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While there are several open source tools available [21] for developing ontologies, we selected Protégé because of its extensibility to a variety of plug-ins that are readily available for integration. It also has the ability to export to different formats including the Ontology Web Language (OWL) (http://www.w3.org/TR/owl-features/), which allows interoperability with other ontologies. |
We have previously described some of the general concepts relating to the IEDB design [1, 2] . More information relating to various aspects of the project can be accessed at http://www.immuneepitope.org/. Herein, we report a detailed description of the novel aspects of the IEDB ontology. In designing our application architecture, we have followed the common system engineering practice of first determining the scope and nature of the data involved. A first essential step is to understand the semantics of the domain and to capture that knowledge in an agreed-upon format. Arranging the domain concepts in a taxonomy is one of the initial organizing steps in the ontology design process. The class hierarchy represents the generalization and specialization relationships between the various classes of objects in a domain [6] . Briefly, classes describe concepts in the domain. Subclasses represent concepts (classes) that are more specific than the superclass and these subclasses can have their own unique properties. Slots represent properties of the classes. For example, in Figure 1 , we see that there is a class named Reference and three more specific subclasses of Reference: Journal Article, Patent Application, and Direct Submission. Figure 1 also shows that the class Epitope has a number of properties (slots) associated with it such as "has Epitope Structure" and "has Epitope Source". |
Our approach for creating the class hierarchy was a topdown development process where we defined each class in a domain and then identified its properties before building the hierarchy. The main classes identified for IEDB are Reference, Epitope Structure, Epitope Source, MHC Binding, Naturally Processed Ligand, T Cell Response, and B Cell Response (Figure 1 ). The Epitope class is the main class that encompasses all the individual concepts that were identified. The individual concepts are related to other classes. The primary relationships use the sub-class relationship or use a property (shown in the figures by the arcs labeled "has") that has a restriction on the type of the value that may fill that slot. |
"Reference" is the class encompassing information related to the data source from which an epitope and its related information are extracted into the IEDB. We have identified three broad subclasses of References that describe where epitope information will be obtained. They are Journal Article, Patent Application, and Direct Submission. The complete listing of slots (fields) encompassed by the Journal Article, Patent Application, and Submission classes are provided in Figure 2 . The Journal Article class refers to manuscripts published in peer-reviewed journals. The Patent Application class captures all the reference fields for a patent application that contain epitope information. The Submission class captures information about sources that contribute data to the IEDB directly. Data deposited by the Large Scale Antibody and T Cell Epitope Discovery contracts [3] and those transferred from other websites fall into this class. |
The Epitope Structure and Epitope Source classes capture intrinsic features of an epitope. The Epitope Structure class captures the physical and chemical features of an epitope. Virtually any molecular structure may provoke an immune response, such as proteins, carbohydrates, DNA, and lipids. In the Epitope Structure class, structural information relating to linear sequences and 2-D structures of the epitope, if available, are catalogued. The Epitope Source class captures the phylogenetic source of an epitope, including species of origin, gene name, protein name, and links to other databases for more detailed information about proteins and genes. Figures 3A and 3B show the listings of properties (slots/fields) encompassing the Epitope Structure and Epitope Source classes. |
The experimental data and information about specific experiments and the methodology utilized are captured in the Assay Information class. The name of the assay used, the type of response measured in the assay, and the readout of the assay are examples of information captured in the Assay Information class. This important class is used as a superclass of several other classes (and thus its properties are inherited by those classes). A complete listing the properties (slots/fields) in the Assay Information class is shown in Figure 3C . |
As with Assay Information, the classes Immunization, Antigen, and Antigen Presenting Cell are used in multiple other class descriptions. Features relating to the induction of the immune response are captured in the Immunization class ( Figure 4A ). It has relationships to other classes like Immunized Species, Immunogen, In vivo Immunization, and In vitro Immunization. Immunized Species contains information relating to the host that is being immunized. The Immunogen class describes the molecules that induce the immune response and an associated carrier molecule, if present. Features relating to how the immunogen was introduced to the immunized species are captured under the In vivo and In vitro Immunization classes. |
Similarly, antigens are defined as the whole molecules that react with the products of an immune response (as opposed to the epitopes which are the specific structures, contained within the antigen that engages the immune receptor). Information relating to the antigen and any associated carrier molecule is captured in the Antigen class ( Figure 4B ). During immune responses, antigen-presenting cells process antigens and present peptide epitopes complexed with MHC molecules. This information is captured in the Antigen Presenting Cells class, which has a relationship to the MHC Molecules and the Source Species classes ( Figure 4C ). The Source Species class describes the species information from which the antigen presenting cells are derived. |
The MHC Binding class captures the details relating to the interaction of the epitope with specific MHC molecules and information relating to the MHC molecule along with any available Epitope-MHC complex structure details. This class also has a slot that is restricted to be an instance of the Assay Information class ( Figure 5A ). |
Overview of IEDB Class Hierarchy Figure 1 Overview of IEDB Class Hierarchy. |
classes. Extrinsic features are context-dependent attributes, being dependent upon specific experimental conditions. The Naturally Processed Ligand class captures data related to epitopes that are naturally processed and presented on the cell surface. This class has properties that are instances of classes including Antigen Presenting Cell, Antigen, and Assay Information ( Figure 5B ). |
The Naturally Processed Ligand class differs from the MHC Binding class in that information related to the antigen that was processed and the cell types in which the processing occurred is represented. MHC Binding class captures data relating to in vitro MHC binding assays, which assess the epitope's binding capacity to the MHC molecule. Hence the MHC Binding class does requires neither the Antigen class not the Antigen Presenting Cells class. In general, naturally processed ligands are assessed in the absence of a T cell response, for example, identified by direct elution from MHC molecules extracted from infected cells or antigen presenting cells. Thus, the Immunization class is not used as a value restriction by the Naturally Processed Ligand class. |
The T Cell Response class captures all of the T cell mediated immunity-related information ( Figure 6A ). It has properties that are of type: Immunization, Effector Cells, Antigen Presenting Cell, Antigen, Assay Information, and Epitope-MHC-TCR Complex. The Effector Cell class describes the cells that are elicited upon immunization and that acquire measurable functions as a result. The B Cell Response class describes antibody responses that are related to the epitope ( Figure 6B ). This class has properties that are of type: Immunization, Antibody Molecule, Antigen, Assay Information, and Antigen-Antibody Complex. Because B cell responses do not require MHC binding and |
There are three classes that capture information about the 3D structure of complexes: Epitope-MHC Complex, Epitope-MHC-TCR Complex, and Antigen-Antibody Complex. The Epitope-MHC Complex, Epitope-MHC-TCR Complex, and Antigen-Antibody Complex classes are used as restrictions on properties of the MHC Binding, T Cell Response, and B Cell Response classes respectively ( Figures 5A, 6A, and 6B) . These Complex classes capture the Protein Data Bank (PDB) Identifier, which provides detailed information about 3D structures. The Protein Data Bank [22, 23] contains approximately 1600 3D structures that are of immunological interest. Other information that is not available in PDB, such as the atom pairs that are involved in the interactions between molecules, the specific residues, the contact area of the molecules, and allosteric effect, is also captured here. |
Each class has numerous slots that capture detailed information associated with epitopes. As mentioned above, a complete list of all the classes, their properties, and relationship, can be found at http://www.immuneep itope.org/ontology/index.html. One of the files provided as supplementary material contains two examples of how two literature references [24, 25] containing epitope information are extracted into the IEDB ontology (additional file 1). Along with the class hierarchy, the IEDB's data dictionary (additional file 2) provides more detailed information about the fields that are defined for the IEDB. The data dictionary contains a textual overview description and a listing of fields that are required to be completed for IEDB entries. The data dictionary also allows database users to provide comments and suggestions to IEDB team to enhance the formal ontology. |
