressure, and prevent heart disease and diabetes. Children with haemophilia may feel restricted from competing in sports through parental concern or pain and difficulty in moving, or they may rebel against such restrictions, thus leaving themselves open to serious injury. Several groups have attempted to classify sports activities with regard to the level of risk involved; however, these are not consistent. It is important to match the child's abilities with the sport in which they want to take part, and suggest alternatives if this is not possible. Prevention of injury should not depend solely on use of factor concentrates.
Inhibitors in haemophilia: pathophysiology
Authors: Saint‐Remy J‐M. R. , Lacroix‐Desmazes S. , Oldenburg J.
Published: October 2004

Summary. Development of inhibitors to coagulation factors is one of the major problems faced by people with haemophilia. Up to a third of patients, following treatment with factor concentrates, will develop an antibody (inhibitor) to that factor, rendering it inactive, and leaving the patient at risk from life‐threatening bleeding. Evidence shows that this immune response is T‐cell‐dependent, but as yet, the epitopes responsible have not been identified. Risk for inhibitor development is highest within the first 50 days of treatment, with reactions being rare after 200 days. The risk is mediated by the major histocompatibility complex class of the patient, and by mutations in the factor VIII genotype, with large deletions conferring greatest risk.
Regulatory challenges to global harmonization and expanded access to concentrates: how will regulators balance the increasing cost of new safety requirements with the desire to increase the availability of affordable product?
Authors: Farrugia A.
Published: October 2004

Summary. The provision of concentrates of the deficient coagulation factors is an essential component of the provision of comprehensive haemophilia care. Their safety, quality and efficacy need to be assured independently of the measures dictated by the market and the individual manufacturers. Over the past 20 years, this assurance has become the role of regulatory authorities, which in the developed world have generated a framework that assesses haemophilia products as medicines in the highest category of risk relative to other therapeutic agents. Systems of official regulation mandating standards and other measures are now coupled with voluntary standards adopted by industry bodies as additional features of a comprehensive nexus of arrangements contributing to product quality and risk minimization. Currently, the regulation of products for haemophilia in less developed economies relies on reference to decisions in the first‐world authorities. This may not always result in optimal outcomes as most of the haemophilia care in the developing world is through local plasma and cryoprecipitate, which are not subject to the oversight of mainstream regulators. Furthermore, the emergence of companies based outside the developed world and seeking to supply the emerging economies of the developing world with haemophilia concentrates has necessitated new strategies for regulation that are independent of the established frameworks. Overall, the principles used by mainstream agencies may be applied in all environments seeking to assure the quality of haemophilia care. Applied properly, they can contribute to maintaining the delivery of a form of therapy that is nowadays amongst the safest in therapeutic practice. A rigid interpretation can seriously impede access to treatment, and therefore the development of independent expertise and appropriate strategies in assuring product safety and quality in the developing world is essential if patient safety and access to products can be assured.
Intranasal antigen targeting to MHC class II molecules primes local IgA and serum IgG antibody responses in mice
Authors: SNIDER D. P. , UNDERDOWN B. J. , MCDERMOTT M. R.
Published: October 2004

Covalent conjugates of hen egg lysozyme (HEL) and anti‐major histocompatibility complex (MHC) class II monoclonal antibodies (mAb) were used to immunize mice intranasally. Three weeks after intranasal (IN) priming, mice responded rapidly to IN challenge with a mixture of HEL and cholera toxin (CT), by producing large titres of anti‐HEL IgA and IgG1 antibody in serum, and IgA antibody in nasal secretions. No secondary response to HEL plus CT occurred in mice that received no priming or mice primed with HEL alone. The secondary serum IgG antibody response was dominated by the IgG1 subclass. HEL combined with CT adjuvant worked much better than HEL alone in producing the secondary response. Control conjugates, containing an IgG isotype‐matched mAb without specificity for mouse tissues, provided poor priming. Mice expressing MHC class II molecules, to which the anti‐MHC II mAb cou ld not bind, produced a weak antibody response compared with those that expressed the appropriate MHC class II molecule. Our results demonstrate that immunotargeting to MHC class II molecules via the IN route allows priming of the local IgA and circulating IgG antibody, and indicate that this technique is a feasible approach for delivery of subunit vaccines in the upper respiratory tract.
Increased adjuvant efficacy in stimulation of antibody responses after macrophage elimination in vivo
Authors: LEENAARS P. P. A. M. , SAVELKOUL H. F. J. , HENDRIKSEN C. F. M. , VAN ROOIJEN N. , CLAASSEN E.
Published: October 2004

To investigate whether macrophages influence the efficacy of adjuvants, we locally eliminated lymph node macrophages with dichloromethylene diphosphonate containing‐liposomes before primary immunization. After macrophage elimination, animals were immunized with a soluble antigen (TNP–KLH; 2,4,6‐trinitrophenyl–keyhole limpet haemocyanin) either in phosphate‐buffered saline (PBS), in complete Freund's adjuvant (CFA), or in Specol. Specol is a water‐in‐oil emulsion, considered to be less aggressive than CFA, but equally effective. A secondary immunization followed with TNP–KLH. In vivo macrophage elimination before Specol/TNP–KLH immunization resulted in increased adjuvant efficacy as measured by (primary) antibody responses. This suggests that the activity of Specol is not primarily mediated through macrophages but rather through other antigen‐presenting cell types (e.g. dendritic cells, B cells, fibroblasts). The overall quality of produced antibodies, in terms of isotype distribution and affinity maturation, remained the same. After primary injection, CFA/TNP–KLH immunization resulted in significantly higher antibody responses in macrophage‐depleted animals and antibody responses did not increase significantly after secondary immunization. However, a booster effect was found when macrophages were not eliminated before CFA/TNP–KLH immunization, suggesting that the presence of macrophages during the first weeks of the primary immune response is essential for the induction of an effective secondary response in CFA immunizations. In conclusion, macrophage depletion before immunization with a soluble T‐cell‐dependent antigen combined with Specol may enhance specific antibody responses without changing the isotype or affinity of the antibodies.
Nitric oxide increased interleukin‐4 expression in T lymphocytes
Authors: CHANG R.‐H. , LIN FENG M.‐H. , LIU W.‐H. , LAI M.‐Z.
Published: October 2004

Nitric oxide (NO) is a regulator of many biological functions including T helper 1 (Th1)/T helper 2 cells balance. It has been demonstrated that NO inhibits the secretion of interleukin‐2 (IL‐2) and interferon‐γ on Th1 cells. Here we showed that, in addition to the suppression of IL‐2 production, NO‐generating agents sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP) increased the secretion of IL‐4 both in Th2 clones and EL4 T cells. The additive effect was dependent on the dose of SNP and SNAP. Augmentation of IL‐4 production was detected with 1 μM SNP, and up to threefold increase in IL‐4 secretion could be observed with higher concentrations of SNP/SNAP. NO also weakly increased the activation of IL‐4 promoter. In contrast, NO markedly inhibited the induction of IL‐2 promoter, which could account for most of the reduction in IL‐2 production. Analysis of the transcriptional elements on IL‐2 and IL‐4 promoters revealed a selective inactivation of NF‐κB and NF‐AT. It is suggested that despite the complex feedback network regulating NO production, the enhanced IL‐4 expression would lead to the expansion of Th2 cells once NO is generated.