The IEDB will be a comprehensive resource pertaining to epitopes of the immune system. By extensively curating both intrinsic and extrinsic features associated with epitopes, the IEDB is expected to provide substantially greater detail about specific epitopes than any other databases presently available. The IEDB will be populated with data derived from three main sources, namely the peer-reviewed literature, patent applications, and direct submission. Epitope data published in the literature and patent applications are curated manually by the IEDB's curation team. Data from already existing epitope databases, whose authors have agreed to share their data, will also be imported into the IEDB. Apart from these, a main data source will be the direct submission of data from the Large-scale Antibody and T Cell Epitope Discovery programs [3] that are funded by NIAID. Presently, fourteen contracts have been awarded under this program, and all of them will submit their data to the IEDB. Direct antibody and T cell epitope submissions will also be sought from the broader research community, with an emphasis on antibody epitopes to NIAID Category A-C pathogens. Because of the large scale of the IEDB project, a formal ontology is critical to ensure consistency in the representation of data. |
Communication between database developers, researchers, analysis tool developers, and team members is crucial, and can be performed in harmony only when a common vocabulary is established. An ontology, which is an explicit formal specification of the terms in the domain and relationships among them, is an effective way to share the knowledge contained in that domain. Accordingly, since the IEDB's domain is epitope-related data, we have created an ontology that captures detailed conceptual structure related to these data. |
The development of this ontology has relevance for the expansion and modification of the epitope knowledge base. Our ontology design defines individual concepts as separate classes and then defined relationships between these classes and other objects in the domain. These classes serve to restrict the values that will describe properties of objects in the database. For example, the species is a separate concept defined in its own class. Depending on the context, this can refer to an immunized species or the species from which antigen presenting cells are derived. Similarly MHC Molecules is defined as a separate class, and it is used as a value restriction by concepts like MHC Binding and Antigen Presenting Cells. Defining concepts as separate classes and using them to restrict the values of properties in other classes facilitates the expansion and modification of our ontology. Adding properties (slots) to concepts is a task easily accomplished when there are well-defined class descriptions that may serve as value restrictions on the properties, and providing that High-level classification of Immunization (A), Antigen (B), and Antigen Presenting Cells (C) class these class descriptions are general enough to apply in all instances. We have ensured in our design that each concept is atomic and that it can be re-used by various classes. |
The development of a formal ontology is valuable to database users and in particular to scientists contributing data to, and downloading data from, the IEDB. We anticipate that the availability of a formal ontology will ensure that a common language and shared understanding of concepts will inspire this type of communication, thus ensuring maximum efficiency and accuracy. The formal ontology developed will most likely require refinement and fine tuning when users provide suggestions and new technologies for performing experiments are discovered. The IEDB website will provide mechanisms for the users to provide suggestions and participate in the enhance- ment of the ontology. The IEDB Data Dictionary has a separate column for the users to provide comments on specific data fields. The IEDB website will also host web forms that will guide users to conform to the ontology definitions when submitting data. Apart from the web forms, an XML schema definition (XSD) will be available on the website for users to inspect and use in their data submission. Users will also be able to download epitope records from the website. |
In the process of developing new ontologies, it is good practice to leverage existing community standards. In our initial analysis, we confirmed that there were no explicit ontologies that efficiently captured epitope details as per the scope of the IEDB program. As mentioned above, we have utilized, as much as possible, inferred ontologies from existing epitope databases. Among the ontologies that we analyzed, IMGT-Ontology and Gene Ontology were the only two formal ontologies that were related to the epitope domain. The IMGT-Ontology was designed for the ImMunoGeneTics database. IMGT is an integrated database specializing in antigen receptors (immunoglobulins and T-cell receptors) and the major histocompatibility complex of all vertebrate species. The ontology developed for this database has specific immunological content, describing the classification and specification of terms needed for immunogenetics. The IEDB does conform to IMGT's standards about receptors and MHC molecule chains in the sense that all the chain names follow IMGT's controlled vocabulary. |
GO provides structured controlled vocabularies for genes, gene products, and sequences annotated for many organisms. The IEDB complements GO in terms of epitopes of immunological interest since GO is incomplete in this area. Antigens, which are primary sources of epitopes, are annotated in GO. Thus, in essence, the IEDB could be utilized to provide an extension of GO for antigens that contain epitope-related information. |
Perhaps the most important element in the development of the IEDB ontology is that, to the best of our knowledge, this represents the first immunological ontology specifically designed to capture both intrinsic biochemical and extrinsic context dependent information. In this respect, it is similar in spirit, but different in approach, from other knowledge resources relating to systems biology. We anticipate that the development of this type of ontology and associated databases might lead to completely new methods for describing and modeling immune responses. Accordingly, this new program might represent a novel tool to assist in the design, testing, and development of new ways to combat infectious diseases and other immune related pathologies such as cancer and autoimmune diseases. |
A complete listing of IEDB's class hierarchy and its properties is available at http://www.immuneepitope.org/ ontology/index.html Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1A(A )and EF1A(B)) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1A(B)>EF1A(A)>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1A(B)>EF1A(A)>S20>β-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1A(A )and EF1A(B )genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon. In real-time RT-PCR, the expression levels of the target genes of interest are estimated on the basis of endogenous controls. Various housekeeping genes, ribosomal RNA (rRNA) and total RNA are most commonly used as references in gene expression analysis today. The purpose of these controls is to remove or reduce differences due to sampling, i.e. differences in RNA quantity and quality. |
The ideal endogenous control should be expressed at a constant level among different tissues of an organism, at all stages of development and should be unaffected by the experimental treatment. It should also be expressed at roughly the same level as the RNA under study [1] . However, data normalization in real-time RT-PCR remains a real problem, especially for absolute quantification [1] . Numerous studies have revealed that no single universal gene has a constant expression level under all developmental or experimental situations. The best choice of reference gene to use as an endogenous control varies, depending on the tissues of interest in the experiment. A large number of genes have for this reason been selected for normalization of mRNA expression data [2, 3] . If the selected reference gene fluctuates randomly between samples, small differences in expression between the genes of interest will be missed. Gene expression coefficient of variation (CV) between different groups of individuals should ideally be as low as possible [4] . In general, the stability of several potential reference genes should be tested in every examined tissue or cell, and under different experimental design [5, 6] . An increasing number of papers are discussing the selection of reference genes in real-time RT-PCR analyses [3, 7] . |
Two of the most commonly used reference genes are those encoding glyceraldehyde-3P-dehydrogenase (GAPDH) and β-actin. Recently, the use of these two genes as endogenous controls has been scrutinized, and several studies have documented that the GAPDH and β-actin genes should be used with caution as controls [2, 8, 9] . GAPDH in mammals is known to play a role in a broad range of cellular mechanisms (for review see Sirover [10] ), including being a key enzyme in glycolysis. Overall, GAPDH mRNA levels might be regulated under a large number of physiological states, and its use as a reference is inappropriate for most experimental conditions. Actin is a major component of the protein scaffold that supports the cell and determines its shape, and is the most abundant intracellular protein in eukaryotic cells. Even though commonly used as a reference, the application of the β-actin gene has recently been characterized as a historical carryover from northern blots and conventional RT-PCR (for a general discussion on the use of 'classic' reference genes like GAPDH and β-actin, see Huggett et al. [7] ). Eukaryotic elongation factor 1A (eEF1A, formerly elongation factor 1 alpha) plays an important role in translation by catalyzing GTP-dependent binding of aminoacyl-tRNA to the acceptor site of the ribosome. However, the protein is involved in a broad diversity of functions and constitutes 1-3% of the total cytoplasmic protein content of the cell. In human, cDNAs of two actively transcribed isoforms have been cloned (eEF1A-1 and eEF1A-2) (for review see Thornton et al. [11] ). Two paralog EF1A genes (A and B) have recently been applied as references in real-time qRT-PCR of Atlantic salmon [12] . It is plausible to assume that the presence of these highly similar genes is a result of a tetraploidization event that occurred in a salmonid ancestor in the comparatively recent past [13, 14] . |
Previously, the 18S rRNA gene was considered to be an ideal internal control in qRT-PCR analysis (Ambion [15] ). Ribosomal RNA constitutes up to 80-90% of total cellular RNA, and several studies have shown that rRNA varies less under conditions that affect the expression of mRNAs (discussed in Bustin & Nolan [16] ). However, questions have been raised against the use of ribosomal RNA genes as references. Vandesompele et al. [5] have stressed the fact that there sometimes might be imbalances in rRNA and mRNA fractions between different samples, making genes encoding ribosomal RNAs unsuitable as references. |
To meet these challenges of accurate interpretation of realtime qRT-PCR data, the authors suggested that an index of the most stable housekeeping genes should be used for normalization, and developed the geNorm VBA applet for Microsoft Excel in this regard [5] . A similar software tool, the BestKeeper, has been developed by Pfaffl et al. [6] . These tools can be used to find the most stable reference genes under different experimental conditions. We used the geNorm software which determines the individual stability of a gene within a pool of genes [5] . The stability is calculated according to the similarity of their expression profile by a pair-wise comparison, using their geometric mean as a normalizing factor. The gene with the highest M, i.e. the least stable gene, is then suggested excluded in a stepwise fashion until the most stable genes are determined, and an index suggested, based on the best genes. geNorm has been used to select the most stable reference genes in several recent studies (e.g. [4, 17, 18] ). |
The aim of this work was to evaluate the usefulness of six potential reference genes in the Atlantic salmon. Salmonid fish are among the most widely studied model fish species in general, and extensive basic information on many different aspects of their biology has been collected [19] . Large-scale DNA-sequencing projects on salmon have been initiated in several laboratories http://www.sal mongenome.no/cgi-bin/sgp.cgi; http://web.uvic.ca/cbr/ grasp/; http://www.abdn.ac.uk/sfirc/salmon/; http:// www.bcgsc.ca/gc/salmon. In this work we selected the two 'classic' reference genes encoding GAPDH and β-actin, two genes encoding 18S rRNA and S20 ribosomal protein and two paralog genes encoding elongation factor 1A (EF1A A and EF1A B ). To evaluate their usefulness as reference genes, RNA from eight tissues of six adult salmon were subjected to real time PCR. The relative transcription levels of the genes were also estimated in four phases of young salmon going through smoltification, in order to check their stabilities under physiological stressful conditions. |
The ranking of the six examined genes analyzed by geNorm is shown in Table 3 . In six tissues (muscle, liver, gills, head kidney, spleen and thymus), the EF1A B gene emerged as the most stable, whereas the EF1A A gene was ranked number one in brain and the β-actin gene was ranked number one in intestine. The 18S rRNA and S20 genes were ranked among the worse genes in all tissues. Not surprisingly, the GAPDH gene was ranked worse in five tissues (liver, head kidney, spleen, brain and thymus), confirming the general skepticism against the use of this gene as reference [7, 16, 20] . Combined, the total ranking reads EF1A B >EF1A A >β-actin>18S rRNA>S20>GAPDH. We did not analyze our data with the Bestkeeper software. Analyzing reference genes in virus infected cells, Radonic et al. [4] concluded that the Bestkeeper tool gave results that slightly deviated from, but nevertheless corresponded to, those obtained using geNorm. |
To be able to evaluate gene stability under stressful conditions, mRNA expression of the selected genes was examined in gills of salmon going through smoltification. Prior to seawater entry, juvenile anadromous salmon undergo a parr-smolt transformation, characterized by behavioral, morphological and physiological changes, known to be challenging for the fish. Physiological alterations include increased seawater tolerance, olfactory sensitivity, metabolic rate, scope for growth and changed hemoglobin and visual pigments [21] . We selected to examine the gills during smoltification, because this tissue plays a major role in ionic and osmotic regulation during adaptation to hyperosmotic seawater. Figure 1 shows the raw Ct values of the studied genes in gills before, during and after smoltification (smoltified in seawater and desmoltified in freshwater). In Figure 2 the same data are presented, but now normalized against an index calculated by geNorm of the three most stable genes (β-actin, EF1A A and EF1A B ). Based on the M values, geNorm ranks the stability of the six genes from 24 fish going through smoltification in the following order: EF1A B >EF1A A >β-actin>S20>18S rRNA>GAPDH ( Figure 3 ). In Figure 1 it can be seen that the 18S gene had the lowest individual raw Ct variation. Most individual raw Ct variation of the studied genes is seen in the presmolt and smolt groups. A characteristic drop in expression can be seen for all genes in the smolt group, compared to the presmolt group. After transfer to seawater, the individual raw Ct variation decreased for all genes. Overall, the raw Ct data suggest that the physiological challenging smoltification process affected the expression of all six genes. When the same data were normalized against an index of the three most stable genes, β-actin, EF1A A and EF1A B , the relative expression levels were altered for all genes. Now the ribosomal 18S gene emerges as the second worse, whereas the two paralog EF1A genes became the most stable. This might have to do with the fact that geNorm will top-rank co-expressed genes [22] , a weakness that has to be considered when evaluating paralog genes likely to be co-regulated. Even though the eEF1A-2 gene has been identified as an important oncogene and has been shown to be differently expressed in human tissues [11] , Hamalainen et al. [23] found the eEF1A-1 gene to be a good reference gene in real-time RT-PCR examinations. A similar finding was reported by Frost and Nilsen [24] in salmon louse, where they showed that the eEF1A and S20 genes were valid candidate references, whereas the 18S rRNA and GAPDH genes were unsuitable. The current findings based on geNorm evaluation question the recommended application of ribosomal genes as references (as suggested for example by Ambion (see reference [15] ), and are in line with earlier warnings against the use of rRNA genes as references [5, 6] . To avoid the normalization of the genes for β-actin, EF1A A and EF1A B against an index partly based on their own expression, the S20 gene was included in the index instead, and the mean normalized expression for these three genes calculated with the new index. The patterns of expression, however, were approximately the same for the three genes as seen in Figure 2 , suggesting that the gene-stability measure M can be used to find the most appropriate reference genes. |
We see a correlation between the A260/230 absorbance on the NanoDrop and the PCR efficiency (data not shown). We tend to get PCR efficiencies that are too high in some samples with low A260/230 ratios. When the samples are treated with DNase solution, the A260/230 ratio usually drops. After DNase treatment, the A260/280 ratio increased from 1.8 to 2.1 (n = 45 samples). At the Table 3 : Evaluation of the usefulness of six potential reference genes in eight tissues of Atlantic salmon ranked by the geNorm software. 1 = best, 6 = worst. Six individuals were analyzed for six genes in eight tissues. qRT-PCR analysis of six genes in gills of six Atlantic salmon going through smoltification; presmolt (before smoltification), smolt (during smoltification), smoltified (finished smoltified in seawater) and desmolt (desmoltification in freshwater) Figure 1 qRT-PCR analysis of six genes in gills of six Atlantic salmon going through smoltification; presmolt (before smoltification), smolt (during smoltification), smoltified (finished smoltified in seawater) and desmolt (desmoltification in freshwater). Numbers indicate raw Ct values. |
qRT-PCR analysis of six genes in gills of six smoltifying Atlantic salmon Figure 2 qRT-PCR analysis of six genes in gills of six smoltifying Atlantic salmon. The same data as in Figure 1 , but now normalized against an index of the three best genes (β-actin, EF1A A and EF1A B ) calculated with the geNorm software. The four groups were analyzed with Kruskal-Wallis test, and if significant, the overall p-value is given in the graphs. For β-actin, there were significant differences between the presmolt and the smoltified group (p < 0.05), the presmolt and the desmolt groups (n<0.01) and between the smolt and desmolt groups (p < 0.05). For EF1A A , there was a significant difference between the presmolt and the desmolt groups (p < 0.01). For EF1A B , there was a significant difference between the smolt and the smoltified groups (p < 0.05). An asterisk denotes significant differences between the groups. |
same time, the A260/230 ratio dropped from 2.4 to 2.1. The DNase treatment therefore adds substances to the RNA solution that increases the absorbance at 230 nm more than it decrease the 260 nm absorbance. The added substance (salt or some other component) may inhibit the RT reaction or the PCR reaction, sometimes called PCR poisoning. We have seen that the A260/230 ratios are quite low in samples that give inadequate PCR efficiency slopes, especially with RNA from head kidney, thymus and intestine tissues, in which the gradient of the standard curve is less than -3.3 ( Table 2 ). The reason one obtain better amplification rate efficiencies with the more diluted samples is because the inhibitor has been diluted below its effective level. The obvious way around this problem is to dilute the amount of cDNA put into the PCR reaction. Alternatively, cleanup columns can be used to purify and concentrate the RNA. |
Transcription levels of the examined genes and the coefficient of variance (CV) in different tissues varied considerably. mRNA levels in tissues are regulated by numerous endogenous and exogenous stimuli [16] . Transcription rates in metabolic active tissues might be up-regulated compared to those of less active tissues, whereas inter-tissue variation in degradation rates of mRNAs, for example, might affect mRNA stability [25] . The results revealed that muscle had the lowest CVs of the studied genes, compared to higher CVs in more active tissues like thymus, head kidney and spleen. In thymus, intestine, head kidney, gills, brain, liver and spleen, the 18S and S20 genes had the lowest CVs, based on raw Ct values. In all tissues, except intestine, the GAPDH gene had the highest CV. Except for thymus, the two elongation factor genes had relatively similar expression in all eight examined tissues. Their expression are most likely co-expressed in the examined tissues, and therefore favored in geNorm calculations [22] . The results also demonstrated that assays optimized for one tissue of an organism do not necessarily work equally well in other tissues. Of the tissues studied in this work, intestine, head kidney and spleen were the most troublesome. |
Our data, based on geNorm calculations, suggest that the Atlantic salmon EF1A genes that have been tested in the present study may be good candidate reference genes. The GAPDH gene seems unsuitable as a reference in quantitative real-time RT-PCR. With regard to the 18S rRNA gene, this must be applied with caution. Tools like the geNorm applet for Microsoft Excel can be useful to help select the most stable genes in various experiments. |
Tissues from 15 individuals were collected (852 ± 702 g, ranging from 254 to 1898 g). These individuals were not separated based on sex, size or sampling time, but treated as one heterogeneous group to examine the width of mRNA expression of the studied genes in eight different organs. This group of fish was handled and fed according to normal aquacultural management, and none of these individuals were exposed to any particular treatment. To examine if physiological stress may alter the gene expression in the gills, a total of 24 individuals were collected before (termed presmolt, 18.3 ± 0.9 g), during (termed smolt, 28.7 ± 3.7 g) and after smoltification. After smoltification, one group was kept and desmoltified in freshwater (termed desmolt FW, 30.0 ± 3.8 g), while the other group was transferred to seawater (termed smoltified SW, 30.2 ± 4.3 g) (n = 6 in each group). The Atlantic salmon examined during smoltification were from the anadromous population "Vosso" of the river Vosso in Southwestern Norway (see Nilsen et al. [27] for details on how these fish were treated). All fish were treated and euthanized according to Norwegian national legislation for laboratory animals. |
Samples from eight organs, i.e. gills, liver, brain, head kidney, spleen, thymus, white muscle and posterior intestine, were dissected out and immediately frozen in cryo tubes in liquid N and stored at -80°C before RNA extraction. The RNA extracted from three spleen and four head kidney tissue samples were of low quality, and we had to redo the sampling from four individuals, These tissue samples Stability of six genes in gills of Atlantic salmon during smoltifi-cation calculated with the geNorm software were stored on RNA later (Ambion) at -20°C before further processing. |
RNA was isolated with phenol-chloroform extraction as described by Chomczynski and Sacchi [28] , and stored in 100 µl RNase-free MilliQ H 2 O. Total RNA was extracted using Trizol reagent (Invitrogen, Life Technologies), according to the manufacturer's instructions. Genomic DNA was eliminated from the samples by DNase treatment according to the manufacturer's description (Ambion). The RNA was then stored at -80°C before further processing. The quality of the RNA was assessed with the NanoDrop ® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). A 260/280 nm absorbance ratio of 1.8 -2.0 indicates a pure RNA sample. The RNA 6000 Nano LabChip ® kit (Agilent Technologies, Palo Alto, CA, USA) was used to evaluate the integrity of the RNA. We used the RNeasy MinElute Cleanup kit from Qiagen to purify our most troublesome samples. With this kit the A260/230 ratio increased on average by 5 % (n = 10). |
The PCR primer and TaqMan MGB probe sequences used for quantification of the genes encoding 18S rRNA, S20 ribosomal protein, β-actin, GAPDH, EF1A A and EF1A B , are shown in Table 1 . Four of these genes, 18S, β-actin, EF1A A and EF1A B , have also been used as references in real-time RT-PCR analyses of Atlantic salmon in other recent studies [12, 28] . The primers amplify PCR products between 57-98 basepairs (bp) long, which is within the range of 50-150 bp as suggested by Applied Biosystems for their Taq-Man assays. qPCR assays were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA) to select appropriate primer and probe sequences from known Atlantic salmon genes. The mRNA sequences encoding S20 ribosomal protein and GAPDH were obtained from GenBank accession numbers BG936672 and BU693999, respectively (exon-exon borders were not considered). The EF1A A assay was based on a cDNA clone that we reported to the GenBank previously (AF321836), whereas the EF1A B assay was based on the EST BG933853. |
An alignment with zebrafish indicated the exon-exon borders [29] . The chosen primers were subsequently used to confirm that the salmon genes contained an intron between the same sites as deduced from the alignment with zebrafish. The PCR products containing the introns were cloned into TOPO vector (Invitrogen) and sequenced (sequences can be provided upon request). PCR primers for β-actin were based on Atlantic salmon BG933897 and designed to span exon-exon borders of this gene, as deduced from corresponding genes in human and zebrafish (NW633959). For 18S rRNA the PCR primers and probe were designed from the Atlantic salmon sequence AJ427629, and placed in a conserved region of the gene based on comparison with the human gene. RNA samples were subjected to DNase treatment to avoid genomic DNA contamination. Amplified PCR products of all actual cDNAs were sequenced to ensure that the correct mRNA sequences were quantified. The fragments were sequenced with BigDye version 3.1 fluorescent chemistry (Applied Biosystems) and run on an ABI PRISM ® 377 DNA apparatus at the University of Bergen Sequencing Facility. |
A two-step real-time RT-PCR protocol was developed to measure the mRNA levels of the studied genes in eight tissues of Atlantic salmon. For evaluation of the potential reference genes, raw Ct values are presented. The geNorm VBA applet for Microsoft Excel was used to determine the most stable genes from the set of tested genes [5] . The Ct values were transformed to quantities using standard curves, according to the geNorm manual. The gene expression stability (M) was calculated with the geNorm applet, and the genes were ranked from best to worst, based on the M value. |
The GraphPad Prism 4.0 software (GraphPad Software, Inc.) was used for the statistical analyses in this work. Linear regression was used to determine PCR efficiency based on dilution curves. Non-parametric Kruskal-Wallis test was used to compare differences among four groups of salmon going through smoltification. |
PAO was responsible for the experiment, data analysis and drafted the manuscript. KKL conducted the real-time RT-PCR analysis, and contributed throughout the experimental process. AEOJ constructed the qPCR assays for two of the genes. TON provided the cDNA from the smoltification experiment. IH participated as a supervisor in the study design, analyses and writing. Relevance of human metapneumovirus in exacerbations of COPD BACKGROUND AND METHODS: Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR. RESULTS: We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10(5 )viral copies/ml in nasal lavage and 1.88 × 10(5 )viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD. CONCLUSION: HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely. Respiratory viruses play an important role in exacerbations of COPD and this has been increasingly recognised since the application of molecular detection methods [1, 2] . The most prevalent viruses detected by polymerase chain reaction so far were respiratory syncytial virus (RSV), Influenza A, Rhinovirus and Parainfluenza 3. Human metapneumovirus (hMPV) is a recently discovered respiratory virus first isolated from a dutch child with lower respiratory tract infection (LRTI) [3] . World wide distribution is probable since it has been isolated in North HMPV has been recognized as a member of the Paramyxoviridae like RSV and it is not only associated with bronchiolitis in most cases, but also with pneumonia, croup and exacerbations of asthma [14, 15] , diseases which share some features with COPD. Up to date reports about hMPV in adults are scarce. In a general Canadian population 14.8% of patients of all age groups with acute respiratory tract infections were hMPV positive. Thirty-three percent of hMPV-infected patients were hospitalized and the hospitalization rates were significantly higher among patients below 5 years and those over 50 years of age [16] . In another prospective cohort of adults, hMPV was detected in 4.5% of all illnesses but also in 4.1% of asymptomatic subjects. HMPV was most prevalent in young adults with children and in frail elderly [17] . HMPV infection can be severe since the virus was isolated from the lungs from a previously healthy man who died from acute pneumonia [18] . The role of hMPV in acute exacerbations of COPD (AE-COPD) has been studied recently in outpatients and only low frequencies have been observed [17, 19] . Up to now the prevalence of hMPV in patients hospitalized with AE-COPD is unknown. Our aim was therefore to investigate the frequency of detection of hMPV in a prospective cohort of hospitalized patients with AE-COPD and to compare these results to patients with stable COPD and to smokers without COPD. |
Three different groups were studied. The first group consisted of hospitalized patients with an acute exacerbation of COPD (AE-COPD), the second group were subjects with stable COPD and the third group was composed of smokers without COPD. The groups were defined as previously published [20] . Briefly AE-COPD patients suffered from COPD as defined by GOLD [21] . Acute exacerbation was characterized by worsening in dyspnea, cough, and expectoration. A routine posterior-anterior chest radiograph was evaluated on admission by expert radiologists to exclude other other reasons for increased symptoms as pneumonia, tuberculosis, pulmonary fibrosis, bronchiectasis, bronchial carcinoma or congestive heart failure.Stable COPD patients did not have an exacerbation within the last 30 days prior to hospital admission and had no changes in therapy within the last 14 days (including inhaled and oral medication) and had been admitted for other medical reasons into departments of internal medicine other than pulmonary care. COPD subjects were recruited in a 2:1 ratio each month in order to prevent seasonal selection bias. Smokers have been smoking more than 10 pack-years, could have chronic symptoms like cough and phlegm but did not report dyspnea and did not have bronchial obstruction (FEV 1 /FVC>70%, FEV 1 >80% predicted). None of the smokers had a history of COPD or asthma, nor was using systemic or topic pulmonary medication. The smokers were recruited either from our smoking cessation initiative or by newspaper advertisement. |
The study was approved by the ethical committee of the Ruhr-University of Bochum, Germany. Written informed consent was obtained from all patients and control subjects before inclusion in the study. |
Clinical evaluation, spirometric tests, nasal lavage, induced sputum, specimen processing and viral ribonucleic acid (RNA) extraction were carried out as described by Rohde et al [2] . Elution volume was 100 µl. cDNA was generated with random-hexamer primers as previously published[2]. |
A hMPV-specific real-time RT-PCR designed and evaluated by Maertzdorf et al was used [22] . Primers and probe are localized within the nucleoprotein gene (NL-N) and the presence of a degenerate base within the probe allows detection of all four genetic lineages of hMPV. |
The assays were performed using the TaqMan ® PCR Core Kit. The final volume was 25 µl containing 500 nM of the forward primer (NL-N-forward (5'-CATATAAGCAT-GCTATATTAAAAGAGTCTC-3')), 250 nM of the reverse primer (NL-N-reverse (5'-CCTATTTCTGCAGCATATTTG-TAATCAG-3')) and 500 nM of the probe (NL-N-probe (5'-FAM-TGYAATGATGAGGGTGTCACTGCGGTTG-TAMRA-3', in which Y is either a C or a T residue). Nuclease-free water was used as negative control and a plasmid containing the N gene of hMPV (kindly provided by James Simon, VIRONOVATIVE, EUR Holding, Erasmus University Rotterdam) was used as a positive control in all PCR runs. Cycling parameters were as follows: 5 min at 95°C, 45 cycles of 30 s at 95°C and 1 min at 60°C. Amplification and detection of RNA from virus isolates or clinical specimens were performed using the GeneAmp ® 5700 Sequence Detection System (Applied Biosystems). The real-time PCR product was cloned with the QIAGEN ® PCR cloning kit (QIAGEN, Hilden, Germany) and this standard plasmid DNA was used for absolute quantification of hMPV viral load. Calculations were performed as previously described for absolute quantification of RSV viral load [23] . |
The primary objective of this study was to compare the frequency of hMPV detection in respiratory specimens between COPD patients with or without an acute exacerbation and smokers without COPD. |
Continuous data were checked for normal distribution using the Kolmogorov-Smirnov test. The data were of non-parametrical distribution and results were expressed as median and range. Differences between groups were assessed by Kruskal-Wallis test. To further analyse significant differences between two individual groups a pair wise comparison by two-sided Mann-Whitney U-test was performed. All significance levels were set to 5%. Data were analysed and processed using SPSS Version 12.0 on a Windows XP operating system. |
A total of 229 subjects were investigated between October 1999 and June 2004: 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. The clinical characteristics and lung function measurements are summarized in table 1. FEV 1 , FEV 1 in % of predicted value and FEV 1 /FVC were normal in smokers, significantly decreased in stable COPD patients (all) and further significantly decreased in AE-COPD patients (all p < 0.05 compared to stable COPD and all p < 0.001 compared to smokers). |
HMPV could be detected in three subjects. All these subjects were AE-COPD patients. The prevalence of hMPV in AE-COPD patients was 2.3%. The virus was simultaneously detected in nasal lavage and induced sputum in one patient only. The viral load was about 100 times higher in nasal lavage than in induced sputum in this patient. Overall the viral load in nasal lavage was about 3.5 times higher compared to induced sputum (for details see table 2). The hMPV positive patients did not differ significantly from other AE-COPD patients when clinical parameters and lung function were analysed. hMPV was detected in the winter season only. |
The main finding of this controlled study investigating the incidence of hMPV in subjects with COPD and smokers without COPD is that this recently discovered respiratory virus was detectable only during exacerbation of COPD. The frequency of detection was very low but in positive cases the viral load was considerable. There was no detection in patients with stable COPD or smokers without COPD. |
Recently Vicente et al [19] reported about the incidence of hMPV in 89 COPD patients. Five patients (5.5%) were hMPV positive. Two of these patients had to be transferred to hospital. Although this was not a controlled study and not all details of the study are available due to the fact that the data were published in form of a letter, these results support our findings. The incidence of hMPV in this and in our study is low compared to other respiratory viruses. In a similar previous study we found that Picornaviruses were detectable in 36% of AE-COPD patients, Influenza A in 25% and Respiratory syncytial virus in 22%[2]. There is another prospective cohort study of adults in which hMPV was detected in 4.5% of all illnesses. HMPV was most prevalent in young adults with children and in frail elderly from long term care facilities [17] . Unfortunately this report does not specify how many of the elderly patients suffered from COPD. In our asymptomatic smokers without COPD hMPV could not be detected. A recent study investigating nasal secretions from adults with and without respiratory illnesses found hMPV in 5 of 146 ill patient and in none of 158 control subjects, strongly supporting our data [24] . A further recent study found hMPV in two out of 111 adult patients (1.8%) who presented to the emergency department for AE-COPD during 2 winter/ spring seasons in Quebec, Canada, also in support of our findings [25] . In a US American study investigating clinical samples collected between 1991 and 1995, hMPV could not be detected at all in 196 patients indicating important geographical and seasonal differences in hMPV prevalence [26] . Taken together the results presented here are in keeping with other studies in adults and add important information on the prevalence of hMPV in hospitalized AE-COPD. |
To our knowledge this is the first study analysing the viral load of hMPV in COPD patients. We found a mean of 6.5 × 10 5 viral copies/ml in nasal lavage and 1.88 × 10 5 viral copies/ml in induced sputum. These values indicate that hMPV may have been the infectious agent triggering exacerbation in these patients. Viral load cut-off values for infectivity in COPD exacerbations have not been studied in detail yet and need further investigation. However, viral loads between 1120 copies/ml in Cytomegalovirus infection in lung-transplant patients [27] and 5.8log 10 copies/ ml in SARS [28] have been considered to indicate infectious disease. Moreover hMPV was only found in acute exacerbation and not in stable disease or in smokers without COPD supporting a triggering role in AE-COPD. |
HMPV infection can be severe since it was isolated from the lungs from a previously healthy man who died from acute pneumonia [18] . Our hMPV positive patients did not differ in their clinical characteristics or lung function from the other AE-COPD patients which does not indicate a more severe course of AE-COPD in these patients. |
Taken together this is the first controlled study on the relevance of hMPV in hospitalized AE-COPD. HMPV was detected in a very low frequency but with noticeable viral load in AE-COPD patients. Given that asymptomatic carriage of hMPV is very unlikely it should be considered as another possible trigger of AE-COPD. Since every AE-COPD has considerable impact on the course of the disease and regional outbreaks of hMPV are possible it should be included into future diagnostic and therapeutic considerations. |
The author(s) declare that they have no competing interests. Bioethical Implications of Globalization: An International Consortium Project of the European Commission The BIG project looks at some of the ethical concerns surrounding globalization and health. T he term "globalization" was popularized by Marshall McLuhan in War and Peace in the Global Village . In the book, McLuhan described how the global media shaped current events surrounding the Vietnam War [1] and also predicted how modern information and communication technologies would accelerate world progress through trade and knowledge development. Globalization now refers to a broad range of issues regarding the movement of goods and services through trade liberalization, and the movement of people through migration. |
Much has also been written on the global effects of environmental degradation, population growth, and economic disparities. In addition, the pace of scientifi c development has accelerated, with both negative and positive implications for global health. Concerns for national health transcend borders, with a need for shared human security and an enhanced role for international cooperation and development [2] . These issues have signifi cant bioethical implications, and thus a renewed academic focus on the ethical dimensions of public health is needed. |
Future developments in science and health policy also require a fi rm grounding in bioethical principles. These core principles include benefi cence; nonmalefi cence (to do no harm); respect for persons and human dignity (autonomy); and attention to equity and social justice. According to the World Health Organization [3] , global ethical approaches should (1) monitor and update ethical norms for research, as necessary; (2) anticipate ethical implications of advances in science and technology for health; (3) apply internationally accepted codes of ethics; (4) ensure that agreed standards guide future work on the human genome; and (5) ensure that quality in health systems and services is assessed and promoted. |
The Bioethical Implications of Globalization (BIG) Project is a 42-month dialogue funded by the European Commission that involves a series of expert panel discussions on four specifi c globalization and health subject areas: (1) mobility of people; |
(2) technological globalization; (3) liberalization of trade; and (4) new global health threats (bioterrorism). In addition, BIG includes a multipleround Delphi Process (Box 1) to solicit input on these issues from a broad, interdisciplinary audience. |
The project's purpose is both to raise short-term, practical considerations about globalization and health and |
Delphi is a group communication technique designed to obtain opinions from a panel of selected experts on specifi c issues through the sending of questionnaires to be completed within a specifi ed time. The experts are contacted individually and they do not know other group participants and their opinions-the aim is to submit the group participants to the same conditions. Participants do not meet personally, thereby avoiding undue infl uence. The process foresees the following points: The process is repeated a number of times, until a convergence of all group members is obtained. The process ends with analysis of the answers and formulation of the fi nal report. |
(Adapted from http://www.bigproject. org/dephi.htm) Thomas E. Novotny*, Emilio Mordini, Ruth Chadwick, J. Martin Pedersen, Fabrizio Fabbri, Reidar Lie, Natapong Thanachaiboot, |
to provide a longer-term, strategic perspective on the four selected public health-related issues. The fi nal conclusions will be presented to a high-level meeting of European Union (EU) policy makers in 2006; these conclusions may then inform future research directions and stimulate additional critical thinking about globalization and its bioethical implications for health policy. This article presents preliminary results from the BIG Project. |
Mobility results from the increasing ease of domestic and international travel as well as from instantaneous access to information through the Internet and other electronic resources. Mobility may involve the pursuit of a better quality of life, development of markets for traded goods and services, return of resources to home countries, and improvement of professional and business networks. |
However, migration may also affect psychological and physical health as a result of confl ict, famine, poverty, and the insuffi cient cultural or economic integration of migrants within their new home society. It may contribute to the spread of infectious diseases across borders ( Figure 1 ). The recent epidemic of SARS was a classic example of an infectious disease propagated through the movement of people across borders; it required attention from the original site to control migration (quarantine) as well as vigilance by secondary sites to protect their populations ( Figure 2 ) [4] . For these and other reasons, the International Organization for Migration is increasingly concerned with migratory patterns and their health consequences in a globalizing world (for an illustration of the emerging confl ict of ideas, see http:⁄⁄www.iom. int and http:⁄⁄www.noborder.org/ iom/index.php). |
Cross-border health commerce is related to mobility. In Europe, this commerce is likely to increase as the EU enlarges to include Eastern and Central European nations. Such commerce may include the movement of health providers from East to West as well as "medical tourism" in pursuit of less costly or more accessible high-quality health care. In addition, international trade in illegal health products and inconsistent regulatory and safety standards for exports may threaten public health, especially in unregulated pharmaceutical markets. |
Ethical concerns may also result from the vast growth in international tourist travel. Such travel now accounts for a twelfth of world trade, supporting an economy the size of a middle-income country [5] . Tourism may provide substantial economic benefi ts to many developing countries, and it may improve cultural understanding among travelers. However, these benefi ts require an ethical concern for the environment and for persons employed in the tourist industry. |
The rights of nations to protect against infectious disease and unsafe medical practices, as well as the rights of human beings displaced by war, traffi cking, and economic and cultural disruption, are critical concerns for health policy makers. Poverty and social disparities are key factors in the growth of global migration. Therefore, it is timely to consider whether mobility is a human right, and whether those who migrate have rights to health care in their new country. These questions should be considered by health policy makers within the ethical contexts of individual autonomy, social justice, nonmalefi cence, and benefi cence. |
Technology drives globalization and in turn is driven by globalization. However, there is considerable ambiguity as to the value of technological globalization, especially for health in low-income countries. The "digital divide" may be important in improving health or income disparities as the electronic revolution provides scientists and health workers in both the developed and developing world with unprecedented access to information. Much could be done to reduce information inequities for the developing world through collective international action, but new global governance mechanisms may be needed to achieve this goal for information technology [6] . |
Interestingly, the Internet is a structural necessity for fi nancial and corporate globalization, but the same technology is used by nongovernmental organizations, political groups, and cultural movements to support grassroots social justice and human rights campaigns against these globalizing corporations. Neither side in this struggle would advocate limitations to the expansion of Internet technology, but both sides need to consider the bioethical implications of increased information access. |
On the other hand, the ethical issues surrounding genomics (with both environmental and human concerns) are quite ambiguous. While there may be signifi cant benefi ts to identifying genetically benefi cial products or genetic determinants of disease, there are also concerns about altering natural environments and about collecting routine genetic information from general populations [7] . |
For example, some experts assert that genetically modifi ed (GM) crops will signifi cantly increase crop yields without requiring any additional farmland, thus preserving valuable rain forests and animal habitats. herbicides, and pesticides. Farmers are not allowed to trade or save GM seed from one harvest to the next, and "terminator technology" (producing grains that are genetically modifi ed so that they cannot be used to generate new crops) is under development. (See http:⁄⁄www.globalissues.org/ EnvIssues/GEFood/Terminator.asp for more information on this technology.) Thus, ethical considerations of distributive justice and benefi cence must be considered in the debate about the global applicability of GM crops. |
For the pharmaceutical and health care industries, genetic testing could provide information about the shape of future markets and the possible tailoring of specifi c pharmacotherapy to genomic susceptibility. For governments, genetic testing may provide predictive information on a population basis that could aid future health care planning. Genetic information might also be similarly used by the insurance industry, but the identifi cation of genetically "high-risk" individuals would likely interfere with their autonomy, in that they may not be able to purchase health insurance. For example, the Apolipoprotein E test may indicate that an individual has two copies of one form (allele e4) of the gene that leads to Alzheimer disease [9] . Could this information be used by insurance companies or possible employers to deny insurability, despite no current adverse health effects? |
In the post-genomic era there is potential to both reduce and increase health inequities, but much will depend on how ethical issues are addressed. If interventions to increase the life span for those with access to high quality health care must compete with expensive investments in genetic research on infectious diseases (which affect the poor most of all), health inequalities may be amplifi ed between those with access and those without access to health care. If research participants or patients in low-income countries have unequal access to information, they may not be properly informed about genetic testing and the counseling needed if adverse genetic information is found. Population-based genomic research may characterize groups of people in such a way that encourages discrimination. Such research may also lead to disputes about ownership of genetic resources from participant populations. Health professionals must have a solid grounding in bioethical issues as they make clinical decisions based on genetic information. However, health policy makers and global governance structures must also be accountable for the potential adverse consequences such decisions might engender. |
One may ask: will genomic science really help developing nations? To what extent can benefi ts be shared? Will pharmaceutical and biotechnology companies invest in poor countries if they can make more money working on therapeutics for high-income countries? Thus, concern for the bioethical issues of social justice and benefi cence arises. Genomics has the potential to be a global public good, but there is considerable uncertainty as to its bioethical justifi cation in all cultures [10] . |
In general, globalization helps liberalize trade through removal of import restrictions and tariffs, through removal of restrictions on trade in services, and through linkage of trade sanctions to the protection of intellectual property rights. All these activities may have an impact on population health. |
Defenders of trade liberalization claim that this process is one of the most effective means of increasing a country's wealth and, by extension, population health. Even if this were always true, there may be specifi c policies that have particularly detrimental effects on health (such as opening markets to trade in manufactured tobacco products). Further, there may be an ethical argument based on social justice against some trade liberalization policies. If, for example, trade liberalization between rich and poor countries produces proportionally more wealth in rich countries compared with poor countries, this may suggest a socially unjust result of liberalization; poor countries' economies may not grow as fast as rich countries' economies in this situation. |
The relationship between wealth and health is actually somewhat controversial: the so-called Preston curves demonstrate a dramatic relationship between health and economic prosperity up to about a Purchasing Power Parity of US$3,000 per capita per year [11] . However, there are cheap, cost-effective approaches to population health (such as vaccination, clean water, and sewage disposal) that may not be affected by the increase in Purchasing Power Parity. These approaches were relatively more important than economic development per se in early 20thcentury interventions in developed countries, and they are likely to be more important for infl uencing health among developing country populations today than simple economic growth. On the other hand, high-intensity technological improvement rather than economic growth may be more important to health in rich countries compared with developing countries. |
The concern for intellectual property rights in trade has been an extraordinarily contentious issue in recent years. Newer drugs that are effective against diseases in resourcepoor but highly impacted countries, such as antiretroviral drugs against HIV, have been prohibitively expensive in these countries, in part because of patent protections. With the Trade-Related Intellectual Property agreement, patent protection became linked to trade policy; if countries in need of cheaper essential drugs did not conform to patent rules, trade retaliation from exporting countries might ensue. However, restrictions on poor countries' responses to legitimate public health emergencies may be unethical on the basis of distributive justice, nonmalefi cence, and benefi cence. Exceptions for public health emergencies (such as HIV/AIDS) under the Trade-Related Intellectual Property agreement include the right to compulsory licensing (local companies produce patented medicines in exchange for a royalty payment to the patent holder) or parallel importing (importing patented drugs sold more cheaply elsewhere) that will make essential medicines more available to highly impacted countries without fear of trade retaliation from the originating country [12] . |
The General Agreement on Trade in Services is a relatively new treaty that covers trade in health services [13] . The agreement has been severely criticized by some, who claim that it increases privatization of health care services and undermines public health care systems. However, given its ambiguities, the actual impact of the agreement on the health sector will be largely determined by the way in which the agreement is further specifi ed in multinational commitments [14] . Social justice, equity, benefi cence, and nonmalefi cence will all come into play in the implementation of this treaty. |
Concerns for security against biological weapons have recently arisen among both poor and wealthy nations. Some, however, question the enormous sums now being spent to address the perceived threats due to bioterrorism even without strong evidence for actual threats. Even without such evidence, global bioethical principles at least suggest the need for a framework for consideration of distributive justice in this arena. |
For example, should a nation with a limited supply of a vaccine against weaponized smallpox offer its stockpiles to a neighboring country that is under direct attack? This case is complicated by the fact that the infection could spread to its own territory. In the case of widespread biological attacks, which global governing agency, country, or other entity would be responsible for global resource allocation? Clearly, risks from bioweapons are trans-border, but resources may be unevenly and inequitably distributed, requiring a bioethically based policy determination on a global basis [15] . |
A further concern with respect to biomedical research is the issue of dual-use technology development for health benefi ts as well as for possible bioweapons. Governments must balance the secrecy necessary for security with the need for disclosure of information that is essential for research and development in health. It is very diffi cult to sequester new knowledge that might be applied to building biological weapons without simultaneously impeding research on defense against those bioweapons and on other benefi cial biomedical advances. Most BIG Project scientists agree that the benefi ts of releasing scientifi c information in general outweigh the risk of its misuse. However, the scientifi c community needs to consider whether new codes of conduct are necessary or whether existing governance is suffi cient to support a bioethical approach to research on possible dualuse technologies. |
Global bioethical challenges require careful theoretical deliberation and practical considerations for international health policies [16] . The BIG Project seeks to guide these processes in four selected areas of interest to the EU, so that the project results may be helpful to policy makers at local, national, and international levels. |
The BIG Project has found that bioethical principles are important in considerations of migration, trade, information technology, genomics, and bioweapons threats. Globalization in these arenas is neither a right nor a wrong process, but it demands careful consideration of bioethical principles including social justice, benefi cence, nonmalefi cence, and individual autonomy. These concerns may not be immediately obvious to health policy makers, and thus the BIG Project results may help clarify the larger goals and purposes of bioethically based health policy development within the EU and elsewhere. More information about the BIG Project can be found at http:⁄⁄www. bigproject.org/project.htm. Public awareness of risk factors for cancer among the Japanese general population: A population-based survey BACKGROUND: The present study aimed to provide information on awareness of the attributable fraction of cancer causes among the Japanese general population. METHODS: A nationwide representative sample of 2,000 Japanese aged 20 or older was asked about their perception and level of concern about various environmental and genetic risk factors in relation to cancer prevention, as a part of an Omnibus Survey. Interviews were conducted with 1,355 subjects (609 men and 746 women). RESULTS: Among 12 risk factor candidates, the attributable fraction of cancer-causing viral and bacterial infection was considered highest (51%), followed by that of tobacco smoking (43%), stress (39%), and endocrine-disrupting chemicals (37%). On the other hand, the attributable fractions of cancer by charred fish and meat (21%) and alcohol drinking (22%) were considered low compared with other risk factor candidates. For most risk factors, attributable fraction responses were higher in women than in men. As a whole, the subjects tended to respond with higher values than those estimated by epidemiologic evidence in the West. The attributable fraction of cancer speculated to be genetically determined was 32%, while 36% of cancer was considered preventable by improving lifestyle. CONCLUSION: Our results suggest that awareness of the attributable fraction of cancer causes in the Japanese general population tends to be dominated by cancer-causing infection, occupational exposure, air pollution and food additives rather than major lifestyle factors such as diet. In Japan, cancer has been recognized as a major component of the overall pattern of disease for decades. Thus, the importance of cancer prevention by lifestyle modification should now be strongly acknowledged. |
Internationally, several studies have estimated the proportion of total cancer deaths attributable to various risk factors based on epidemiologic evidence [1, 2] , and various international guidelines and recommendations derived from these have appeared [3] [4] [5] [6] . Not surprisingly, domestic guidelines and recommendations for cancer prevention in Japan such as the 'Twelve recommendations for cancer prevention [7]' and 'Healthy People Japan 21 [8] ' have been significantly influenced by these reports. |
Public awareness of risk factors in relation to cancer prevention has been surveyed in only a few countries [9, 10] , and results have demonstrated poor awareness. Other studies focusing on specific cancers only have also appeared [11] [12] [13] [14] . However, none of these studies quantitatively evaluated public awareness of the attributable fraction of individual risk factors. |
In Japan, it appears that most people are aware of the major risk factors of cancer. Although we are unaware of any published evidence, however, public knowledge and information on cancer prevention now seems influenced largely by the mass media and other sources, rather than by information provided directly by health professionals, resulting in a distorted picture of causation. Cancer control policy therefore urgently requires a clarification of the discrepancies which now exist between ideal levels of public concern about risk factors and the current reality, particularly public health policy makers in their formulation of cancer control measures. To address this need, the present study was designed to provide information on awareness of the attributable fraction of cancer causes among the Japanese general population. Since we are interested in quantitatively estimating the awareness of preventability, we placed special emphasis on gauging awareness by attributable fraction of cancer. |
The study was conducted as a part of an omnibus survey in December, 2003, by commission to a polling agency. The omnibus survey is a monthly multipurpose cross-sectional survey which includes public opinion research, social research, scientific research, market research, and others. Using a stratified two-stage sampling method, a total of 2,000 people aged 20 or older were randomly selected as study subjects, from 160 districts selected from area units representing 12 geographical blocks (Hokkaido, Tohoku, Kanto, Keihin, Koshinetu, Hokuriku, Tokai, Kinki, Hanshin, Chugoku, Shikoku, Kyushu) and 3 types of city scale (14 metropolises, other cities, towns and villages) in proportion to the population distribution as at March 2002. After an initial visit to obtain oral informed consent and schedule a visit for the interview, the survey was conducted by face-to-face interview using trained interviewers in each district. The omnibus survey does not collect any personally identifiable information such as name, date of birth or address details at interview. For the present report, we obtained the electronic data file for the relevant interview component, with no personal identifiers. Ethical approval was not applicable to the present study under the Japanese ethical guidelines for epidemiologic studies, which comply with the declaration of Helsinki. |
Among the 2,000 people selected for survey (977 men, 1,023 women), interviews were successfully obtained The questionnaire of this survey comprised questions on the awareness of various environmental and genetic risk factors in relation to cancer prevention by enquiring about the attributable fraction of cancer. Fractions were: 1) 12 risk factor candidates, namely alcoholic beverages, unbalanced diet, use of food additives and pesticide chemicals, charred fish and meat, tobacco smoking, obesity, physical inactivity, endocrine-disrupting chemicals, air pollution such as diesel emissions, occupational exposure, cancer-causing viral and bacterial infection, and stress; 2) genetic factors in general; and 3) the preventable fraction of cancer occurrence by lifestyle modification [see Additional file 1]. |
The first question asked about the preventable fraction of cancer which would result in Japan if each factor were completely and totally eliminated, using the fine categories of <5%, 5 to <10%, 10 to <15%, 15 to <20%, 20 to <25%, 25 to <30%, 30 to <40%, 40 to <50%, 50 to <60%, 60 to <70%, 70 to <80%, 80 to <90%, and 90 to 100%. These categories were exhibited together on a pie chart. |
These risk factor candidates were selected with reference to previous international and domestic recommendations and guidelines [1] [2] [3] [4] [5] [6] [7] [8] . The second question asked about the fraction of cancer genetically predetermined using the same categories as the first, while the third asked about the preventable fraction of cancer by modification of lifestyle using estimation of an actual percent value. In addition to these questions, subjects were also asked about their smoking and drinking practices, and occupational and educational status. |
Mean values of the attributable fractions were calculated for each risk factor of cancer and compared by demographic and habitual smoking and drinking status. For analyses, the mid-values of each category were assigned for categorical variables. All analyses were performed using Stata statistical software, S/E Version 8 [15] . |
A total of 1,355 (67.8%) subjects responded to the survey, with a higher response rate in women (72.9%) than in men (62.3%). Response rate was lower in the 20s age strata than in the other age groups, but no trend to an increase in response rate with increasing age was observed. Overall, no significant difference in area and age distribution was seen between the sampled population and survey respondents. Response rate tended to be lower among subjects who reside in the Kanto region and in cities other than the 14 metropolises than among other subjects ( Table 1) . |
Characteristics of the 1,355 respondents (609 men, 746 women) are presented in Table 2 . The proportion of current smokers was 44% in men and 15% in women, and decreased with age in both genders. In female subjects aged in their 20s, 26% currently smoke and 49% drink alcohol beverages at least 4 times a week. |
Awareness of the attributable fraction of cancer causes among the Japanese general population is presented in Table 3 . Among the 12 risk factor candidates, the attributable fraction was considered highest for cancer-causing viral and bacterial infection (51.3%), followed by tobacco smoking (43.0%), stress (39.0%), and endocrine-disrupting chemicals (37.1%). In contrast, the attributable fraction of charred fish and meat (21.4%) and alcohol drinking (21.7%) were considered low compared with other risk factor candidates. The attributable fraction of other risk factor candidates such as occupational exposure, air pollution, food additives and pesticides, unbalanced diet, obesity and physical activity ranked between the high and low fractions. The attributable fraction responses tended to be higher in women than in men, and were increased among inhabitants of larger cities and in homemakers and decreased in those engaged in agriculture, forestry and fisheries. In contrast, risk factor candidate rankings were similar by gender, age group, city scale, and educational and occupational status. In men, those who neither smoke nor drink tended to consider the preventive fraction of the risk factors higher than those who both smoke and drink, whereas in women, the former subjects considered the values lower than the latter. |
The speculated fraction of cancer which is genetically determined was 31.5% as an average (Table 3 ). This fraction was higher in current heavy smokers and former drinkers, and lower in homemakers and students. On the other hand, an average 35.5% of cancer were considered preventable by lifestyle improvement, with this ratio being higher in homemakers, former smokers, and never and former drinkers. |
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